Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

Dette projekt har været finansieret delvist med føderale midler fra det nationale Institut for allergi og smitsomme sygdomme, National Institutes of Health, Department of Health og Human Services, under CEIRS kontrakt P01AI097092-04S1 (P.E.L.).

Eksperimenter præfabrikerede i dette håndskrift var gjort følgende Icahn School of Medicines institutionelle kodeks for etik og forordninger.
1. angivelse af Influenza Virus Hemagglutinin via Transfektion eller infektion
Transfektion:
<ol><li>Plade menneskelige embryonale nyre (HEK 293T) celler med en tæthed på 2 x 104 celler/brønd i en hvid vævskultur behandlet 96-brønd plade og lad det sidde i 4 h i et 37 °C ...

<ol>
	<li>Hobson, D., Curry, R. L., Beare, A. S., Ward-Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+serum+haemagglutination-inhibiting+antibody+in+protection+against+challenge+infection+with+influenza+A2+and+B+viruses.">The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses.</a> J Hygiene. 70 (04), 767-777 (1972).</li><li>Throsby, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosubtypic+neutralizing+monoclonal+antibodies+cross-protective+against+H5N1+and+H1N1+recovered+from+human+IgM++memory+B+cells.">Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.</a> PloS One. 3 (12), e3942 (2008).</li><li>Ekiert, D. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+solution+to+group+2+influenza+virus+neutralization.">A common solution to group 2 influenza virus neutralization.</a> PNAS. 111 (1), 445-450 (2014).</li><li>Dreyfus, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Conserved+Protective+Epitopes+on+Influenza+B+Viruses.">Highly Conserved Protective Epitopes on Influenza B Viruses.</a> Science. 337 (6100), 1343-1348 (2012).</li><li>Krammer, F., Palese, P., Steel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Universal+Influenza+Virus+Vaccine+Design+and+Antibody+Mediated+Therapies+Based+on+Conserved+Regions+of+the+Hemagglutinin.">Advances in Universal Influenza Virus Vaccine Design and Antibody Mediated Therapies Based on Conserved Regions of the Hemagglutinin.</a> Curr Top Microb and Immunol. , (2014).</li><li>Impagliazzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stable+trimeric+influenza+hemagglutinin+stem+as+a+broadly+protective+immunogen.">A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.</a> Science. , (2015).</li><li>Nachbagauer, R., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Microbiology+and+Infection.">Clinical Microbiology and Infection.</a> Clin Microb Infect. , 1-7 (2017).</li><li>Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+universal+influenza+virus+vaccine+approaches.">Novel universal influenza virus vaccine approaches.</a> Current opinion in virology. 17, 95 (2016).</li><li>Steel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+vaccine+based+on+the+conserved+hemagglutinin+stalk+domain.">Influenza virus vaccine based on the conserved hemagglutinin stalk domain.</a> mBio. 1 (1), (2010).</li><li>Krammer, F., Pica, N., Hai, R., Margine, I., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+hemagglutinin+influenza+virus+vaccine+constructs+elicit+broadly+protective+stalk-specific+antibodies.">Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies.</a> J Virol. 87 (12), 6542-6550 (2013).</li><li>Krammer, F., Pica, N., Hai, R., Tan, G. S., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+Stalk-Reactive+Antibodies+Are+Boosted+following+Sequential+Infection+with+Seasonal+and+Pandemic+H1N1+Influenza+Virus+in+Mice.">Hemagglutinin Stalk-Reactive Antibodies Are Boosted following Sequential Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice.</a> J Virol. 86 (19), 10302-10307 (2012).</li><li>Hai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+viruses+expressing+chimeric+hemagglutinins:+globular+head+and+stalk+domains+derived+from+different+subtypes.">Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes.</a> J Virol. 86 (10), 5774-5781 (2012).</li><li>Dunand, C. J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+Neutralizing+and+Non-Neutralizing+Human+H7N9+Influenza+Vaccine-Induced+Monoclonal+Antibodies+Confer+Protection.">Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.</a> Cell Host and Microbe. 19 (6), 800-813 (2016).</li><li>Tan, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly-Reactive+Neutralizing+and+Non-neutralizing+Antibodies+Directed+against+the+H7+Influenza+Virus+Hemagglutinin+Reveal+Divergent+Mechanisms+of+Protection.">Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.</a> PLoS Path. 12 (4), e1005578 (2016).</li><li>DiLillo, D. J., Tan, G. S., Palese, P., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+hemagglutinin+stalk-specific+antibodies+require+Fc&#947;R+interactions+for+protection+against+influenza+virus+in+vivo.">Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc&#947;R interactions for protection against influenza virus in vivo.</a> Nat Med. 20 (2), 143-151 (2014).</li><li>DiLillo, D. J., Palese, P., Wilson, P. C., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+anti-influenza+antibodies+require+Fc+receptor+engagement+for+in+vivo+protection.">Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.</a> J Clin Invest. 126 (2), 605-610 (2016).</li><li>Potter, C. W., Oxford, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+immunity+to+influenza+infection+in+man.">Determinants of immunity to influenza infection in man.</a> Brit Med Bull. 35 (1), 69-75 (1979).</li><li>Memoli, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Antihemagglutinin+and+Antineuraminidase+Antibodies+as+Correlates+of+Protection+in+an+Influenza+A/H1N1+Virus+Healthy+Human+Challenge+Model.">Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A/H1N1 Virus Healthy Human Challenge Model.</a> mBio. 7 (2), e00417 (2016).</li><li>Jegaskanda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+cross-reactive+antibody-dependent+cellular+cytotoxicity+toward+2009+pandemic+influenza+A+virus+subtype+H1N1.">Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.</a> J Infect Dis. 208 (7), 1051-1061 (2013).</li><li>Jegaskanda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-Reactive+Influenza-Specific+Antibody-Dependent+Cellular+Cytotoxicity+Antibodies+in+the+Absence+of+Neutralizing+Antibodies.">Cross-Reactive Influenza-Specific Antibody-Dependent Cellular Cytotoxicity Antibodies in the Absence of Neutralizing Antibodies.</a> J Immunol. 190 (4), 1837-1848 (2013).</li><li>Jegaskanda, S., Wheatley, A. K., Kent, S. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+activation+of+Fc-mediated+effector+functions+by+influenza+virus+hemagglutinin+antibodies+requires+two+points+of+contact.">Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact.</a> PNAS. , 201613225 (2016).</li><li>Benjamin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Broadly+Neutralizing+Human+Monoclonal+Antibody+Directed+against+a+Novel+Conserved+Epitope+on+the+Influenza+Virus+H3+Hemagglutinin+Globular+Head.">A Broadly Neutralizing Human Monoclonal Antibody Directed against a Novel Conserved Epitope on the Influenza Virus H3 Hemagglutinin Globular Head.</a> J Virol. 88 (12), 6743-6750 (2014).</li><li>Ekiert, D. C., Kashyap, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-neutralization+of+influenza+A+viruses+mediated+by+a+single+antibody+loop.">Cross-neutralization of influenza A viruses mediated by a single antibody loop.</a> Nature. 488 (7417), 526-532 (2013).</li><li>He, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+Neutralizing+Anti-Influenza+Virus+Antibodies:+Enhancement+of+Neutralizing+Potency+in+Polyclonal+Mixtures+and+IgA+Backbones.">Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones.</a> J Virol. 89 (7), 3610 (2015).</li><li>Kristensen, A. 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Requirement for antibody and non-T lymphocytes.</a> J Immunol (Baltimore, Md. : 1950). 119 (6), 2100-2106 (1977).</li><li>Corrales-Aguilar, E., Trilling, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+detecting+virus-specific+antibodies+triggering+activation+of+Fc&#947;+receptors.">A novel assay for detecting virus-specific antibodies triggering activation of Fc&#947; receptors.</a> J Immunol Meth. 387 (1-2), 21-35 (2013).</li><li>de Vries, R. 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Metoden beskrevet her tillader brugeren at måle en HA-specifikke monoklonale antistof evne til at engagere de murine FcγRIV. Target antigen, influenza virus hemagglutinin, udtrykkes på overfladen af celler efter infektion med virus eller Transfektion af plasmid DNA. Analysen er yderligere modtagelig hen til benytter forskellige overflade-udtrykt virale proteiner i kombination med andre FcγRs (mennesker eller murine). Derudover har vi brugt sera prøver for at vurdere antistoffer i forbindelse med polyklonale evne til...

Immunitet, som den sæsonbestemte influenzavaccine er traditionelt blevet vurderet af den hemagglutination hæmning (HI) analyse1, hvilke måler tilstedeværelsen af antistoffer rettet mod webstedet receptor binding af hemagglutinin. Disse HI-aktive antistoffer kan overdrage sterilisering immunitet, men er generelt smalle i deres bredde af beskyttelse, giver immunitet til kun en udvalgt nogle stammer af influenzavirus. Isolering og karakterisering af bredt reaktive monoklonale antistoffer, der genkender hemagglutinin (HA) tyder på at udvikle en universal influenzavaccine er inden for rækkevidde2,3,4, 5 , 6 , 7 , 8. en af de vigtigste mål for en universal influenzavaccine er at fremkalde en stærk antistofrespons mod de bevarede dele af influenza virus9,10,11,12 , 13 , 14 , 15 , 16. stort set reaktive antistoffer, der anerkender disse bevaret epitoper, består af både neutraliserende og ikke-neutraliserende antistoffer17,18, har vist sig at kræve Fc-FcγR interaktioner for optimal beskyttelse i vivo og Fremhæv bidrag af Fc-medieret immunitet over for influenza virus19,20samlede immunresponset.
Korrelerer beskyttelse er afgørende for vurderingen af immunitet over for infektion. Disse målinger giver mulighed for forskere og klinikere til at vurdere effekten af vacciner, betydningen af data og behandlingsforløbet. Den eneste etablerede korrelat af beskyttelse mod influenza virusinfektion er hemagglutination hæmning assay. En titer på 1:40 er forbundet med en 50% reduktion i risikoen for sygdom1,21 – og afspejler tilstedeværelsen af antistoffer, der hæmmer agglutination af målretning receptor bindende stedet ligger på den kugleformede hoved af HA. Det skal dog også bemærkes, at T-celle svar kan være en bedre korrelat af beskyttelse mod influenza virusinfektion hos ældre. Interessant nok, tyder en nylig rapport på, at neuraminidasetypen hæmning aktivitet kan være en bedre indikator for immunitet over for influenza22. Heldigvis, typisk in vitro- undersøgelser såsom neutralisering eller neuraminidase hæmning assays kan måle HA stilk - eller neuraminidase-specifikke antistoffer. De fleste af disse konventionelle in vitro- assays, imidlertid kun tages hensyn til funktionen af antigen bindende region af antistoffet og måler ikke rolle i regionen Fc. Derudover bidrag af ikke-neutraliserende antistoffer, som beskytter via Fc receptor engagement i vivo er ikke opdaget17,18. For at måle af antistoffer, der inducerer Fc-medieret immunitet som antistof afhængig celle medieret cytotoksicitet (ADCC) ydede beskyttelse, der er behov for en robust i vitro assay.
Metoden beskrevet nedenfor vurderer murine monoklonale antistoffer evne til at fremkalde Fc-medieret funktioner ved hjælp af en genetisk modificeret Jurkat cellelinje udtrykker en aktivering murine skrive 1 FK, FcγRIV. Antistof engagement af FcγR transduces intracellulær signalering at udløsere nukleare faktor af aktiverede T-celler-medieret luciferase aktivitet. Analysen har flere fordele frem for traditionelle teknikker, der kræver isolering og kultur af primære effektor celler og brugen af flowcytometri hen til opdager aktivisering af Fc-medieret effektor funktion19,23,24 ,25,26. Først kan protokollen beskrevet her nemt tilpasses til at optage forskellige viral mål, FcγRs og antistoffer fra forskellige arter (menneskelige og murine). Andet brugen af en modificeret Jurkat celle linje at udtrykke en FcγR med et luciferase reporter gen under en nuklear faktor af aktiverede T-celle (NFAT) giver mulighed for et stort format assay, der nemt kan analyseres ved hjælp af en Pladelæser måling luminescens. Her, erstattes den traditionelle aktivering af primære effektor celler med induktion af NFAT-luciferase ved antistof engagement i en FcγR på overfladen af cellen Jurkat. Denne 96-brønd plade format assay giver mulighed for måling af op til fire forskellige prøver i tre med syv fortyndinger – et antal stikprøver, der kunne være besværligt, ved hjælp af traditionelle teknikker. Der er yderligere punkter at overveje, med denne analyse, der kan påvirke design af eksperimenter og fortolkning af data. Viral målet skal udtrykkes på celleoverfladen i rækkefølge for antistof anerkendelse. For at afbøde dette, kan målet antigen (f.eks. en intern viral protein) belægges direkte på overfladen af en plade. Men dette ikke endnu er blevet grundigt testet. Derudover den modificerede Jurkat-cellelinie udtrykker kun én type af aktivering FcγR og udtrykker ikke nogen hæmmende FcγRs, mens primær cellelinjer udtrykke alle receptorer i en fysiologisk kontekst.
Vi har tidligere brugt dette assay for at demonstrere at epitopspecificitet et polyklonalt led spiller en afgørende rolle i reguleringen af Fc-medieret effektor funktion og at optimal aktivering af antistof-afhængige celle-medieret reaktion kræver to punkter i Kontakt27,28. Her, beskriver vi en metode, der vurderer evne til stilk-specifikke monoklonale antistoffer til at engagere sig og aktivere den murine aktivering FcγRIV. Selvom der er undtagelser29,30, et stort antal bredt reaktive antistoffer målrette hemagglutinin antigent bevarede stilk-regionen og dermed spille en vigtig rolle i udviklingen af en universel influenza vaccine.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">A549 cells&#160;</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-185</td>
			<td style="width:353px;">Adenocarcinomic human alveolar basal epithelial cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">HEK 293T cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CRL-3216</td>
			<td>Human embryonic kidney cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Lipofectamine 2000</td>
			<td style="width:141px;">Life Technologies&#160;</td>
			<td style="width:184px;">11668019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Opti-MEM Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">51985-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White tissue culture treated 96-well plate</td>
			<td>Corning, Inc.&#160;</td>
			<td>3917</td>
			<td>Assay plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">10X MEM</td>
			<td style="width:141px;">Gibco</td>
			<td style="width:184px;">11430-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing murine FcgRIV</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">M1201</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7010</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Jurkat cell expressing human FcgRIIa</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G9901</td>
			<td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luminometer</td>
			<td>BioTek</td>
			<td>Synergy H1 Multi-Mode reader</td>
			<td>Luminescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bio-Glo Luciferase Assay System</td>
			<td style="width:141px;">Promega</td>
			<td style="width:184px;">G7940</td>
			<td>Contains luciferase assay buffer and luciferase assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Madin Darby canine kidney cells</td>
			<td style="width:141px;">ATCC</td>
			<td style="width:184px;">CCL-34</td>
			<td>Canine kidney cells</td>
		</tr>
	</tbody>
</table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

Antistoffer spiller en afgørende rolle i at kombinere de medfødte og adaptive immunrespons mod viral patogener gennem deres antigen bindende domæner og Fc-regioner. Her, vi beskriver, hvordan at måle aktivering af Fc effektor funktion af monoklonale antistoffer rettet mod influenza virus hemagglutinin med brug af en genetisk manipuleret Jurkat cellelinje udtrykker en aktivering af type 1 Fc-FcγR. ved hjælp af denne metode, den bidrag af specifikke Fc-FcγR interaktioner tillagt immunoglobuliner kan bestemmes ved hjælp af en i vitro assay.

Vi vist, at en stilk-specifikke mAb, 6F12, men ikke hovedet-specifikke en hoved-specifikke mAb, PY102, var i stand til at aktivere primære naturlige dræberceller ved upregulating CD107a og interferon γ19. For at modellere en stilk-specifikke antistof evne til at aktivere primære menneskelige NK celler, viser vi, at en stilk-specifikke mAb, 6F12, håndfast kan aktivere cellen modificerede Jurkat udtrykker den murine FcγRIV af ~ 14-fold. På den anden side gør hoved-specifikke mAb, PY102 og ko...

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Vi beskriver en metode til at måle aktivering af Fc-medieret effektor funktion af antistoffer der er rettet mod influenza virus hemagglutinin. Denne analyse kan også tilpasses for at vurdere monoklonale antistoffer eller polyklonale sera rettet mod andre virale overflade glykoproteiner evne til at fremkalde Fc-medieret immunitet.

En metode til at vurdere Fc-medieret effektor funktion induceret af Influenza Hemagglutinin specifikke antistoffer

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Research

JoVE Journal

Immunology and Infection

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

High-throughput Detection Method for Influenza Virus

High-throughput Detection Metode til Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In vitro assay at evaluere virkningen af immunoregulerende Pathways på HIV-specifikke CD4 T-celle effektorfunktion

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay at vurdere Patogen induceret Neutrophil Trans-epitel Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

Ekspression af funktionelle Rekombinant hæmagglutinin og neuraminidase Proteiner fra Novel H7N9 influenza virus ved hjælp baculovirusekspressionssystemet

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Måling Attachment og internalisering af influenza A-virus i A549-celler ved flowcytometri

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In vitro Demontering af influenza A-virus capsider ved gradientcentrifugering

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Anvendelse fluorescensresonansenergioverførsel (FRET) til at undersøge Effector translokationseffektivitet af<em&gt; Coxiella burnetii</em&gt; Under siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Fluorescensbaseret neuraminidase-inhiberingsassay i vurdering af den følsomhed over influenzavira til neuraminidaseinhibitoren Class of Antivirale

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Generation af undslippe varianter af neutraliserende Influenza Virus monoklonale antistoffer

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...