Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein
Identifieur interne : 000A27 ( Pmc/Corpus ); précédent : 000A26; suivant : 000A28Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein
Auteurs : T. A. Ozharovskaia ; O. V. Zubkova ; I. V. Dolzhikova ; A. S. Gromova ; D. M. Grousova ; A. I. Tukhvatulin ; O. Popova ; D. V. Shcheblyakov ; D. N. Scherbinin ; A. S. Dzharullaeva ; A. S. Erokhova ; M. M. Shmarov ; S. Y. Loginova ; S. V. Borisevich ; B. S. Naroditsky ; D. Y. Logunov ; A. L. GintsburgSource :
- Acta Naturae [ 2075-8251 ] ; 2019.
Abstract
The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 during the first Middle East respiratory syndrome (MERS) outbreaks. MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality rate of ~35.5%. Currently, there are no registered vaccines or means of therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein plays the most important role in the viral life cycle (virus internalization). The S protein is an immunodominant antigen and the main target for neutralizing antibodies. In the present study, the immunogenicities of five different forms of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein, the full-length S glycoprotein with the transmembrane domain of the G glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5) were used as delivery vehicles. Vaccination with all of the developed rAd5 vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral immune response was induced after the animal had been vaccinated with the membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing antibodies among all vaccinated animals. The most significant cellular immune response was induced after vaccination of the animals with the full-length S (rAd5-S). These investigations suggest that the full-length S and the membrane form of the RBD (RBD-G) are the most promising vaccine candidates among all the studied forms of S glycoprotein.
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PubMed: 31024747
PubMed Central: 6475872
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein</title><author><name sortKey="Ozharovskaia, T A" sort="Ozharovskaia, T A" uniqKey="Ozharovskaia T" first="T. A." last="Ozharovskaia">T. A. Ozharovskaia</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Zubkova, O V" sort="Zubkova, O V" uniqKey="Zubkova O" first="O. V." last="Zubkova">O. V. Zubkova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Dolzhikova, I V" sort="Dolzhikova, I V" uniqKey="Dolzhikova I" first="I. V." last="Dolzhikova">I. V. Dolzhikova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Gromova, A S" sort="Gromova, A S" uniqKey="Gromova A" first="A. S." last="Gromova">A. S. Gromova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Grousova, D M" sort="Grousova, D M" uniqKey="Grousova D" first="D. M." last="Grousova">D. M. Grousova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Tukhvatulin, A I" sort="Tukhvatulin, A I" uniqKey="Tukhvatulin A" first="A. I." last="Tukhvatulin">A. I. Tukhvatulin</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Popova, O" sort="Popova, O" uniqKey="Popova O" first="O." last="Popova">O. Popova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Shcheblyakov, D V" sort="Shcheblyakov, D V" uniqKey="Shcheblyakov D" first="D. V." last="Shcheblyakov">D. V. Shcheblyakov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Scherbinin, D N" sort="Scherbinin, D N" uniqKey="Scherbinin D" first="D. N." last="Scherbinin">D. N. Scherbinin</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Dzharullaeva, A S" sort="Dzharullaeva, A S" uniqKey="Dzharullaeva A" first="A. S." last="Dzharullaeva">A. S. Dzharullaeva</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Erokhova, A S" sort="Erokhova, A S" uniqKey="Erokhova A" first="A. S." last="Erokhova">A. S. Erokhova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Shmarov, M M" sort="Shmarov, M M" uniqKey="Shmarov M" first="M. M." last="Shmarov">M. M. Shmarov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Loginova, S Y" sort="Loginova, S Y" uniqKey="Loginova S" first="S. Y." last="Loginova">S. Y. Loginova</name><affiliation><nlm:aff id="AF2">«48 Central Research Institute», Ministry of Defense of Russian Federation, Oktyabrskaya Str. 11, 141306 , Sergiev Posad, Russia</nlm:aff></affiliation></author><author><name sortKey="Borisevich, S V" sort="Borisevich, S V" uniqKey="Borisevich S" first="S. V." last="Borisevich">S. V. Borisevich</name><affiliation><nlm:aff id="AF2">«48 Central Research Institute», Ministry of Defense of Russian Federation, Oktyabrskaya Str. 11, 141306 , Sergiev Posad, Russia</nlm:aff></affiliation></author><author><name sortKey="Naroditsky, B S" sort="Naroditsky, B S" uniqKey="Naroditsky B" first="B. S." last="Naroditsky">B. S. Naroditsky</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Logunov, D Y" sort="Logunov, D Y" uniqKey="Logunov D" first="D. Y." last="Logunov">D. Y. Logunov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Gintsburg, A L" sort="Gintsburg, A L" uniqKey="Gintsburg A" first="A. L." last="Gintsburg">A. L. Gintsburg</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31024747</idno><idno type="pmc">6475872</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475872</idno><idno type="RBID">PMC:6475872</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000A27</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A27</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein</title><author><name sortKey="Ozharovskaia, T A" sort="Ozharovskaia, T A" uniqKey="Ozharovskaia T" first="T. A." last="Ozharovskaia">T. A. Ozharovskaia</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Zubkova, O V" sort="Zubkova, O V" uniqKey="Zubkova O" first="O. V." last="Zubkova">O. V. Zubkova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Dolzhikova, I V" sort="Dolzhikova, I V" uniqKey="Dolzhikova I" first="I. V." last="Dolzhikova">I. V. Dolzhikova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Gromova, A S" sort="Gromova, A S" uniqKey="Gromova A" first="A. S." last="Gromova">A. S. Gromova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Grousova, D M" sort="Grousova, D M" uniqKey="Grousova D" first="D. M." last="Grousova">D. M. Grousova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Tukhvatulin, A I" sort="Tukhvatulin, A I" uniqKey="Tukhvatulin A" first="A. I." last="Tukhvatulin">A. I. Tukhvatulin</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Popova, O" sort="Popova, O" uniqKey="Popova O" first="O." last="Popova">O. Popova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Shcheblyakov, D V" sort="Shcheblyakov, D V" uniqKey="Shcheblyakov D" first="D. V." last="Shcheblyakov">D. V. Shcheblyakov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Scherbinin, D N" sort="Scherbinin, D N" uniqKey="Scherbinin D" first="D. N." last="Scherbinin">D. N. Scherbinin</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Dzharullaeva, A S" sort="Dzharullaeva, A S" uniqKey="Dzharullaeva A" first="A. S." last="Dzharullaeva">A. S. Dzharullaeva</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Erokhova, A S" sort="Erokhova, A S" uniqKey="Erokhova A" first="A. S." last="Erokhova">A. S. Erokhova</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Shmarov, M M" sort="Shmarov, M M" uniqKey="Shmarov M" first="M. M." last="Shmarov">M. M. Shmarov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Loginova, S Y" sort="Loginova, S Y" uniqKey="Loginova S" first="S. Y." last="Loginova">S. Y. Loginova</name><affiliation><nlm:aff id="AF2">«48 Central Research Institute», Ministry of Defense of Russian Federation, Oktyabrskaya Str. 11, 141306 , Sergiev Posad, Russia</nlm:aff></affiliation></author><author><name sortKey="Borisevich, S V" sort="Borisevich, S V" uniqKey="Borisevich S" first="S. V." last="Borisevich">S. V. Borisevich</name><affiliation><nlm:aff id="AF2">«48 Central Research Institute», Ministry of Defense of Russian Federation, Oktyabrskaya Str. 11, 141306 , Sergiev Posad, Russia</nlm:aff></affiliation></author><author><name sortKey="Naroditsky, B S" sort="Naroditsky, B S" uniqKey="Naroditsky B" first="B. S." last="Naroditsky">B. S. Naroditsky</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Logunov, D Y" sort="Logunov, D Y" uniqKey="Logunov D" first="D. Y." last="Logunov">D. Y. Logunov</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author><author><name sortKey="Gintsburg, A L" sort="Gintsburg, A L" uniqKey="Gintsburg A" first="A. L." last="Gintsburg">A. L. Gintsburg</name><affiliation><nlm:aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</nlm:aff></affiliation></author></analytic><series><title level="j">Acta Naturae</title><idno type="ISSN">2075-8251</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in
2012 during the first Middle East respiratory syndrome (MERS) outbreaks.
MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality
rate of ~35.5%. Currently, there are no registered vaccines or means of
therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein
plays the most important role in the viral life cycle (virus internalization).
The S protein is an immunodominant antigen and the main target for neutralizing
antibodies. In the present study, the immunogenicities of five different forms
of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein,
the full-length S glycoprotein with the transmembrane domain of the G
glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S
glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane
domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human
IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5)
were used as delivery vehicles. Vaccination with all of the developed rAd5
vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral
immune response was induced after the animal had been vaccinated with the
membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the
glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing
antibodies among all vaccinated animals. The most significant cellular immune
response was induced after vaccination of the animals with the full-length S
(rAd5-S). These investigations suggest that the full-length S and the membrane
form of the RBD (RBD-G) are the most promising vaccine candidates among all the
studied forms of S glycoprotein.
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Acta Naturae</journal-id><journal-id journal-id-type="iso-abbrev">Acta Naturae</journal-id><journal-id journal-id-type="publisher-id">ActaNaturae</journal-id><journal-title-group><journal-title>Acta Naturae</journal-title></journal-title-group><issn pub-type="ppub">2075-8251</issn><publisher><publisher-name>A.I. Gordeyev</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31024747</article-id><article-id pub-id-type="pmc">6475872</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject><subj-group><subject>Molecular Biology</subject></subj-group></subj-group></article-categories><title-group><article-title>Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ozharovskaia</surname><given-names>T. A.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Zubkova</surname><given-names>O. V.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Dolzhikova</surname><given-names>I. V.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Gromova</surname><given-names>A. S.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Grousova</surname><given-names>D. M.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Tukhvatulin</surname><given-names>A. I.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Popova</surname><given-names>O.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Shcheblyakov</surname><given-names>D. V.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Scherbinin</surname><given-names>D. N.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Dzharullaeva</surname><given-names>A. S.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Erokhova</surname><given-names>A. S.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Shmarov</surname><given-names>M. M.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Loginova</surname><given-names>S. Y.</given-names></name><xref ref-type="aff" rid="AF2"></xref></contrib><contrib contrib-type="author"><name><surname>Borisevich</surname><given-names>S. V.</given-names></name><xref ref-type="aff" rid="AF2"></xref></contrib><contrib contrib-type="author"><name><surname>Naroditsky</surname><given-names>B. S.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib><contrib contrib-type="author"><name><surname>Logunov</surname><given-names>D. Y.</given-names></name><xref ref-type="aff" rid="AF1"></xref><email>logunov@gamaleya.org</email></contrib><contrib contrib-type="author"><name><surname>Gintsburg</surname><given-names>A. L.</given-names></name><xref ref-type="aff" rid="AF1"></xref></contrib></contrib-group><aff id="AF1">Federal Research Centre of Epidemiology and Microbiology named after Honorary Academician N. F. Gamaleya, Ministry of Health of Russian Federation, Gamaleya Str. 18, 123098, Moscow, Russia</aff><aff id="AF2">«48 Central Research Institute», Ministry of Defense of Russian Federation, Oktyabrskaya Str. 11, 141306 , Sergiev Posad, Russia</aff><pub-date pub-type="ppub"><season>Jan-Mar</season><year>2019</year></pub-date><volume>11</volume><issue>1</issue><fpage>38</fpage><lpage>47</lpage><history><date date-type="received"><day>30</day><month>10</month><year>2018</year></date></history><permissions><copyright-statement>Copyright ® 2019 National Research University Higher School of Economics.</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0/"><license-p>This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is
properly cited.
</license-p></license></permissions><abstract><p>The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in
2012 during the first Middle East respiratory syndrome (MERS) outbreaks.
MERS-CoV causes an acute lower-respiratory infection in humans, with a fatality
rate of ~35.5%. Currently, there are no registered vaccines or means of
therapeutic protection against MERS in the world. The MERS-CoV S glycoprotein
plays the most important role in the viral life cycle (virus internalization).
The S protein is an immunodominant antigen and the main target for neutralizing
antibodies. In the present study, the immunogenicities of five different forms
of the MERS-CoV S glycoprotein were compared: the full-length S glycoprotein,
the full-length S glycoprotein with the transmembrane domain of the G
glycoprotein of VSV (S-G), the receptor-binding domain (RBD) of the S
glycoprotein, the membrane-fused RBD (the RBD fused with the transmembrane
domain of the VSV G glycoprotein (RBD-G)), and the RBD fused with Fc of human
IgG1 (RBD-Fc). Recombinant vectors based on human adenoviruses type 5 (rAd5)
were used as delivery vehicles. Vaccination with all of the developed rAd5
vectors elicited a balanced Th1/Th2 response in mice. The most robust humoral
immune response was induced after the animal had been vaccinated with the
membrane-fused RBD (rAd5-RBD-G). Only immunization with membrane forms of the
glycoprotein (rAd5-S, rAd5-S-G, and rAd5-RBD-G) elicited neutralizing
antibodies among all vaccinated animals. The most significant cellular immune
response was induced after vaccination of the animals with the full-length S
(rAd5-S). These investigations suggest that the full-length S and the membrane
form of the RBD (RBD-G) are the most promising vaccine candidates among all the
studied forms of S glycoprotein.
</p></abstract><kwd-group kwd-group-type="author"><kwd>Middle East respiratory syndrome</kwd><kwd>MERS</kwd><kwd>MERS-CoV</kwd><kwd>glycoprotein</kwd><kwd>adenoviral vector</kwd><kwd>immunity</kwd></kwd-group></article-meta></front><body><sec><title>INTRODUCTION</title><p>The Middle East respiratory syndrome (MERS) is an acute inflammatory
respiratory infection that was first identified in Saudi Arabia in June 2012.
The causative agent of MERS was officially called the Middle East respiratory
syndrome coronavirus (MERS-CoV) in 2013
[<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R2" ref-type="bibr">2</xref>].
MERS-CoV is a single-stranded RNA virus belonging to the Coronaviridae family, genus
Betacoro navirus. Dromedary camels are the natural reservoir of MERS-CoV; viral
transmission to humans occurs during consumption of unpasteurized camel milk;
airborne transmission is also possible
[<xref rid="R3" ref-type="bibr">3</xref>-<xref rid="R5" ref-type="bibr">5</xref>].
</p><p>To date, a total of 2,260 laboratory-confirmed cases of MERS-CoV infection have
been reported, including 803 deaths [<xref rid="R6" ref-type="bibr">6</xref>,
<xref rid="R7" ref-type="bibr">7</xref>]. Due to its high mortality rate (~
35.5%) [<xref rid="R6" ref-type="bibr">6</xref>], combined with a wide
distribution of the reservoir and absence of effective preventive drugs or
treatment, WHO experts classify MERS-CoV as a virus with the potential to cause
a pandemic [<xref rid="R8" ref-type="bibr">8</xref>]. Therefore, vaccine
development is necessary in order to stave off such a pandemic.
</p><p>The main protective MERS-CoV antigen is the S glycoprotein presented as a
trimer on the virus surface. The S glycoprotein plays an important role in the
virus’ internalization into the cell [<xref rid="R9" ref-type="bibr">9</xref>].
The S glycoprotein is subdivided into two subunits: the S1
subunit containing the receptor-binding domain (RBD), and the S2 subunit
responsible for the fusion of the virus and cell membrane
[<xref rid="R10" ref-type="bibr">10</xref>-<xref rid="R13" ref-type="bibr">13</xref>].
These features make the S protein an important target for MERS-CoV vaccine
development [<xref rid="R14" ref-type="bibr">14</xref>-<xref rid="R17" ref-type="bibr">17</xref>]. The RBD of the MERS-CoV S glycoprotein is a key target
for the development of preventive and therapeutic means against MERS [<xref rid="R18" ref-type="bibr">18</xref>-<xref rid="R20" ref-type="bibr">20</xref>],
because the RBD mediates the interaction between MERS-CoV and the receptor DPP4
on the cell surface.
</p><p>In order to develop an effective MERS-CoV vaccine, it is important to
understand which form of the glycoprotein to include in the vaccine to provide
protection against MERS. There are data that demonstrate the immunogenicity of
various forms of the MERS-CoV glycoprotein
[<xref rid="R21" ref-type="bibr">21</xref>-<xref rid="R25" ref-type="bibr">25</xref>].
However, the question of which form is preferable for a vaccine remains open, since an
antigen panel has not been tested under the same conditions. In order to
address this knowledge gap, we constructed five recombinant vectors based on
human adenovirus type 5 (rAd5) expressing different forms of the MERS-CoV S
glycoprotein:
</p><p>– the full-length S glycoprotein localized in the endoplasmic reticulum
(ER);
</p><p>– two secreted variants of the receptor binding domain of the MERS-CoV S
glycoprotein containing the alkaline phosphatase leader peptide (the RBD and
the RBD fused with Fc of human IgG1 to increase stability and immunogenicity
[<xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R19" ref-type="bibr">19</xref>]); and
</p><p>– two transmembrane (TM) forms localized in the plasma membrane of the
cell: either the full-length S or the RBD with the TM domain of the vesicular
stomatitis virus (VSV) G glycoprotein. Because the full-length S glycoprotein
is localized in the ER [<xref rid="R26" ref-type="bibr">26</xref>], we
constructed an S-G variant with a deleted ER localization signal.
</p><p>We chose a platform based on the recombinant viral vectors rAd5 for delivering
the S glycoprotein, because such vectors can efficiently deliver the transgene
to multiple cell types [<xref rid="R27" ref-type="bibr">27</xref>, <xref rid="R28" ref-type="bibr">28</xref>], their genome has been fully
characterized, they are able to grow to high titers [<xref rid="R29" ref-type="bibr">29</xref>], and they can induce a strong humoral and cellular immune
response [<xref rid="R30" ref-type="bibr">30</xref>, <xref rid="R31" ref-type="bibr">31</xref>].
</p><p>The present study compares the humoral and cellular immune responses induced by
vaccination of mice with rAd5 carrying different forms of the MERS-CoV S
glycoprotein.
</p></sec><sec><title>EXPERIMENTAL</title><p><bold>Cell lines</bold></p><p>The HEK293 and A549 cell lines were obtained from the Russian collection of
vertebrate cell lines (Russia). The HEK293T and Vero E6 cells were obtained
from ATCC (USA). All cells were cultured in DMEM (Dulbecco’s modified
Eagle medium) supplemented with 10% fetal bovine serum at 37°C with 5%
CO<sub>2</sub>.
</p><p><bold>Construction of recombinant adenoviral particles expressing different
forms of the MERS-CoV S glycoprotein</bold></p><p>The MERS-CoV S glycoprotein amino acid sequences of strains (2015–2017)
were obtained from the NCBI database [<xref rid="R32" ref-type="bibr">32</xref>]. The consensus sequence of the MERS-CoV S glycoprotein was
made on the basis of amino acid sequences using the Geneious® 10.2.3
software. The nucleotide sequences of different glycoprotein forms were
optimized for expression in mammalian cell lines and synthesized by Evrogen JSC
(Russia). Five recombinant plasmids (pAd5-S, pAd5- S-G, pAd5-RBD, pAd5-RBD-G,
and pAd5-RBD-Fc) were generated. rAd5 was obtained according to the procedure
described previously [<xref rid="R33" ref-type="bibr">33</xref>].
</p><p><bold>Generation of lentiviral particles pseudotyped with MERS-CoV S
glycoprotein (pseudoviruses)</bold></p><p>HEK293T cells were seeded in 15-cm culture Petri dishes and co-transfected with
three plasmids (pCMVΔR8,2; pLV-CMV-EGFP; pCMV-MERS-CoV-S) to obtain
pseudoviruses. Seventy-two hours later, the supernatants were collected,
filtered, divided into aliquots, and stored at –80°C. Vero E6 cells
were used for titrating the pseudoviruses. The titer of the pseudotyped virus
was determined in terms of focus-forming units (FFUs).
</p><p><bold>Evaluating the expression of different forms of the MERS-CoV S
glycoprotein by western blotting</bold></p><p>HEK293 cells were seeded in 35 mm Petri dishes and incubated overnight to 70%
confluence. Then, rAd5 were added to the cells at 100 PFUs/cell. rAd5-null was
used as a control virus. After 24 h, the expression of different forms of the
MERS-CoV S glycoprotein was evaluated by western blotting using S-specific
antibodies (40069-RP02, Sino Biological, China) and antibodies specific to
rabbit IgG (NA934V, GE, Great Britain). Expression of the membrane-fused forms
of the glycoprotein (S, S-G, RBD-G) was detected in cell lysates prepared using
the Cell Culture Lysis Reagent (Promega, USA). Expression of the secreted
versions of the glycoprotein (RBD, RBD-Fc) was evaluated in the culture medium.
Lysate samples were loaded onto wells (10 μg of total protein in a volume
of 10 μl/well). Samples of the medium were loaded in volume 10
μl/well.
</p><p><bold>Laboratory animals</bold></p><p>All animal experiments were performed in strict accordance with the
recommendations of the National Standard of the Russian Federation (GOST R
53434–2009; Principles of Good Laboratory Practice). Six-week-old female
C57/BL6 mice (18–20 g) were obtained from the Pushchino Breeding Facility
(Russia). The mice had free access to water and food. The mice were housed in
an ISOcage system (Tecniplast, Italy).
</p><p><bold>Immunization and serum samples collection</bold></p><p>The mice were randomly distributed into groups (n = 5 per group for the
analysis of the humoral immune response and n = 9 per group for the analysis of
the T-cell response) and intramuscularly vaccinated with the obtained
recombinant adenoviral particles at a dose of 108 v.p./mouse in a total volume
of 0.1 mL. Serum specimens were collected on day 21 post-vaccination for
detection of S-specific IgG antibodies.
</p><p><bold>Determination of antibody titers in mouse serum samples using
enzyme-linked immunosorbent assay (ELISA)</bold></p><p>Glycoprotein-specific antibody titers in mouse serum samples were determined by
enzyme-linked immunosorbent assay (ELISA). The following recombinant proteins
were used for analysis: the S glycoprotein (40069- V08B; Sino Biological) and
the RBD (40071-V08B1; Sino Biological). Non-specific antibody binding sites
were blocked with PBS with 0.1% Tween 20 (PBST) containing 5% fat-free milk
(A0830; AppliChem, Spain). The serum samples were titrated with two-fold serial
dilutions in PBST containing 3% fat-free milk. The following anti-mouse IgG
horseradish peroxidase-conjugated secondary antibodies were used for detection:
for the total IgG titer, NXA931 (GE Healthcare, USA); for IgG1, ab97240 (Abcam,
UK); for IgG2a, ab97245 (Abcam, UK); for IgG2b, ab97250 (Abcam, UK); and for
IgG3, ab97260 (Abcam, UK). A tetramethylbenzidine solution (Research Institute
of Organic Semiproducts and Dyes, Russia) was used as a visualizing reagent.
The reaction was stopped by adding 1M H<sub>2</sub>SO<sub>4</sub>, and the
optical density was measured at 450 nm (OD<sub>450</sub>) using a Multiscan FC
spectrophotometric plate reader (Thermo Fisher Scientific). The IgG titer was
determined as the maximum serum dilution in which the OD<sub>450</sub> value of
a serum sample from an immunized animal exceeded that of the control animal
serum sample more than twofold.
</p><p><bold>Pseudovirion-based neutralization assay (PsVNA) </bold></p><p>The pseudovirion-based neutralization assay (PsVNA) was performed as described
previously [<xref rid="R34" ref-type="bibr">34</xref>]. Briefly,
heat-inactivated serum samples were diluted 1:10, 1:40, 1:160, and 1:640. These
samples were then mixed with an equal volume of DMEM containing 105 FFUs/ml of
the pseudovirions. The mixture was incubated at 37°C for 1 h, then
inoculated onto a Vero E6 cell monolayer and incubated at 37°C for 42 h.
The number of EGFP fluorescent cell focuses was counted. The pseudovirion
neutralization titer of serum samples from an immunized animal was determined
as the maximum dilution where 50% reduction of EGFP fluorescent cell focuses
compared with the serum samples of intact (non-immunized) animals was
determined.
</p><p><bold>Analysis of T-cell response (lymphocyte proliferation assay) </bold></p><p>Mice were euthanized on day 8 post-vaccination, and their spleens were
collected. The spleens were homogenized by passage through a 100 μm sieve
in sterile PBS. Splenocytes were isolated by Ficoll (1.09 g/mL; PanEco, Russia)
density gradient centrifugation (800 <italic>g </italic>for 30 min). For T-cell
proliferation assay, the splenocytes were stained with carboxyfluorescein using
a succinimidyl ester (CFSE) tracer kit (Invitrogen, USA) according to the
procedure described previously [<xref rid="R35" ref-type="bibr">35</xref>]. The
cells were seeded in 96-well plates (2 × 10<sup>5</sup> cells/well) in a
complete RPMI1640 medium re-stimulated with the recombinant MERS-CoV S protein
(40071-V08B1; Sino Biological) at 1 μg/well. After 3 days, the cells were
harvested, washed with PBS, stained with antibodies specific to CD3, CD4, and
CD8: allophycocyanin (APC)-labelled anti-CD3, APC–Cy7-labelled anti-CD8,
and phycoerythrin-labelled anti-CD4 (BD Biosciences, USA), and fixed in 1%
paraformaldehyde. Proliferating CD4+ and CD8+ T lymphocytes were determined in
the cell mixture using a BD FACS Aria III flow cytometer (BD Biosciences). The
resulting percentage of proliferating cells (X) was determined using the
formula X = %st – %, where %st is the percentage of proliferating cells
after splenocyte re-stimulation with the recombinant MERS-CoV S glycoprotein,
and % is the percentage of proliferating cells in the absence of splenocyte
re-stimulation (intact cells).
</p><p><bold>Analysis of interferon gamma (IFN-γ) production </bold></p><p>Splenocytes were isolated on day 15 post-vaccination using the procedure
described above. The cells were seeded in 96-well plates (2 ×
10<sup>5</sup> cells/well) in a RPMI1640 medium, followed by re-stimulation
with recombinant MERS-CoV S (40071-V08B1; Sino Biological) at a concentration
of 1 μg/well. Forty-eight h post-treatment, the culture supernatants were
collected. The concentration of IFN-γ in the supernatants was measured by
ELISA using a commercial kit (mouse IFN-γ ELISA kit; Invitrogen) according
to the manufacturer’s instructions. The increase in IFN-γ
concentration was determined using the formula X = Cst /Cint, where X is the
fold increase in IFN-γ concentration, Cst is the IFN-γ concentration
in the medium from the stimulated cells (pg/ml), and Cint is the IFN-γ
concentration in the medium from the non-stimulated (intact) cells (pg/ml).
</p><p><bold>Statistical analysis </bold></p><p>The statistical analysis was performed using the GraphPad 7.0 software
(GraphPad Software, USA). When analysing data from unpaired samples, either the
Student’s t-test for independent samples or the Mann–Whitney
U<italic>-</italic>test was used depending on the data distribution normality.
Distribution normality was determined using the generalized
D’Agostino–Pearson test.
</p></sec><sec><title>RESULTS</title><p><bold>Generation of rAd5 vectors </bold></p><p>In order to determine and compare the immunogenicities of different forms of
the MERS-CoV S protein, we constructed five rAd5 vectors: rAd5-S, rAd5-S-G,
rAd5-RBD, rAd5-RBD-G, and rAd5-RBD-Fc. The schemes for the target transgenes in
the rAd5 genomes are shown
in <italic><xref ref-type="fig" rid="F1">Fig. 1A</xref></italic>.
Expression levels of different forms of the MERS-CoV S
glycoprotein were evaluated by western blot analysis
(<italic><xref ref-type="fig" rid="F1">Fig. 1B</xref></italic>).
In the samples of the full-length glycoprotein (rAd5-S and rAd5-S-G),
the S protein was detected as two polypeptides
(<italic><xref ref-type="fig" rid="F1">Fig. 1B</xref></italic>),
with the upper band representing the
glycosylated form of the S protein (~230–250 kDa), and the lower band
(~100 kDa) representing the S1 subunit resulting from S-protein cleavage by
host-cell proteases. The molecular weights of the bands were higher than those
calculated according to the nucleotide sequences, being indicative of tentative
protein glycosylation [<xref rid="R14" ref-type="bibr">14</xref>, <xref rid="R16" ref-type="bibr">16</xref>, <xref rid="R36" ref-type="bibr">36</xref>,
<xref rid="R37" ref-type="bibr">37</xref>, <xref rid="R38" ref-type="bibr">38</xref>]. A single polypeptide specifically recognized by the
antibody was detected in the RBD (RBD, RBD-G and RBD-Fc) samples
(<italic><xref ref-type="fig" rid="F1">Fig. 1B</xref></italic>),
its molecular weight being ~25 kDa, ~30 kDa, and ~55 kDa, respectively.
The molecular weights of the polypeptides based on the RBD corresponded
to the calculated weights.
</p><fig id="F1" orientation="portrait" position="float"><label>Fig. 1</label><caption><p><italic>A </italic>– Schematic structures of target transgenes in the
rAd5-S, rAd5-S-G, rAd5-RBD, rAd5-RBD-G, and rAd5-RBD-Fc genomes. Cmv – a
promoter of the E1 region of human cytomegalovirus; G – the gene of the G
glycoprotein of the vesicular stomatitis virus; LP – the leader-peptide
sequence directing protein secretion; pA – polyadenylation signal; RBD
– the receptor-binding domain of MERS-CoV S glycoprotein; Spike –
MERS CoV S glycoprotein. <italic>B </italic>– Western blot analysis of
the MERS-CoV S glycoprotein variants expressed by each rAd5. Lane <italic>1
</italic>– lysate from control Ad5-null cells; lane <italic>2
</italic>– intact cells; lane <italic>3 </italic>– lysate from
rAd5-S cells; lane <italic>4 </italic>– lysate from rAd5-S-G cells; lane
<italic>5 </italic>– the medium from the rAd5- RBD cell culture; lane
<italic>6 </italic>– lysate from rAd5-RBD-G cells; lane <italic>7
</italic>– the medium from the rAd5-RBD-Fc cell culture
</p></caption><graphic xlink:href="AN20758251-11-1-038-g001"></graphic></fig><p><bold>rAd5 expressing different MERS-CoV S glycoprotein variants induce a
humoral immune response </bold></p><p>Mice were intramuscularly immunized with single doses of rAd5-S, rAd5-S-G,
rAd5-RBD, rAd5-RBD-G, and rAd5-RBD-Fc (108 v.p. per mouse). Serum samples were
collected three weeks after the immunization, and the titers of antibodies
specific to S protein and RBD were analyzed
(<italic><xref ref-type="fig" rid="F2">Fig. 2</xref></italic>).
No glycoprotein-specific IgG was detected in the serum samples from mice in the
control groups (non-immunized mice and those immunized with rAd5-null). The
highest titer of IgG specific to S glycoprotein was detected in the group
immunized with rAd5-RBD-G [geometric mean titer (GMT) was 356,055; the 95%
confidence interval (CI) was 139,042–911,772]. The lowest titer of IgG
specific to the S glycoprotein was observed in the group immunized with
rAd5-RBD-Fc [GMT: 29,407; 95% CI: 11,455–75,492]
(<italic><xref ref-type="fig" rid="F2">Fig. 2A</xref></italic>).
ELISA for RBD-specific IgG antibodies showed that rAd5-RBD-G
[GMT: 89,144; 95% CI: 60,665–130,994] and rAd5-RBD [GMT: 58,831; 95% CI:
40,024–86,424] were the constructs with the highest immunogenicity, while
rAd5-RBD-Fc [GMT: 6,400; 95% CI: 2,230–18,364] had the lowest
immunogenicity. No significant differences in RBD-specific IgG titers were
detected between rAd5-RBD and rAd5- RBD-G
(<italic><xref ref-type="fig" rid="F2">Fig. 2B</xref></italic>).
</p><fig id="F2" orientation="portrait" position="float"><label>Fig. 2</label><caption><p>Glycoprotein-specific IgG titers in the blood serum of immunized animals. The
figure shows IgG titers: (<italic>A</italic>) specific to the MERS-CoV S
glycoprotein and (<italic>B</italic>) specific to the RBD. Scatter plots show
the geometric mean titer (GMT) and 95% confidence interval (CI) for each group
(n = 5 mice/group). Asterisks indicate significant intergroup differences in
IgG titers. * <italic>p </italic> < 0.05, rAd5-RBD-Fc is compared with other
groups; ** <italic>p </italic> < 0.05, rAd5-RBD, and rAd5-RBD-G are compared
with other groups, except for rAd5-RBD and rAd5-RBD-G (the Mann–Whitney U
test)
</p></caption><graphic xlink:href="AN20758251-11-1-038-g002"></graphic></fig><p>There are four IgG isotypes known in mice to be responsible for identification
and clearance of many antigens: IgG1, IgG2a, IgG2b, and IgG3
[<xref rid="R39" ref-type="bibr">39</xref>]. Determination of the titers of IgG
isotypes three weeks post-immunization showed that all four IgG isotypes were
detected in all vaccinated animals
(<italic><xref ref-type="fig" rid="F3">Fig. 3</xref></italic>). For the IgG1,
IgG2a, and IgG2b isotypes in immunized animals, titers were as follows for each
group: rAd5-S (GMT: 409,600, 409,600, and 89,144, respectively); rAd5-S-G (GMT:
54,0470, 470,506, and 155,209, respectively); rAd5-RBD (GMT: 540,470,713,155,
and 135,118, respectively); and rAd5-RBD-G (GMT: 356,578, 713,155, and 204,800,
respectively). We observed no significant intergroup difference in isotype
titers. Following vaccination with rAd5-RBD-Fc, IgG1, IgG2a, and IgG2b, the
titers were significantly lower than those in the other groups (GMT:102,400,
89,144, and 12,800, respectively). The IgG3 titers did not differ significantly
between the groups (rAd5-S, rAd5-S-G, rAd5-RBD, rAd5-RBD-G, and rAd5-RBD-Fc;
GMT: 33,779, 14,703, 51,200, 33,779, and 5572, respectively). Hence, according
to these findings, the IgG1 and IgG2a isotypes make the greatest contribution
to the total titer of glycoprotein-specific IgG.
</p><fig id="F3" orientation="portrait" position="float"><label>Fig. 3</label><caption><p>Analysis of IgG antibody isotypes in mice after immunization with rAd5
expressing different forms of the MERS-CoV S glycoprotein. The figure shows the
titers of IgG1 (<italic>A</italic>), IgG2a (<italic>B</italic>), IgG2b
(<italic>C</italic>), and IgG3 (<italic>D</italic>) specific to the MERS-CoV S
glycoprotein in the serum samples of immunized animals. Scatter plots show the
geometric mean titer (GMT) and 95% confidence interval (CI) for each group (n =
5). Asterisks indicate significant intergroup differences in IgG titers. *
<italic>p </italic> < 0.05, Mann–Whitney U test
</p></caption><graphic xlink:href="AN20758251-11-1-038-g003"></graphic></fig><p><bold>rAd5 expressing membrane forms of the MERS-CoV S glycoprotein elicit the
production of neutralizing antibodies in mice </bold></p><p>Determination of the titers of neutralizing antibodies (in the
pseudovirion-based neutralization assay) showed that all mice immunized with
rAd5-S, rAd5- S-G, and rAd5-RBD-G generated neutralizing antibodies
(<italic><xref ref-type="fig" rid="F4">Fig. 4</xref></italic>)
with the GMT of 1:121, 1:160 and 1:70, respectively;
no significant differences in PsVNA titers were observed (<italic>p
</italic>> 0.05). In the rAd5-RBD group, neutralizing antibodies were
detected only in three mice while no neutralizing antibodies were detected in
the rAd5-RBD-Fc group and in intact animals. Hence, the results of the
conducted experiment showed that only immunization with rAd5 expressing the
membrane forms of the glycoprotein (S, S-G, RBD-G) leads to the generation of
neutralizing antibodies.
</p><fig id="F4" orientation="portrait" position="float"><label>Fig. 4</label><caption><p>The titers of neutralizing antibodies in the blood serum of immunized animals.
The neutralization assay was performed using lentiviral particles pseudotyped
with MERS-CoV S glycoprotein. Scatter plots show the geometric mean titer (GMT)
and 95% confidence interval (CI) for each group (n = 5). Asterisks indicate no
significant intergroup differences in the titers of neutralizing antibodies. *
<italic>p </italic> > 0.05, Mann–Whitney U test
</p></caption><graphic xlink:href="AN20758251-11-1-038-g004"></graphic></fig><p><bold>rAd5 expressing the MERS-CoV S protein variants elicit the T-cell
response </bold></p><fig id="F5" orientation="portrait" position="float"><label>Fig. 5</label><caption><p>The study of lymphoproliferative activity of splenocytes in immunized mice. The
figure shows the levels (%) of proliferating CD4+ and CD8+T cells re-stimulated
by the MERS-CoV S protein on day 8 post-vaccination. Scatter plots show the
median lymphoproliferative activity of re-stimulated cells (%) with 95% CIs for
each group from one representative experiment (n = 6 mice/group). Asterisks
indicate significant differences in the percentage of proliferating cells
between vaccinated and intact animals. * <italic>p </italic> < 0.05,
Mann–Whitney U test
</p></caption><graphic xlink:href="AN20758251-11-1-038-g005"></graphic></fig><p>The post-vaccination cellular immune response was evaluated using two methods:
according to the number of proliferating T cells and according to IFN-γ
production by T cells in response to glycoprotein re-stimulation. The
full-length MERS-CoV S protein was used for re-stimulation, since it contains
the largest number of epitopes and is present in MERS-CoV particles. The
proliferation assay of CD4+ cells on day 8 post-vaccination
(<italic><xref ref-type="fig" rid="F5">Fig. 5</xref></italic>,
left-side panel) showed that the highest lymphoproliferative
activity was observed in the rAd5-S group (2.10%), while the lowest one was
detected in the rAd5-RBD-Fc group (0.25%). Significant differences in the
lymphoproliferative response of CD4+ cells between the groups of immunized and
intact animals were observed in the rAd5-S (2.10%) and rAd5-S-G (1.63%) groups.
CD8+ cells proliferation assay
(<italic><xref ref-type="fig" rid="F5">Fig. 5</xref></italic>, right-side panel)
showed that the highest lymphoproliferative response was observed in the rAd5-S
group (1.90%), while the lowest one was detected in the rAd5-RBD-Fc group
(0.35%). Significant differences in the lymphoproliferative response of CD8+
cells between the groups of immunized and intact animals were observed in the
rAd5-S (1.90%), rAd5-S-G (1.15%), and rAd5-RBD (0.55%) groups. An analysis of
IFN-γ production by splenocytes after the MERS-CoV S glycoprotein
re-stimulation also showed that the strongest cellular immune response
developed in groups of animals immunized with rAd5-S and rAd5-S-G
(<italic><xref ref-type="fig" rid="F6">Fig. 6</xref></italic>):
IFN-γ secretion increased as compared to that for intact
cells 15.12 ± 0.43-fold and 10.14 ± 0.97-fold, respectively.
</p></sec><sec><title>DISCUSSION</title><p>Currently, there are no specific prophylactic or therapeutic agents against the
Middle East respiratory syndrome in the world. Intensive research focusing on
the development of vaccines against this disease is being conducted in the USA,
Germany, South Korea, and other countries [<xref rid="R40" ref-type="bibr">40</xref>, <xref rid="R41" ref-type="bibr">41</xref>]. Several
candidate vaccines based on MERS-CoV glycoprotein are known: recombinant viral
vectors based on the recombinant vaccinia virus, adenovirus, measles virus and
others; DNA vaccines; combined candidate vaccines based on DNA and recombinant
protein; and candidate vaccines based on virus-like particles and recombinant
proteins [<xref rid="R22" ref-type="bibr">22</xref>, <xref rid="R38" ref-type="bibr">38</xref>, <xref rid="R41" ref-type="bibr">41</xref>-<xref rid="R46" ref-type="bibr">46</xref>].
</p><p>The key in vaccine development is antigen selection. Most of the developed
vaccines against MERS are based on the application of different forms of the
MERS-CoV glycoprotein (the full-length S, the S1 subunit, and RBD) [<xref rid="R14" ref-type="bibr">14</xref>, <xref rid="R15" ref-type="bibr">15</xref>,
<xref rid="R16" ref-type="bibr">16</xref>, <xref rid="R18" ref-type="bibr">18</xref>, <xref rid="R22" ref-type="bibr">22</xref>, <xref rid="R24" ref-type="bibr">24</xref>, <xref rid="R47" ref-type="bibr">47</xref>-<xref rid="R55" ref-type="bibr">55</xref>], which is the
main target for neutralizing antibodies. However, the question that still
remains open is which form to choose for the development of an effective
vaccine? It is known that the full-length S glycoprotein ensures 100%
protection against lethal infection caused by MERS-CoV in animals [<xref rid="R44" ref-type="bibr">44</xref>]. However, some authors have expressed
concern about the use of full-length MERS-CoV S in the vaccine. Thus, it has
been reported that a vaccine based on a full-length glycoprotein of the severe
acute respiratory syndrome (SARS) coronavirus (which, like MERS-CoV, belongs to
the genus Betacoronavirus) induces immunopathology in the lungs of a vaccinated
organism because of the strong antibody response to the SARS-CoV glycoprotein
and weak T cell (Th2-skewed) immune response [<xref rid="R56" ref-type="bibr">56</xref>, <xref rid="R57" ref-type="bibr">57</xref>].
</p><p>Glycoprotein modifications were for the most part based on the fact that the
receptor-binding domain of the glycoprotein was included in the antigen.
Various studies showed immunogenicity of the S1 subunit, the RBD or the RBD
fused with Fc of human IgG1 (RBD-Fc) [<xref rid="R15" ref-type="bibr">15</xref>, <xref rid="R18" ref-type="bibr">18</xref>, <xref rid="R19" ref-type="bibr">19</xref>, <xref rid="R49" ref-type="bibr">49</xref>,
<xref rid="R58" ref-type="bibr">58</xref>]. Studies focused on protection of
drugs based on RBD (subunit vaccines) showed that RBD vaccination protected ~
80% of animals from MERS-CoV, despite the high titers of neutralizing
antibodies capable of blocking the interaction between the virus and the DPP4
receptor on the cell surface [<xref rid="R42" ref-type="bibr">42</xref>, <xref rid="R59" ref-type="bibr">59</xref>]. The lack of 100% protection seems to be
related to the need to develop a cellular immune response, as well as the need
to block fusion of the viral and cell membranes, which is mediated by the S2
subunit. Furthermore, application of subunit-based vaccines, as well as
inactivated ones, prevents the emergence of a balanced Th1/Th2 response and
often leads to the development of Th2-skewed immunity [<xref rid="R22" ref-type="bibr">22</xref>, <xref rid="R42" ref-type="bibr">42</xref>]. In the case of
MERS, its development can lead to lung immunopathologies [<xref rid="R48" ref-type="bibr">48</xref>]. Therefore, it is important to take into account the fact
that the vaccine-induced immunity must be Th1/Th2-balanced when developing an
anti-MERS vaccine.
</p><p>Numerous antigens based on the MERS-CoV S glycoprotein have been studied.
However, these antigens, under the same conditions (using the same antigen
delivery platform), have not been compared directly. In the present study,
direct comparison of the immunogenicities of five different forms of the
MERS-CoV S glycoprotein under the same conditions was carried out; rAd5 was
used for delivery. Strong antibody (mainly IgG1 and IgG2a) and T-cell immune
responses developed in animals vaccinated with rAd5 expressing various forms of
the MERS-CoV S glycoprotein. Each of the rAd5 variants allowed for the
emergence of a balanced Th1/Th2 response, which is one of the key aspects in
the development of an anti-MERS vaccine. In a study focused on the intensity of
the humoral immune response, the membrane form of the RBD (rAd5-RBD-G) elicited
a more powerful IgG response than the other studied forms. It was also
demonstrated that only the membrane forms of the MERS-CoV glycoprotein (rAd5-S,
rAd5-S-G, and rAd5-RBD-G) stimulate the production of neutralizing antibodies.
In the investigation of cellular immune response intensity, the forms of the
full-length MERS-CoV glycoprotein (rAd5-S and rAd5-S-G) were characterized by
the strongest immunogenicity.
</p><fig id="F6" orientation="portrait" position="float"><label>Fig. 6</label><caption><p>The increase in the concentration of IFN-γ in the media of splenocytes
from immunized mice after re-stimulation with the recombinant full-length
MERS-CoV S protein. Scatter plots show the median (95% CI) increase (fold
change) in IFN-γ production following re-stimulation for each group from
one representative experiment (n = 3 mice/group). Asterisks indicate
significant differences in IFN-γ production between the cells taken from
vaccinated and intact animals. * <italic>p </italic> < 0.05, Student’s
t-test for independent samples
</p></caption><graphic xlink:href="AN20758251-11-1-038-g006"></graphic></fig><p>To sum up, the findings obtained in our study suggest that, among all the
studied forms of the MERS-CoV S glycoprotein, the full-length S glycoprotein
and the membrane form of RBD (RBD-G) are the most promising candidates for
inclusion in a vaccine.
</p></sec><sec><title>CONCLUSIONS</title><p>The immunogenicities of five forms of MERS-CoV S glycoprotein in mice were
compared in this study. A platform based on the recombinant adenoviral vectors
rAd5 was used for antigen delivery. The studies have yielded the following
results:
</p><p>– The most powerful humoral immune response was observed in animals
immunized with the membrane-bound form of the RBD (rAd5-RBD-G);
</p><p>– Only the membrane forms of MERS-CoV glycoprotein (rAd5-S, rAd5-S-G and
rAd5-RBD-G) induced the generation of neutralizing antibodies in all vaccinated
mice;
</p><p>– The most significant cellular immune response developed after
immunization of animals with the full-length glycoprotein (rAd5-S); and
</p><p>– Vaccination of mice with all developed rAd5 vectors elicited a balanced
Th1/Th2 response.
</p></sec></body><back><ack><p>This work was supported by the Ministry of Health of the Russian Federation
(State Assignment no. 056-00108-18-00).
</p><p>The authors declare that there is no conflict of interest regarding the
publication of this article.
</p></ack><glossary><def-list><title>Abbreviations</title><def-item><term id="G01">95% CI</term><def><p>95% confidence interval</p></def></def-item><def-item><term id="G02">APC</term><def><p>allophycocyanin; DPP4 – dipeptidyl peptidase 4</p></def></def-item><def-item><term id="G03">Fc</term><def><p>fragment crystallizable</p></def></def-item><def-item><term id="G04">FFU</term><def><p>focus-forming units</p></def></def-item><def-item><term id="G05">rAd5</term><def><p>recombinant vector based on adenovirus type 5</p></def></def-item><def-item><term id="G06">RBD</term><def><p>receptor-binding domain of MERS-CoV S glycoprotein</p></def></def-item><def-item><term id="G07">RBD-Fc</term><def><p>receptor-binding domain of MERS-CoV S glycoprotein fused with Fc of human IgG1</p></def></def-item><def-item><term id="G08">RBD-G</term><def><p>receptor-binding domain of MERS-CoV S glycoprotein fused with the transmembrane domain of the VSV G glycoprotein</p></def></def-item><def-item><term id="G09">S</term><def><p>MERS-CoV glycoprotein</p></def></def-item><def-item><term id="G10">S1, S2</term><def><p>domains of MERS-CoV S glycoprotein</p></def></def-item><def-item><term id="G11">S-G</term><def><p>full-length S glycoprotein with the transmembrane domain of the G glycoprotein of VSV</p></def></def-item><def-item><term id="G12">Th</term><def><p>T helper</p></def></def-item><def-item><term id="G13">VSV</term><def><p>vesicular stomatitis virus</p></def></def-item><def-item><term id="G14">MERS</term><def><p>Middle East respiratory syndrome</p></def></def-item><def-item><term id="G15">MERS-CoV</term><def><p>Middle East respiratory syndrome coronavirus</p></def></def-item><def-item><term id="G16">PFU</term><def><p>plaque-forming unit</p></def></def-item><def-item><term id="G17">v.p.</term><def><p>viral particles</p></def></def-item><def-item><term id="G18">GMT</term><def><p>geometric mean titer</p></def></def-item><def-item><term id="G19">IFNγ</term><def><p>interferon gamma</p></def></def-item><def-item><term id="G20">TM</term><def><p>transmembrane domain</p></def></def-item><def-item><term id="G21">SARS</term><def><p>severe acute respiratory syndrome</p></def></def-item><def-item><term id="G22">SARS-CoV</term><def><p>severe acute respiratory syndrome coronavirus</p></def></def-item><def-item><term id="G23">PBS</term><def><p>phosphate-buffered saline</p></def></def-item><def-item><term id="G24">PBST</term><def><p>PBS supplemented with 0.1% Tween 20</p></def></def-item><def-item><term id="G25">ER</term><def><p>endoplasmic reticulum</p></def></def-item></def-list></glossary><ref-list><title>References</title><ref id="R1"><label>1</label><element-citation publication-type="journal"><source>Virology Journal</source><year>2013</year><volume>87</volume><issue>14</issue><fpage>7790</fpage><lpage>7792</lpage><name><surname>de Groot</surname><given-names>R.J.</given-names></name><name><surname>Baker</surname><given-names>S.C.</given-names></name><name><surname>Baric</surname><given-names>R.S.</given-names></name><name><surname>Brown</surname><given-names>C.S.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name><name><surname>Enjuanes</surname><given-names>L.</given-names></name><name><surname>Fouchier</surname><given-names>R.A.M.</given-names></name><name><surname>Galiano</surname><given-names>M.</given-names></name><name><surname>Gorbalenya</surname><given-names>A.E.</given-names></name><name><surname>Memish</surname><given-names>Z.A.</given-names></name></element-citation></ref><ref id="R2"><label>2</label><element-citation publication-type="journal"><source>N. 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