Name: utilizing the antigen capsid-incorporation strategy for the development of adenovirus serotype 5-vectored vaccine approaches
Uploaded: 2015-05-06T14:00:00
Description: Watch this Scientific Journal Video about Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches at JoVE.com

Dette arbejde blev støttet delvist af National Institutes of Health tilskud 5T32AI7493-20 og 5R01AI089337-03. De finansieringskilderne havde ingen rolle i studie design, indsamling og analyse af data, beslutning om at offentliggøre, eller forberedelse af manuskriptet.

University of Alabama i Birmingham Institutional Animal brug og Omsorgsudvalget godkendt brugen af ​​mus som beskrevet heri under den godkendte protokol nummer 101.109.272. 1. Genetisk Konstruktion af et Modified Plasmid pAd5 / H5-HVR1-Kwas-HVR5-His 6 med antigenet capsidet-Indbygning strategi <ol><li> Konstruktion af shuttle plasmidet HVR1-Kwas-HVR5-Hans 6 / pH5S <ol><li> Bestil et plasmid indeholdende den syntetiserede DNA-sekvens HVR1-Kwas mellem restriktionsen...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+innate+and+pre-existing+immunity+on+adenovirus+therapy.">The influence of innate and pre-existing immunity on adenovirus therapy.</a> Journal of cellular biochemistry. 108, 778-790 (2009).</li><li>Sumida, S. 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=Neutralizing+antibodies+and+CD8++T+lymphocytes+both+contribute+to+immunity+to+adenovirus+serotype+5+vaccine+vectors.">Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors.</a> Journal of virology. 78, 2666-2673 (2004).</li><li>Sumida, S. 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=Neutralizing+antibodies+to+adenovirus+serotype+5+vaccine+vectors+are+directed+primarily+against+the+adenovirus+hexon+protein.">Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein.</a> Journal of immunology. 174, 7179-7185 (2005).</li><li>Bradley, R. 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Anvendelsen af den traditionelle transgen strategi for Ad5 modifikation for udviklingen af vacciner er blevet reduceret primært som følge af flaskehalsen forbundet med Ad5 PEI 4,6. Denne flaskehals kan delvis formindskes ved anvendelse af alternative Antigen capsidet-Indbygning strategi (figur 1A), da denne strategi kan unddrage neutralisering af Ad5 NABS ved at erstatte neutraliserende epitoper af Ad5 med antigener af interesse, og letter generation af robuste immunitet til de inkorporered...

Human adenovirus (Ad) er en mellemstor, ikke-kappeklædte virus med en tyvefladet nukleokapsid indeholder en dobbeltstrenget DNA genom. Annoncen tilhører Adenoviridae familien, med en klassifikation i syv grupper (A til G). Hver gruppe indeholder virus af forskellige serotyper. Heraf adenovirus serotype 5 (Ad5) fra gruppe C har været den mest omfattende undersøgt og er det mest ansøgte vectored tilgange såsom genterapi og vaccination. Den traditionelle transgen strategi er blevet udviklet og anvendt til Ad5 modifikationer, som er karakteriseret ved forskydningen af ​​virus tidlige gener med et gen af ​​interesse, og den fokuserede ekspression af genet af interesse i en vært. Eksempler er konstruktionen af pENV9 / Ad5hrΔE3 ved at erstatte tidlige gen 3 (E3) med rev-genet fra Simian Immunodeficiency Virus 1, opførelse af AdCMVGag ved at erstatte tidlige gen 1 (E1) med gag-genet af human immundefektVirus (HIV) 2, og opførelsen af Ad lacZ ved at erstatte E1 med lacZ gen 3. Den brede anvendelse af den traditionelle transgen strategi for Ad5 afhænger af følgende fortjenester: den brede vifte af værter for Ad5 den mulige gen engineering på virus og virus formering, store indkvartering af fremmed gen indsatsen og sikkerhed Ad5 4,5. De reducerede effektiviteter af Ad5-vectored kliniske behandlinger ved brug af denne strategi har dog været en vigtig flaskehals, som er blevet kortlagt til primært forbundet med Ad5 PEI, da Ad5 er så udbredt blandt de fleste børn og voksne 4,6. At overvinde de store hindringer for Ad5 det primære mål er at udvikle en alternativ strategi omgå Ad5 PEI. Medfødte immunitet 7, adaptiv immunitet såsom neutraliserende antistoffer (NABS) 8-10 og CD8 + T-cellereaktioner 10 imod Ad5 er blevet vist at contribute til Ad5 PEI, med Ad5 NABS synes at spille den dominerende rolle i bidragene til Ad5 PEI 10,11. Desuden Ad5 nabs målepitoper placeret i capsidproteiner, herunder det store protein hexon, fiber og penton base. Heraf hexon er den vigtigste mål for Ad5 NABS 8,11-13. Baseret på disse resultater, er der indført en innovativ Antigen capsidet-Indbygning-strategi. Denne roman strategi fremhæver udskiftning eller inkorporering af proteiner af interesse på Ad5 capsidproteiner, der skifter eller masker Ad5 neutraliserende epitoper, der fører til nedsat anerkendelse fra NABS og effektive Ad5 vektor forvaltninger. Det er bemærkelsesværdigt, at denne strategi er konkurrencedygtige, fordi det også kan hjælpe værter fremkalde robust humoral immunitet og potent cellulær immunitet ved direkte at præsentere antigener af interesse til immunsystemet 4,14,15. Baseret på denne strategi, kan den molekylære kloning og rekombinant Ad viral vektor redning strukturelt opdelti fire hovedtrin: (a) fremstilling af genet af interesse fragment ved enten polymerasekædereaktion (PCR) eller syntese; (B) ligering af gen-fragment i en shuttle-plasmid, der indeholder genet af interesse fragment og homologe arme til en adenovirus backbone; (C) den homologe rekombination af co-transformation shuttle plasmid indeholdende genet af interesse fragmentet med det lineariserede plasmid backbone pAd5 / ΔH5 (GL) 16; (D) transfektion af lineariseret rekombinant adenoviral plasmid for at redde den rekombinante Ad-vektoren indarbejdet med antigener af interesse. Vores gruppe og nogle andre har udvidet denne alternative annonce inkorporering strategi for Ad vektoriserede vaccine udvikling mod forskellige infektiøse patogener. Vi rapporteret dannelsen af en rekombinant Ad vektor Ad-HVR1-LGS-His 6 -V3 ved at inkorporere et His-mærket HIV-1-antigenet V3 ind HVR1 locale af Ad5 hexon (hexon5). Dette genererede vektor udløste strrespons ong humoral immunrespons specifikt for V3 epitopen 4. Vi rapporterede også udviklingen af Ad5 / HVR2-MPER-L15ΔE1 ved at inkorporere HIV-1 membran proksimale ektodomæne region (MPER) i HVR2 locale af hexon5 2. Derudover har Dr. Zhou gruppe anvendes fordelene ved denne annonce inkorporering strategi for at udvikle Ad serotype 3 (Ad3) vektoriserede vacciner, dvs.., Frembringelsen af virale vektor R1SP70A3 ved inkorporering af et neutraliserende epitop SP70 af Enterovirus 71 i HVR1 af Ad3 hexon (hexon3). R1SP70A3 genereret stærke NABS og IFN-γ produktion specifikke for epitopen SP70, som fører til den høje beskyttelse mod Enterovirus 71 udfordring 15. Med henblik på teknisk reference, vores undersøgelse udnyttede den kvalitative Antigen capsidet-Indbygning strategi om at fokusere på dannelsen af et divalent Ad5 vektor Ad5 / H5-HVR1-Kwas-HVR5-His 6 ved at inkorporere en HIV-1-antigen i HVR1 enda Hans-tag ind HVR5 af hexon5. Den genererede virusvektor blev også immunologisk evalueret. Antigenet capsid--Indbygning strategi kunne udnyttes til udvikling af Ad5-vectored vaccination tilgange mod forskellige infektionssygdomme.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1x DPBS</td>
			<td>Thermo Scientific</td>
			<td>SH30256.01</td>
			<td>for cell spliting&#160;</td>
		</tr>
		<tr>
			<td>10x DPBS</td>
			<td>Thermo Scientific</td>
			<td>SH30378.02</td>
			<td>for dialysis buffer preparation</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>SIGMA</td>
			<td>G5516-1L</td>
			<td>for dialysis buffer preparation</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>BIO-RAD</td>
			<td>161-0301</td>
			<td>for virus lysis buffer preparation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Thermo Scientific</td>
			<td>SH30910.03</td>
			<td>component of culture medium</td>
		</tr>
		<tr>
			<td>100X Non-Essential Amino Acids&#160;</td>
			<td>Thermo Scientific</td>
			<td>SH30238.01</td>
			<td>component of culture medium</td>
		</tr>
		<tr>
			<td>200mM/L L-glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td>component of culture medium</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin solution&#160;</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td>component of culture medium</td>
		</tr>
		<tr>
			<td>DMEM with high glucose</td>
			<td>Thermo Scientific</td>
			<td>SH30081.01</td>
			<td>for HEK293 cell culture</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle</td>
			<td>SIGMA</td>
			<td>M5650</td>
			<td>for HeLa cell culture</td>
		</tr>
		<tr>
			<td>phenol:chloroform:isoamyl alcohol&#160;</td>
			<td>SIGMA</td>
			<td>P3803-100ML</td>
			<td>for large size of DNA purification</td>
		</tr>
		<tr>
			<td>cesium chloride&#160;</td>
			<td>Research Products International Corp.</td>
			<td>C68050</td>
			<td>for virus purificiation</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cellgro</td>
			<td>25-060-CI</td>
			<td>for CsCl solution preparation</td>
		</tr>
		<tr>
			<td>HEK293</td>
			<td>ATCC</td>
			<td>51-0036</td>
			<td>for virus rescue and upscale</td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>for neutralization assay</td>
		</tr>
		<tr>
			<td>T-25 flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td>for cell culture&#160;</td>
		</tr>
		<tr>
			<td>T-75 flask</td>
			<td>CORNING</td>
			<td>430641</td>
			<td>for cell culture&#160;</td>
		</tr>
		<tr>
			<td>T-175 flask</td>
			<td>Thermo Scientific</td>
			<td>159910</td>
			<td>for cell culture&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>BECKMAN</td>
			<td>NA</td>
			<td>for virus purification</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tube</td>
			<td>BECKMAN</td>
			<td>344059</td>
			<td>for virus purification</td>
		</tr>
		<tr>
			<td>dialysis cassette&#160;</td>
			<td>Thermo Scientific</td>
			<td>66380</td>
			<td>for virus dialysis</td>
		</tr>
		<tr>
			<td>ELISA plate</td>
			<td>Thermo Scientific</td>
			<td>442404</td>
			<td>for ELISA</td>
		</tr>
		<tr>
			<td>human anti-gp41 (2F5) mAb</td>
			<td>NIH AIDS Reagent Program</td>
			<td>1475</td>
			<td>for immunological assays</td>
		</tr>
		<tr>
			<td>mouse anti-His tag mAb</td>
			<td>GenScript</td>
			<td>A00186</td>
			<td>for immunological assays</td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>CORNING</td>
			<td>46-003-CR</td>
			<td>for transformation</td>
		</tr>
		<tr>
			<td>PCR master mix solution</td>
			<td>QIAGEN</td>
			<td>201445</td>
			<td>for PCR</td>
		</tr>
		<tr>
			<td>Animal lancet (point length at 5mm)</td>
			<td>MEDIpoint</td>
			<td></td>
			<td>for mice bleeding&#160;</td>
		</tr>
		<tr>
			<td>Biophotometer</td>
			<td>Eppendorf</td>
			<td></td>
			<td>for virus physical titer titration</td>
		</tr>
	</tbody>
</table>

utilizing the antigen capsid-incorporation strategy for the development of adenovirus serotype 5-vectored vaccine approaches

Adenovirus serotype 5 (Ad5) er blevet grundigt modificeret med traditionelle transgene metoder til udvikling af vacciner. De reducerede effektiviteter af disse traditionelt modificerede Ad5 vektorer i kliniske forsøg kunne primært korreleret med Ad5 allerede eksisterende immunitet (PEI) blandt det flertal af befolkningen. At fremme Ad5-vektoriserede vaccine udvikling ved at løse bekymring Ad5 PEI, har den innovative Antigen capsidet-Indbygning strategi været ansat. Ved merit i denne strategi, Ad5-vectored vi først konstrueret hexon shuttle plasmidet HVR1-Kwas-HVR5-Hans 6 / pH5S ved subkloning af hypervariable region (HVR) 1 af hexon i en tidligere konstrueret shuttle plasmid HVR5-Hans 6 / pH5S, som havde His6 tag indarbejdes i HVR5. Denne HVR1 DNA-fragment indeholdende en HIV-epitop ELDKWAS blev syntetiseret. HVR1-Kwas-HVR5-Hans 6 / pH5S blev derefter lineariseret og co-transformeret med lineariseret backbone plasmid pAd5 / ΔH5 (GL),til homolog rekombination. Dette rekombineret plasmid pAd5 / H5-HVR1-Kwas-HVR5-His 6 blev transficeret ind i celler for at generere den virale vektor Ad5 / H5-HVR1-Kwas-HVR5-His 6. Denne vektor blev valideret til at have kvalitative fitness angivet ved viral fysisk titer (VP / ml), infektiøs titer (IP / ml) og tilsvarende VP / IP-forhold. Både HIV-epitop og His-tag 6 var overflade-eksponeret på Ad5 capsid, og beholdt epitop-specifikke antigenicitet af deres egne. En neutralisation assay indikerede evne denne divalente vektor at omgå neutralisering af Ad5-positive sera in vitro. Mus immunisering demonstreret frembringelse af robuste humoral immunitet specifikke for HIV epitopen og His6. Dette proof-of-principle undersøgelse foreslået, at protokollen er knyttet til Antigen capsidet-Indbygning strategi kunne indpasses anvendes til generering af Ad5-vectored vacciner ved at ændre forskellige kapsidproteiner. Denne protokol kan endda være fDERLIGERE modificeret til frembringelse af sjældne serotype adenovirus-vectored vacciner.

Antigenet capsid-Inkorporering strategien (figur 1A) blev anvendt til at generere den divalente Ad5 virusvektor Ad5 / H5-HVR1-Kwas-HVR5-His 6. For det første shuttle plasmidet HVR1-Kwas-HVR5-His 6 / pH5S blev konstrueret ved subkloning HVR1-Kwas fragment i den tidligere konstruerede shuttle-plasmid HVR5-His 6 / pH5S 17. For det andet plasmid pAd5 / H5-HVR1-Kwas-HVR5-His 6 14 blev konstrueret ved en homolog rekombination mellem det fragme...

Watch this Scientific Journal Video about Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches at JoVE.com

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Her præsenterer vi en protokol til at generere en proof-of-principle divalente adenovirus type 5 (Ad5) vektor Ad5 / H5-HVR1-Kwas-HVR5-His 6 ved at udnytte Antigen capsidet-Indbygning-strategi. Denne vektor blev påvist at udvise kvalitativ egnethed skal muligheden undslippe Ad5-positive sera in vitro, og antigeniciteten samt immunogenicitet til de inkorporerede antigener.

Udnytte den Antigen capsidet-Indbygning strategi for udvikling af adenovirusserotype 5-vektoriserede Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

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