Stalking influenza by vaccination with pre-fusion headless HA mini-stem
Identifieur interne : 000287 ( Pmc/Checkpoint ); précédent : 000286; suivant : 000288Stalking influenza by vaccination with pre-fusion headless HA mini-stem
Auteurs : Sophie A. Valkenburg [Hong Kong] ; V. Vamsee Aditya Mallajosyula [Inde] ; Olive T. W. Li [Hong Kong] ; Alex W. H. Chin [Hong Kong] ; George Carnell [Royaume-Uni] ; Nigel Temperton [Royaume-Uni] ; Raghavan Varadarajan [Inde] ; Leo L. M. Poon [Hong Kong]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.
Url:
DOI: 10.1038/srep22666
PubMed: 26947245
PubMed Central: 4780079
Affiliations:
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Valkenburg</name><affiliation wicri:level="3"><nlm:aff id="a1"><institution>HKU-Pasteur Research Pole, School of Public Health, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong</institution>, Hong Kong</nlm:aff><country xml:lang="fr">Hong Kong</country><wicri:regionArea>, Chicago</wicri:regionArea><placeName><settlement type="city">Chicago</settlement><region type="state">Illinois</region></placeName></affiliation><affiliation wicri:level="3"><nlm:aff id="a2"><institution>Center of Influenza Research and School of Public Health, The University of Hong Kong</institution>, Hong Kong</nlm:aff><country xml:lang="fr">Hong Kong</country><wicri:regionArea>, Chicago</wicri:regionArea><placeName><settlement type="city">Chicago</settlement><region type="state">Illinois</region></placeName></affiliation></author><author><name sortKey="Mallajosyula, V Vamsee Aditya" sort="Mallajosyula, V Vamsee Aditya" uniqKey="Mallajosyula V" first="V. Vamsee Aditya" last="Mallajosyula">V. Vamsee Aditya Mallajosyula</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Molecular Biophysics Unit, Indian Institute of Science</institution>, Bangalore,<country>India</country></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Li, Olive T W" sort="Li, Olive T W" uniqKey="Li O" first="Olive T. W." last="Li">Olive T. W. Li</name><affiliation wicri:level="3"><nlm:aff id="a2"><institution>Center of Influenza Research and School of Public Health, The University of Hong Kong</institution>, Hong Kong</nlm:aff><country xml:lang="fr">Hong Kong</country><wicri:regionArea>, Chicago</wicri:regionArea><placeName><settlement type="city">Chicago</settlement><region type="state">Illinois</region></placeName></affiliation></author><author><name sortKey="Chin, Alex W H" sort="Chin, Alex W H" uniqKey="Chin A" first="Alex W. H." last="Chin">Alex W. H. Chin</name><affiliation wicri:level="3"><nlm:aff id="a2"><institution>Center of Influenza Research and School of Public Health, The University of Hong Kong</institution>, Hong Kong</nlm:aff><country xml:lang="fr">Hong Kong</country><wicri:regionArea>, Chicago</wicri:regionArea><placeName><settlement type="city">Chicago</settlement><region type="state">Illinois</region></placeName></affiliation></author><author><name sortKey="Carnell, George" sort="Carnell, George" uniqKey="Carnell G" first="George" last="Carnell">George Carnell</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Viral Pseudotype Unit, School of Pharmacy, University of Kent</institution>, Kent,<country>United Kingdom</country></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Temperton, Nigel" sort="Temperton, Nigel" uniqKey="Temperton N" first="Nigel" last="Temperton">Nigel Temperton</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Viral Pseudotype Unit, School of Pharmacy, University of Kent</institution>, Kent,<country>United Kingdom</country></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Varadarajan, Raghavan" sort="Varadarajan, Raghavan" uniqKey="Varadarajan R" first="Raghavan" last="Varadarajan">Raghavan Varadarajan</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Molecular Biophysics Unit, Indian Institute of Science</institution>, Bangalore,<country>India</country></nlm:aff><country xml:lang="fr">Inde</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Poon, Leo L M" sort="Poon, Leo L M" uniqKey="Poon L" first="Leo L. M." last="Poon">Leo L. M. Poon</name><affiliation wicri:level="3"><nlm:aff id="a2"><institution>Center of Influenza Research and School of Public Health, The University of Hong Kong</institution>, Hong Kong</nlm:aff><country xml:lang="fr">Hong Kong</country><wicri:regionArea>, Chicago</wicri:regionArea><placeName><settlement type="city">Chicago</settlement><region type="state">Illinois</region></placeName></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Gerhard, W" uniqKey="Gerhard W">W. Gerhard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name></author><author><name sortKey="Tefsen, B" uniqKey="Tefsen B">B. Tefsen</name></author><author><name sortKey="Shi, Y" uniqKey="Shi Y">Y. Shi</name></author><author><name sortKey="Gao, G F" uniqKey="Gao G">G. F. Gao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Skehel, J J" uniqKey="Skehel J">J. J. Skehel</name></author><author><name sortKey="Wiley, D C" uniqKey="Wiley D">D. C. Wiley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Skehel, J J" uniqKey="Skehel J">J. J. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26947245</article-id><article-id pub-id-type="pmc">4780079</article-id><article-id pub-id-type="pii">srep22666</article-id><article-id pub-id-type="doi">10.1038/srep22666</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Stalking influenza by vaccination with pre-fusion headless HA mini-stem</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Valkenburg</surname><given-names>Sophie A.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Mallajosyula</surname><given-names>V. Vamsee Aditya</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Olive T. W.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chin</surname><given-names>Alex W. H.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Carnell</surname><given-names>George</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Temperton</surname><given-names>Nigel</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Varadarajan</surname><given-names>Raghavan</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Poon</surname><given-names>Leo L. M.</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>HKU-Pasteur Research Pole, School of Public Health, HKU Li Ka Shing Faculty of Medicine, The University of Hong Kong</institution>, Hong Kong</aff><aff id="a2"><label>2</label><institution>Center of Influenza Research and School of Public Health, The University of Hong Kong</institution>, Hong Kong</aff><aff id="a3"><label>3</label><institution>Molecular Biophysics Unit, Indian Institute of Science</institution>, Bangalore,<country>India</country></aff><aff id="a4"><label>4</label><institution>Viral Pseudotype Unit, School of Pharmacy, University of Kent</institution>, Kent,<country>United Kingdom</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>varadar@mbu.iisc.ernet.in</email></corresp><corresp id="c2"><label>b</label><email>llmpoon@hku.hk</email></corresp></author-notes><pub-date pub-type="epub"><day>07</day><month>03</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>6</volume><elocation-id>22666</elocation-id><history><date date-type="received"><day>20</day><month>11</month><year>2015</year></date><date date-type="accepted"><day>15</day><month>02</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016, Macmillan Publishers Limited</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Macmillan Publishers Limited</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Design of HA mini-stem from HPAI H5N1 A/Viet Nam/1203/2004 HA.</title><p><bold>(A)</bold> All full-length H5N1 HA sequences isolated from human hosts (n = 215) were analyzed and the conservation score (Q-score) obtained from a multiple sequence alignment was mapped onto a monomer of the surface representation of the H5 HA A/Viet Nam/1203/2004 trimer (PDB ID: 2FK0), and the remaining HA in light gray. <bold>(B)</bold> Conservation analysis was extended to include all avian influenza H5 HA sequences (n = 2427) <bold>(C)</bold> The conserved HA stem fragments included in H5F (cartoon representation). One monomer was colored based on the Q-score. The second monomer was colored as described in (<bold>E</bold>), whilst the third monomer is shown in light gray. Dashed lines represent connecting linkers. Schematic of <bold>(D)</bold> full-length H5 HA and ‘headless’ HA stem-fragment, H5F. Intra-subunit (solid lines, black) and inter-subunit (solid line, blue) disulfide bonds are shown. The polybasic cleavage site (blue), fusion peptide (tan), trans-membrane domain (dark green) and the cytoplasmic tail (orange) are indicated. Red brackets () are the start/stop sites of each subunit in the crystal structure (PDB ID: 2FK0). The crystal structure numbering was followed here. Dashed lines indicate linkers. A synthetic trimerization motif, foldon (light green), was appended at the C-terminus of the disulfide-free HA mini-stem to promote oligomerization. The expression vector derived N-terminal His-tag (dark blue) that facilitates a facile, one-step Ni-NTA affinity purification is also indicated. <bold>(E)</bold> Sequence of H5F colored according to the schematic in panel <bold>(D)</bold>. Site-specific mutations (bold and underlined) were introduced to prevent protein aggregation. Aspartate (<bold>D</bold>) mutations (bold, italics and underlined) were introduced to destabilize the low-pH conformation of HA. <bold>(F)</bold> The sequence conservation in the HA stem fragments between the vaccine candidates and influenza virus challenge strains was determined by pairwise sequence alignment (<ext-link ext-link-type="uri" xlink:href="http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/">http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/</ext-link>). The structural similarity (main chain) between HA stem fragments across different influenza virus strains was calculated by a topology independent comparison of modeled structures using the CLICK server (<ext-link ext-link-type="uri" xlink:href="http://mspc.bii.a-star.edu.sg/minhn/output/143882270252.html">http://mspc.bii.a-star.edu.sg/minhn/output/143882270252.html</ext-link>). <bold>(A–C)</bold> were rendered using PyMOL.</p></caption><graphic xlink:href="srep22666-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>The HA mini-stem vaccines elicit broad, cross-reactive neutralizing Abs in mice.</title><p>The binding of sera (total IgG) to HA proteins was probed by ELISA/BLI, from day 21 post primary (pooled n = 8) or secondary vaccine dose (pooled n = 20). <bold>(A–C)</bold> The sera Abs (total IgG) were captured using Protein G (ProG) biosensors and the kinetics of binding to HA proteins was determined using an Octet RED96 instrument. Lower equilibrium dissociation constant (<italic>K</italic><sub>D</sub>) indicates higher affinity. Convalescent sera from mice that recovered from a sub-lethal H5N2 (Wigeon/07) or H1N1 (pdm Ca/09) virus challenge was used as positive controls. The BLI determined sera reactivity profile of primary and secondary <bold>(A)</bold> H5F and <bold>(B)</bold> H1F immune sera (n.b. indicates no detectable binding). <bold>(C)</bold> The BLI determined sera reactivity profile of secondary immune sera against a panel of HAs from influenza A groups 1 and 2, and influenza B using BLI. <bold>(D)</bold> Immune sera competition with the bnAb CR6261 for binding to H1 HA (pdm Ca/09). CR6261 was captured on amine reactive biosensors and the interaction with full-length H1 HA (pdm Ca/09) was monitored by BLI using the Octet RED96 instrument. The binding of H1-HA (pdm Ca/09) protein to CR6261 was also probed after incubation (30 mins) of the protein with different sera dilutions (as indicated). The decreased binding of H1 HA (pdm Ca/09) to the immobilized CR6261 in the presence of vaccine sera indicated the existence of competing antibodies. Pooled serum (n = 5–20 mice) was used, experiments were repeated two to three times and performed in duplicate. (<bold>E</bold>) Vaccine sera tested in direct ELISA specific for different recombinant HA proteins.</p></caption><graphic xlink:href="srep22666-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>HA mini-stem immunization provides robust protection against lethal heterologous virus challenge in mice.</title><p>Female BALB/c mice were vaccinated twice at 3 weeks interval by the intramuscular (i.m.) route with 20 μg of HA mini-stem adjuvanted with Addavax. PBS with Addavax (PBS + A) vaccinated mice were used as a negative control. Vaccinated mice were challenged 21 days after the secondary immunization with <bold>(A)</bold> HPAI H5N1 (Indo/05, 30TCID<sub>50</sub>), <bold>(B)</bold> H1N1 (pdm Ca/09, 1.36 × 10<sup>6</sup> TCID<sub>50</sub>) or <bold>(C)</bold> H3N2 (HK/68, 2.5 × 10<sup>6</sup> TCID<sub>50</sub>) virus. Survival and symptom severity were monitored for 14 days post virus challenge (n = 5). Lung viral loads at day 3 and day 7 were determined by standard TCID50 on MDCK cells (n = 3–6). <bold>(D)</bold> Passive transfer of immune sera (day 21 after the secondary immunization, heat inactivated, pooled n = 20 mice). 500 μl of serum was given intraperitoneally (i.p.) prior to HPAI H5N1 (Indo/05, 30TCID<sub>50</sub>) virus challenge of naïve BALB/c mice (n = 8). Survival, symptom severity (n = 5), and day 7 viral loads (n = 3) were determined. Statistics for survival was determined by a log-rank test. Statistics for symptom severity and lung viral loads versus PBS + A controls were determined Student’s t-test. (P-values: <sup>#</sup><0.05, <sup>*</sup><0.01, <sup>**</sup><0.001).</p></caption><graphic xlink:href="srep22666-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>HA-stem specific neutralizing antibodies are CD4 dependent and do not augment viral entry.</title><p><bold>(A)</bold> The virus neutralization potency of ‘headless’ HA stem immunized mice sera was tested in a standard microneutralization assay against a panel of influenza viruses, and the lowest sera dilution able to neutralize 100TCID<sub>50</sub> of a given virus is reported. Naïve mouse sera was used as a negative control, and convalescent sera from H1N1 (pdm Ca/09) or H5N2 (LPAI, Wigeon/07) virus challenge were used as positive controls. Experiments were performed at least 3 times. <bold>(B)</bold><italic>In vitro</italic> neutralization activity of HA mini-stem vaccine serum against a panel of H5 pseduotyped virus particles. IC<sub>50</sub> titer is the reciprocal of sera dilution at which half-maximal neutralization was observed. IC<sub>50</sub> titer of zero indicates no detectable neutralization. <bold>(C)</bold> Antibody dependent enhancement was assessed by H1N1 (pdm Ca/09) or H3N2 (HK/68) infection of Raji cells in the presence of vaccine sera, convalescent sera or naïve mouse sera for 6 hours. Influenza NP expression of Raji cells was assessed by flow cytometry. % NP expression is shown, experiment was perfomred twice, in triplicate.</p></caption><graphic xlink:href="srep22666-f4"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Hong Kong</li><li>Inde</li><li>Royaume-Uni</li></country><region><li>Illinois</li></region><settlement><li>Chicago</li></settlement></list><tree><country name="Hong Kong"><region name="Illinois"><name sortKey="Valkenburg, Sophie A" sort="Valkenburg, Sophie A" uniqKey="Valkenburg S" first="Sophie A." last="Valkenburg">Sophie A. Valkenburg</name></region><name sortKey="Chin, Alex W H" sort="Chin, Alex W H" uniqKey="Chin A" first="Alex W. H." last="Chin">Alex W. H. Chin</name><name sortKey="Li, Olive T W" sort="Li, Olive T W" uniqKey="Li O" first="Olive T. W." last="Li">Olive T. W. Li</name><name sortKey="Poon, Leo L M" sort="Poon, Leo L M" uniqKey="Poon L" first="Leo L. M." last="Poon">Leo L. M. Poon</name><name sortKey="Valkenburg, Sophie A" sort="Valkenburg, Sophie A" uniqKey="Valkenburg S" first="Sophie A." last="Valkenburg">Sophie A. Valkenburg</name></country><country name="Inde"><noRegion><name sortKey="Mallajosyula, V Vamsee Aditya" sort="Mallajosyula, V Vamsee Aditya" uniqKey="Mallajosyula V" first="V. Vamsee Aditya" last="Mallajosyula">V. Vamsee Aditya Mallajosyula</name></noRegion><name sortKey="Varadarajan, Raghavan" sort="Varadarajan, Raghavan" uniqKey="Varadarajan R" first="Raghavan" last="Varadarajan">Raghavan Varadarajan</name></country><country name="Royaume-Uni"><noRegion><name sortKey="Carnell, George" sort="Carnell, George" uniqKey="Carnell G" first="George" last="Carnell">George Carnell</name></noRegion><name sortKey="Temperton, Nigel" sort="Temperton, Nigel" uniqKey="Temperton N" first="Nigel" last="Temperton">Nigel Temperton</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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