Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S
Identifieur interne : 000662 ( Pmc/Corpus ); précédent : 000661; suivant : 000663Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S
Auteurs : Martin C. Langenmayer ; Anna-Theresa Lülf-Averhoff ; Silvia Adam-Neumair ; Robert Fux ; Gerd Sutter ; Asisa VolzSource :
- Biologicals [ 1045-1056 ] ; 2018.
Abstract
Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the
Url:
DOI: 10.1016/j.biologicals.2018.05.004
PubMed: 29759890
PubMed Central: 7128986
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S</title><author><name sortKey="Langenmayer, Martin C" sort="Langenmayer, Martin C" uniqKey="Langenmayer M" first="Martin C." last="Langenmayer">Martin C. Langenmayer</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Lulf Averhoff, Anna Theresa" sort="Lulf Averhoff, Anna Theresa" uniqKey="Lulf Averhoff A" first="Anna-Theresa" last="Lülf-Averhoff">Anna-Theresa Lülf-Averhoff</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Adam Neumair, Silvia" sort="Adam Neumair, Silvia" uniqKey="Adam Neumair S" first="Silvia" last="Adam-Neumair">Silvia Adam-Neumair</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation></author><author><name sortKey="Fux, Robert" sort="Fux, Robert" uniqKey="Fux R" first="Robert" last="Fux">Robert Fux</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Sutter, Gerd" sort="Sutter, Gerd" uniqKey="Sutter G" first="Gerd" last="Sutter">Gerd Sutter</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Volz, Asisa" sort="Volz, Asisa" uniqKey="Volz A" first="Asisa" last="Volz">Asisa Volz</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29759890</idno><idno type="pmc">7128986</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128986</idno><idno type="RBID">PMC:7128986</idno><idno type="doi">10.1016/j.biologicals.2018.05.004</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000662</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000662</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S</title><author><name sortKey="Langenmayer, Martin C" sort="Langenmayer, Martin C" uniqKey="Langenmayer M" first="Martin C." last="Langenmayer">Martin C. Langenmayer</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Lulf Averhoff, Anna Theresa" sort="Lulf Averhoff, Anna Theresa" uniqKey="Lulf Averhoff A" first="Anna-Theresa" last="Lülf-Averhoff">Anna-Theresa Lülf-Averhoff</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Adam Neumair, Silvia" sort="Adam Neumair, Silvia" uniqKey="Adam Neumair S" first="Silvia" last="Adam-Neumair">Silvia Adam-Neumair</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation></author><author><name sortKey="Fux, Robert" sort="Fux, Robert" uniqKey="Fux R" first="Robert" last="Fux">Robert Fux</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Sutter, Gerd" sort="Sutter, Gerd" uniqKey="Sutter G" first="Gerd" last="Sutter">Gerd Sutter</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author><author><name sortKey="Volz, Asisa" sort="Volz, Asisa" uniqKey="Volz A" first="Asisa" last="Volz">Asisa Volz</name><affiliation><nlm:aff id="aff1">Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">German Center for Infection Research (DZIF), Munich Partner Site, Germany</nlm:aff></affiliation></author></analytic><series><title level="j">Biologicals</title><idno type="ISSN">1045-1056</idno><idno type="eISSN">1095-8320</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the <italic>in vivo</italic> distribution of a recombinant MVA candidate vaccine against the Middle Eastern Respiratory Syndrome (MVA-MERS-S) in mice. Intramuscularly inoculated mice were necropsied at different time points and examined by histology, immunohistochemistry and real-time PCR. We detected inflammation and myonecrosis at the parenteral site and hyperplasia of the draining lymph nodes. MVA-MERS-S did not result in detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. Real-time PCR analysis of >240 tissue samples detected MVA-DNA predominantly at the injection site and in the draining lymph nodes, and suggested continuous clearance of the candidate vaccine during the observation period. Levels of parenteral site inflammation and hyperplasia of draining lymph nodes were considered in line with immunological responses to vaccine inoculation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Mayr, A" uniqKey="Mayr A">A. Mayr</name></author><author><name sortKey="Hochstein Mintzel, V" uniqKey="Hochstein Mintzel V">V. Hochstein-Mintzel</name></author><author><name sortKey="Stickl, H" uniqKey="Stickl H">H. Stickl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Volz, A" uniqKey="Volz A">A. Volz</name></author><author><name sortKey="Sutter, G" uniqKey="Sutter G">G. Sutter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Verheust, C" uniqKey="Verheust C">C. Verheust</name></author><author><name sortKey="Goossens, M" uniqKey="Goossens M">M. Goossens</name></author><author><name sortKey="Pauwels, K" uniqKey="Pauwels K">K. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biologicals</journal-id><journal-id journal-id-type="iso-abbrev">Biologicals</journal-id><journal-title-group><journal-title>Biologicals</journal-title></journal-title-group><issn pub-type="ppub">1045-1056</issn><issn pub-type="epub">1095-8320</issn><publisher><publisher-name>International Alliance for Biological Standardization. Published by Elsevier Ltd.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29759890</article-id><article-id pub-id-type="pmc">7128986</article-id><article-id pub-id-type="publisher-id">S1045-1056(18)30137-4</article-id><article-id pub-id-type="doi">10.1016/j.biologicals.2018.05.004</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Langenmayer</surname><given-names>Martin C.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Lülf-Averhoff</surname><given-names>Anna-Theresa</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Adam-Neumair</surname><given-names>Silvia</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Fux</surname><given-names>Robert</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Sutter</surname><given-names>Gerd</given-names></name><email>gerd.sutter@lmu.de</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Volz</surname><given-names>Asisa</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Institute for Infectious Diseases and Zoonoses, LMU Munich, Germany</aff><aff id="aff2"><label>b</label>German Center for Infection Research (DZIF), Munich Partner Site, Germany</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author. Institute for Infectious Diseases and Zoonoses, Veterinaerstrasse 13, D-80539 Munich, Germany. <email>gerd.sutter@lmu.de</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>6</day><month>7</month><year>2018</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>7</month><year>2018</year></pub-date><pub-date pub-type="epub"><day>6</day><month>7</month><year>2018</year></pub-date><volume>54</volume><fpage>58</fpage><lpage>62</lpage><history><date date-type="received"><day>13</day><month>3</month><year>2017</year></date><date date-type="rev-recd"><day>14</day><month>3</month><year>2018</year></date><date date-type="accepted"><day>4</day><month>5</month><year>2018</year></date></history><permissions><copyright-statement>© 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>International Alliance for Biological Standardization</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract id="abs0010"><p>Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the <italic>in vivo</italic> distribution of a recombinant MVA candidate vaccine against the Middle Eastern Respiratory Syndrome (MVA-MERS-S) in mice. Intramuscularly inoculated mice were necropsied at different time points and examined by histology, immunohistochemistry and real-time PCR. We detected inflammation and myonecrosis at the parenteral site and hyperplasia of the draining lymph nodes. MVA-MERS-S did not result in detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. Real-time PCR analysis of >240 tissue samples detected MVA-DNA predominantly at the injection site and in the draining lymph nodes, and suggested continuous clearance of the candidate vaccine during the observation period. Levels of parenteral site inflammation and hyperplasia of draining lymph nodes were considered in line with immunological responses to vaccine inoculation.</p></abstract><kwd-group id="kwrds0010"><title>Keywords</title><kwd>Viral vector</kwd><kwd>Poxvirus vaccine</kwd><kwd>Vaccinia virus MVA</kwd><kwd>Biodistribution</kwd><kwd>MERS</kwd><kwd>Immunohistochemistry</kwd><kwd>PCR</kwd></kwd-group></article-meta></front><body><sec id="sec1"><label>1</label><title>Introduction</title><p id="p0010">Modified Vaccinia Virus Ankara (MVA) is a highly attenuated vaccinia virus serving as a well-established viral vector system used for developing vaccines against infectious diseases [<xref rid="bib1" ref-type="bibr">1</xref>,<xref rid="bib2" ref-type="bibr">2</xref>]. MVA is largely replication-deficient in mammalian cells, but grows well in chicken embryo fibroblasts [<xref rid="bib3" ref-type="bibr">3</xref>,<xref rid="bib4" ref-type="bibr">4</xref>]. Non-replicating MVA vaccines have an excellent safety profile in preclinical models using irradiated rabbits, immunosuppressed macaques, SCID and immunocompetent mice, and in clinical testing in humans using different inoculation routes [<xref rid="bib5" ref-type="bibr">[5]</xref>, <xref rid="bib6" ref-type="bibr">[6]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>, <xref rid="bib8" ref-type="bibr">[8]</xref>, <xref rid="bib9" ref-type="bibr">[9]</xref>].</p><p id="p0015">A recombinant MVA vaccine expressing the full length spike protein of Middle Eastern Respiratory Syndrome Coronavirus (MVA-MERS-S) proved to be suitable for production at an industrial scale, immunogenic, and protective against MERS-CoV infections in mice and dromedaries [<xref rid="bib10" ref-type="bibr">10</xref>,<xref rid="bib11" ref-type="bibr">11</xref>]. Here we wished to generate additional preclinical data on the <italic>in vivo</italic> distribution of MVA-MERS-S in order to prepare for the evaluation of the MVA-MERS-S vector vaccine in a first-in-man phase I clinical trial. This ‘biodistribution’ of a candidate vaccine is important to estimate risks potentially associated with an <italic>in vivo</italic> application of the virus. Relevant aspects include monitoring for signs of virus replication and for potential side effects of the vaccination in the selected animal model. For instance, a dispersion of virus from the parenteral site of inoculation to excretory organs could be indicative of possible viral shedding and spillover into the general environment. Although MVA has been investigated as a safe vector vaccine candidate for years, there are not many studies regarding distribution in sites peripheral to the administration site.</p><p id="p0020">In this study, we assessed the distribution of MVA-MERS-S after intramuscular application in the mouse model using histology, immunohistochemistry, and PCR to detect virus-associated lesions, viral and recombinant antigen <italic>in situ,</italic> and viral DNA.</p></sec><sec id="sec2"><label>2</label><title>Materials & methods</title><sec id="sec2.1"><label>2.1</label><title>Animals</title><p id="p0025">C57BL/6N mice (6–10 weeks old) were maintained under specified pathogen-free conditions. Animals were allowed to adjust to the facilities (one week) before vaccination experiments were performed and had free access to food and water. Experiments were in compliance with the German regulations for animal experimentation (Animal Welfare Acts).</p><p id="p0030">Mice were inoculated by intramuscular injection with 10<sup>7</sup> (PCR-study) or 10<sup>8</sup> plaque-forming units (pfu, histology-study) of MVA-MERS-S or a control recombinant MVA-GFP-mCherry [<xref rid="bib12" ref-type="bibr">12</xref>] or PBS into the thigh. Mice were monitored daily for signs of disease and were euthanized and necropsied at different time points (<xref rid="tbl1" ref-type="table">Table 1</xref>).<table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Distribution studies of MVA-MERS-S using qPCR and histology.</p></caption><alt-text id="alttext0020">Table 1</alt-text><table frame="hsides" rules="groups"><thead><tr><th colspan="13"><bold>A</bold> C57BL/6N mice<hr></hr></th></tr><tr><th rowspan="2">Group</th><th>Treatment<hr></hr></th><th colspan="2">Animals<hr></hr></th><th colspan="8">Necropsy, animals per time point post inoculation<hr></hr></th><th rowspan="2">Organs</th></tr><tr><th>Single i.m. inoculation</th><th>♀</th><th>♂</th><th>16 h</th><th>24 h</th><th>2 d</th><th>3 d</th><th>4 d</th><th>6 d</th><th>7 d</th><th>21 d</th></tr></thead><tbody><tr><td rowspan="2" valign="middle">qPCR<hr></hr></td><td align="left">MVA-MERS-S</td><td align="left">10</td><td align="left">17</td><td align="left">–</td><td align="left">6</td><td align="left">–</td><td align="left">6</td><td align="left">–</td><td align="left">–</td><td align="left">6</td><td align="left">9</td><td rowspan="2" valign="middle">See Part B<hr></hr></td></tr><tr><td align="left">PBS<hr></hr></td><td align="left">10<hr></hr></td><td align="left">3<hr></hr></td><td align="left">–<hr></hr></td><td align="left">4<hr></hr></td><td align="left">–<hr></hr></td><td align="left">3<hr></hr></td><td align="left">–<hr></hr></td><td align="left">–<hr></hr></td><td align="left">3<hr></hr></td><td align="left">3<hr></hr></td></tr><tr><td rowspan="3" valign="middle">Histology</td><td align="left">MVA-MERS-S</td><td align="left">1</td><td align="left">5</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">–</td><td align="left">–</td><td rowspan="3" valign="middle">See <xref rid="fig1" ref-type="fig">Fig. 1</xref></td></tr><tr><td align="left">MVA-GFP-mCherry</td><td align="left">1</td><td align="left">5</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">1</td><td align="left">–</td><td align="left">–</td></tr><tr><td align="left">PBS</td><td align="left">4</td><td align="left">–</td><td align="left">–</td><td align="left">1</td><td align="left">–</td><td align="left">1</td><td align="left">–</td><td align="left">2</td><td align="left">–</td><td align="left">–</td></tr></tbody></table><table frame="hsides" rules="groups"><thead><tr><th colspan="9"><bold>B</bold> Organ real-time PCR results<hr></hr></th></tr><tr><th></th><th colspan="8" align="center">Time point/Vaccine<hr></hr></th></tr><tr><th><hr></hr></th><th colspan="2" align="center">24 h<hr></hr></th><th colspan="2" align="center">3 d<hr></hr></th><th colspan="2" align="center">7 d<hr></hr></th><th colspan="2" align="center">21 d<hr></hr></th></tr><tr><th>Organ/Tissue</th><th>MVA-MERS-S</th><th>PBS</th><th>MVA-MERS-S</th><th>PBS</th><th>MVA-MERS-S</th><th>PBS</th><th>MVA-MERS-S</th><th>PBS</th></tr></thead><tbody><tr><td align="left">Gonads</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr><tr><td align="left">Kidneys</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr><tr><td align="left">Liver</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">1/6<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr><tr><td align="left">Lung</td><td align="left">1/6<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">1/9<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td align="left">0/3</td></tr><tr><td align="left">Rectum + Feces</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr><tr><td align="left">Spleen</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr><tr><td align="left">Urinary Bladder</td><td align="left">0/6</td><td align="left">0/4</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/6</td><td align="left">0/3</td><td align="left">0/9</td><td align="left">0/3</td></tr></tbody></table><table-wrap-foot><fn id="tbl1fna"><label>a</label><p id="ntpara0010">Cycle threshold (Ct) > 36.</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="sec2.2"><label>2.2</label><title>Necropsy, qPCR, histology and immunohistochemistry</title><p id="p0035">A full necropsy was performed and organ samples for PCR and histology/immunohistochemistry were taken. An orthopoxvirus-specific TaqMan-PCR-assay was used to detect viral-DNA [<xref rid="bib13" ref-type="bibr">13</xref>]. Samples (<xref rid="tbl1" ref-type="table">Table 1</xref>A) were frozen at −80 °C until DNA was isolated using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. For real-time PCR (qPCR) the QuantiTect probe PCR kit (Qiagen) was used. A quantification cycle cut-off ≤38 was regarded specific.</p><p id="p0040">For histology, tissues (<xref rid="fig1" ref-type="fig">Fig. 1</xref>) were paraformaldehyde-fixed, paraffin-embedded, cut and sections were stained with hemalum-eosin. Skull, rectum and urinary bladder were taken from 48 h on. Skull and whole thigh were decalcified.<fig id="fig1"><label>Fig. 1</label><caption><p>Organ lesions and antigen distribution: Depicted are cumulative results from histological analysis of sampled organs in sections stained with hemalum-eosin and immunohistochemistry. Total number of organs/tissues sampled (white bars), organs/tissues with lesions (grey bars), lesions with VACV-antigen detection (black bars). A MVA-MERS-S inoculated mice. B MVA-GFP-mCherry inoculated mice.</p></caption><alt-text id="alttext0010">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0045">Anti-VACV-immunohistochemistry (IHC) was performed on all tissues as previously described [<xref rid="bib14" ref-type="bibr">14</xref>] with heat-induced epitope-retrieval. Anti-MERS-S-IHC was performed using a polyclonal rabbit serum (1:1000; Sino Biological, 100208 RP-02) without epitope-retrieval. IHC positive and negative controls were included.</p></sec></sec><sec id="sec3"><label>3</label><title>Results</title><sec id="sec3.1"><label>3.1</label><title>Histological analyses</title><sec id="sec3.1.1"><label>3.1.1</label><title>MVA-MERS-S inoculated animals</title><p id="p0050">Macroscopic examination revealed swelling of the left thigh with muscular pallor, mild edema and hemorrhage, and swelling of draining lymph nodes. Macroscopic lesions in other organs attributable to MVA inoculation were not observed.</p><p id="p0055">Histologically, at all time points, there was absence of lesions attributable to MVA inoculation in any tissue other than the parenteral site and draining lymph nodes (<xref rid="fig1" ref-type="fig">Fig. 1</xref>). At the early time points, there was separation of muscle fibers through inoculum and edema, muscle fiber degeneration and necrosis at the parenteral site. Interstitial areas, adjacent adipose tissue and inoculum were predominantly infiltrated by many neutrophils. At the late time points there was evidence of muscular regeneration, characterized by myoblast tubes and internalized nuclei and many predominantly mononuclear infiltrates in the interstitia and surrounding the inoculum. The draining lymph nodes revealed increased paracortical lymphocytic cellularity (hyperplasia).</p><p id="p0060">VACV antigen was detected only at the parenteral site. Other tissues including draining lymph nodes revealed no VACV antigen. VACV antigen was located in the cytoplasm of spindle cells in the interstitium (<xref rid="fig2" ref-type="fig">Fig. 2</xref>) and phagocytic cells (macrophages and neutrophils) in the interstitium or surrounding inoculum. MERS-S antigen was detected in MVA-MERS-S inoculated animals only at the parenteral site in the cytoplasm of similar spindle cells with a comparable distribution pattern (<xref rid="fig2" ref-type="fig">Fig. 2</xref>). Draining lymph nodes displayed no specific MERS-S antigen-staining.<fig id="fig2"><label>Fig. 2</label><caption><p><bold>A</bold> PBS inoculated mouse (72 h), left thigh, VACV-immunohistochemistry, no signal. <bold>B</bold> MVA-MERS-S inoculated mouse (16 h), left thigh, VACV-antigen in spindle cells (center, brown color) and inoculum (bottom right). <bold>C</bold> MVA-GFP-mCherry inoculated mouse (48 h), left thigh, VACV-antigen in similar spindle cells. <bold>D</bold> MVA-MERS-S inoculated mouse (24 h), left thigh, cytoplasmic MERS-S-antigen in interstitial spindle cell. Bars = 50 μm.</p></caption><alt-text id="alttext0015">Fig. 2</alt-text><graphic xlink:href="gr2_lrg"></graphic></fig></p></sec><sec id="sec3.1.2"><label>3.1.2</label><title>MVA-GFP-mCherry inoculated animals</title><p id="p0065">The distribution and severity of lesions were remarkably similar to the MVA-MERS-S inoculated animals. Macroscopic and histologic examinations revealed similar findings in the parenteral site and draining lymph nodes. Acute inflammation, characterized by edema and many infiltrating neutrophils with myofiber degeneration and necrosis were prevalent at the early time points. At later time points, the infiltrate became predominantly mononuclear and there was evidence of muscle regeneration. Macroscopic and microscopic lesions in other organs attributable to MVA inoculation were not observed at any time point. VACV-antigen was only detected at the parenteral site within the cytoplasm of phagocytic and spindle cells (<xref rid="fig2" ref-type="fig">Fig. 2</xref>). Other tissues including draining lymph nodes revealed no VACV antigen.</p></sec><sec id="sec3.1.3"><label>3.1.4</label><title>Saline inoculated animals</title><p id="p0070">Macroscopic examination revealed no lesions. Histological analysis displayed mild injection-associated lesions at the parenteral site consisting of band-like degeneration of single muscle fibers, regeneration at later time points and focal scarce interstitial leukocytic infiltrates. The subiliac lymph node of one animal revealed mildly increased paracortical cellularity. Histological changes attributable to inoculation were not detected in any other tissue in any animal. A specific reaction product was not observed in either of the two IHCs.</p></sec></sec><sec id="sec3.2"><label>3.2</label><title>PCR analyses</title><p id="p0075">MVA-DNA was detected only in MVA-MERS-S-inoculated mice at the parenteral site (Ct-range: 19–36) and draining lymph nodes (Ct-range: 33–35) at days 1, 3 and 7 days post inoculation. Only in three animals, single organs were positive (<xref rid="tbl1" ref-type="table">Table 1</xref>B).</p></sec></sec><sec id="sec4"><label>4</label><title>Discussion</title><p id="p0080">This study was done to contribute to the development of the vaccine candidate MVA-MERS-S towards clinical phase I testing. Here, we report data supporting the biodistribution assessment of MVA-MERS-S after intramuscular application in the mouse model. With regard to systemic spread, potential excretion and side effects we analyzed tissue samples by histology, immunohistochemistry and qPCR.</p><p id="p0085">As determined by histology, the candidate vaccine did not produce generalized lesions or organ lesions peripheral to the parenteral site. MERS-S- and VACV-antigen remained restricted to the parenteral site and were not observed in draining lymph nodes. Anti-VACV-IHC did not reveal antigen in any of the analyzed peripheral tissues underlining the incompetence of MVA to result in generalized spreading. Inoculation of mice with the control virus MVA-GFP-mCherry produced a reaction pattern indistinguishable from MVA-MERS-S underlining the maintenance of a well confined vector virus infection. Moreover, the limited distribution of MVA-MERS-S was further confirmed by the results of the qPCR analysis detecting relevant amounts of MVA-DNA only at the injection site and in draining lymph nodes. In three animals, high Ct-values of MVA-DNA were obtained in lung and liver. These PCR signals can probably be attributed to marginal quantities of vector DNA, which was dislodged from the parenteral site. Similar studies also detected minimal amounts of vector DNA in single organs, however, attempts of MVA re-isolation were negative [<xref rid="bib6" ref-type="bibr">6</xref>,<xref rid="bib8" ref-type="bibr">8</xref>]. Therefore, it seems unlikely that these signals represent viable virions.</p><p id="p0090">Importantly, MVA-DNA was not found in excretory organs (kidneys, rectum including feces and urinary bladder including urine). Thus, the risk of MVA excretion and spillover of the genetically modified organism into the general environment is regarded negligible.</p><p id="p0095">Other studies using recombinant MVA candidate vaccines also report locally confined lesions even in severely immunosuppressed animals. While MVA-DNA can remain detectable by PCR for several weeks after inoculation, attempts to isolate infectious virus proved to be negative. Histology in either case did not reveal peripheral lesions associated with MVA inoculation [<xref rid="bib5" ref-type="bibr">5</xref>,<xref rid="bib8" ref-type="bibr">8</xref>].</p><p id="p0100">In this study, the observed local inflammation infiltrating the inoculum and adjacent tissue is not regarded as undesired reaction but part of the physiologic immune reaction against the vaccine virus. The detection of VACV antigen in phagocytic cells supports an efficient <italic>in vivo</italic> targeting of antigen presenting cells following the inoculation of MVA vector vaccines. The degree and extent of inflammation and myonecrosis in this study is in accordance with the ratio of inoculum volume in relation to parenteral site (mouse muscle) volume, which is expected to be considerably smaller in human vaccinees. The hyperplasia observed in the draining lymph nodes again reflects the immune competence of the host and is characteristic for an early response to inflammation at the draining site [<xref rid="bib15" ref-type="bibr">15</xref>].</p><p id="p0105">We conclude that a single intramuscular injection of equivalents of a human dose of MVA-MERS-S in C57BL/6 mice is without lesions in tissues other than the parenteral site. Further, viral-DNA was not found in other peripheral organs, with the exception of very low copy numbers detected in single samples. 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contributions of our vaccine research group, notably Sylvia Jany and our animal caretaker team.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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