Konformationel Evaluering af HIV-1 trimere kappeglycoproteinerne hjælp af en celle-ELISA-assay
Summary
Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.
Abstract
HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.
Introduction
Human immundefekt virus type 1 (HIV-1) indrejse, medieret af de trimere virale kappeglycoproteiner (ENV) er det første skridt i den infektiøse cyklus. Som den eneste udsatte viralt antigen, der præsenteres på overfladen af virioner Env trimeren fremkalder neutraliserende og ikke-neutraliserende antistoffer. Som sådan, det repræsenterer en interessant kandidat til vaccine immunogen design. Men vaccination forsøg med Env i opløselige eller rekombinante former fremkaldte reaktioner med kun minimal effektivitet mod de fleste primært HIV-1-isolater 1-3. Ikke desto mindre, delvis effekt observeret i RV144 vaccineforsøg 4 fornyet interesse for HIV-1 Env som et immunogen kandidat. Dette blev bekræftet af en nylig undersøgelse, der beskriver, at vaccine-fremkaldte anti-Env antistoffer var tilstrækkelig til at frembringe en vis grad af beskyttelse mod SIV og hiv udfordringer 5.
Efter at være blevet syntetiseret i det endoplasmatiske reticulum, Env glycoprotei forstadiet, gp160, gennemgår forskellige post-translationelle modifikationer, der er kritiske for sin evne til at sætte gang virale fusionsprocessen. Env-forstadium skal folde korrekt og associeret i trimerer før blive spaltet i sine ekstra cytoplasmiske gp120 og transmembrane gp41 underenheder 6-10, med ikke-kovalente interaktioner opretholde gp120-gp41-kontakt. Den inficerede celle maskiner er også ansvarlig for kraftigt glycosylere Env, der omfatter omkring 50% af dens samlede masse 11,12. Det resulterende kompleks struktur giver mulighed Env at være konformationelt fleksible 13,14, men samtidig give en metastability, som menes at tillade Env at tilpasse og skjule bestemte højimmunogene epitoper, som ellers ville blive udsat 15-19, fremhæve betydningen for bedre at forstå de forskellige konformationer samplet af den indfødte Env trimeren.
Til dato har adskillige teknikker blevet udviklet og med succes anvendt til at undersøge Env konformationel lmanges. Men de varierer i deres begrænsninger, idet der ofte er begrænset til bestemte Env sammenhænge. For eksempel overfladeplasmonresonans eller immunopræcipitationsanalyser hjælp kropsbygning specifikke monoklonale antistoffer (mAb'er), er afhængige enten monomere opløselige eller solubiliserede Env molekyler, som vides at være immunogenetically forskellige fra deres trimere former 20,21. Nylige undersøgelser tyder også på, at spaltning påvirker Env konformationer resulterer i eksponering af epitoper primært genkendes af neutraliserende antistoffer 14,22,23.
Her beskriver vi i detaljer en metode, der giver mulighed for hurtig og let bestemmelse af konformationen af cellulært udtrykt Env trimerer 18,24-26. Efter transient transfektion Env i en human adhærerende cellelinje bindingen af Env-specifikke antistoffer påvises ved anvendelse af en simpel kemiluminescens reaktion. Denne teknik kan også anvendes til at karakterisere den konformationelle præference konformationsbegrænsende DEPENdent antistoffer. Således dette assay giver en robust og meget fleksibel påvisningsmetode.
Protocol
Representative Results
Discussion
Dette assay er optimeret til at detektere interaktionen af specifikke mAb'er med HIV-1 trimer Env udtrykt på celleoverfladen. Når protokollen er blevet etableret, kan den bruges ved medium til høj produktivitet med lave samlede materielle omkostninger og små mængder af antistoffer. Eftersom dette assay er transfektion-baseret, kan det let tilpasses til coekspression af cellulære proteiner, såsom CD4 for at undersøge deres virkninger på Env konformation.
Men transfektion ba…
Declarações
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. James Robinson for hans generøse gave af A32, 17b, 48d, og C11-mAb'er. PGT 121 blev opnået gennem NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (Kat # 12343). Dette arbejde blev støttet af en Canada Foundation for Innovation Program Leader # 29866, ved en CIHR operativsystem # 257792 ved en FRQS Etablering af unge videnskabsmand indrømme # 24639 til AF og ved en CRCHUM kontinuum tilskud samt ved en CIHR katalysator tilskud # 126630 til AF og MR. AF er modtageren af en FRSQ Chercheur Boursier Junior 1 fællesskab # 24639. MV blev understøttet af en CIHR ph.d.-forskning Award # 291485.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| HOS cells | ATCC | CRL-1543 | |
| White Opaque Tissue Culture Plate, 96 well, Flat Bottom | BD | 353296 | |
| Polyethylenimine, linear, 25000 MW | Polysciences | 23966 | Prepared in 1mg/ml solution |
| Dulbecco's Modified Eagle Medium | Invitrogen | 11995 | |
| Goat Anti-Human IgG, Peroxidase Conjugated | Pierce | 31413 | |
| Enhanced Chemiluminescence Substrate | PerkinElmer | NEL105001EA | |
| TriStar LB 941, Plate Reader | Berthold Technologies |
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