Conformational Evaluering av HIV-1 Trimeric kappeglykoproteiner Ved hjelp av en Cell-basert ELISA-analysen
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
Humant immunsviktvirus type 1 (HIV-1) oppføring, mediert av de trimere proanthocyanidiner viral kappeglykoproteiner (konv) er det første trinnet av den smittsomme syklus. Som den eneste synlige viralt antigen presentert på overflaten av virioner, utløser Env trimer nøytraliserende og nonneutralizing antistoffer. Som sådan representerer den en interessant kandidat for vaksine immunogen design. Men vaksinasjonsforsøk med konvolutt i løselig eller rekombinante former fremkalte responser med bare minimal effekt mot mest primære HIV-1-isolater 1-3. Likevel, delvis effekt observert i RV144 vaksineforsøk fire fornyet interesse for HIV-1 konvolutt som en immunogen kandidat. Dette ble bekreftet av en fersk undersøkelse som beskriver at vaksine fremkalte anti-konv antistoffer var tilstrekkelig til å generere en viss grad av beskyttelse mot SIV og HIV utfordrer fem.
Etter å ha blitt syntetisert i endoplasmatiske retikulum, ENV glycoprotei forløperen, gp160, gjennomgår en rekke post-translasjonelle modifikasjoner som er kritiske for sin evne til drivstoff virale fusjonsprosessen. ENV forløper må kaste riktig og advokatfullmektig i trimers før de hugges til sine ekstra-cytoplasmatiske GP120 og transmembrane gp41 subenheter 6-10, med ikke-kovalente interaksjoner opprettholde den gp120-gp41 liaison. Den infiserte cellen maskiner er også ansvarlig for tungt glykosylere konvolutt, som består av ca 50% av den totale massen 11,12. Den resulterende kompleks struktur tillater konvolutt å være konformasjonelt fleksibel 13,14, samtidig som det gir en metastability som er tenkt å tillate konvolutt å tilpasse seg og skjule visse svært immunogene epitoper som ellers ville bli utsatt 15-19, fremhever viktigheten av å bedre forstå de ulike konformasjoner samplet av de innfødte konvolutt trimer.
Hittil har flere teknikker blitt utviklet og brukt til å studere konvolutt conformational lmanges. Men, de varierer i sine begrensninger, blir ofte begrenset til bestemte konv sammenhenger. For eksempel, overflate-plasmonresonans eller Immunoutfellingsunder analyser ved hjelp conformation spesifikke monoklonale antistoffer (mAbs), avhengig enten av monomere oppløselige eller oppløseliggjorte Env-molekyler som er kjent for å være immunogenetically forskjellig fra deres trimere proanthocyanidiner 20,21. Nyere studier tyder også på at cleavage påvirker konv conformations resulterer i eksponeringen av epitoper hovedsakelig anerkjent av nonneutralizing antistoffer 14,22,23.
Her beskriver vi i detalj en fremgangsmåte som tillater rask og enkel bestemmelse av konformasjon av cellulært uttrykt Env-trimerer 18,24-26. Etter transient transfeksjon av Env i en human cellelinje vedheftende binding av env-spesifikke antistoffer blir detektert ved hjelp av en enkel kjemiluminescens reaksjon. Denne teknikken kan også brukes til å karakterisere conformational preferanse for konformasjon-DEPENdent antistoffer. Således gir denne analysen en robust og meget fleksibelt påvisningsmetode.
Protocol
Representative Results
Discussion
Denne analysen er optimalisert for å detektere interaksjonen av spesifikke mAbs med HIV-1 trimeric Env uttrykt på celleoverflaten. Når protokollen har blitt etablert, kan den brukes ved middels til høye hastigheter med lave totale materialkostnader og små mengder av antistoffer. Siden denne analysen er transfeksjon basert, kan det lett bli tilpasset for coexpression av cellulære proteiner som CD4 for å studere deres virkninger på konvolutt konformasjon.
Men transfeksjon base av denne…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi takker Dr. James Robinson for hans generøse gaven av A32, 17b, 48d, og C11 mAbs. PGT 121 ble innhentet gjennom NIH AIDS Reagens Program, Division of AIDS, NIAID, NIH (Cat # 12343). Dette arbeidet ble støttet av en Canada Foundation for Innovation Program Leader # 29866, av en CIHR drifts # 257792, av en FRQS Etablering av Young Scientist gi # 24639 til AF og ved en CRCHUM kontinuum stipend samt av en CIHR katalysator stipend # 126630 til AF og MR. AF er mottaker av en FRSQ Chercheur Boursier Junior 1 fellesskap # 24639. MV ble støttet av en CIHR Doktorgrads 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 |
References
- Bures, R., et al. Immunization with recombinant canarypox vectors expressing membrane-anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1. AIDS Res. Human Retroviruses. 16, 2019-2035 (2000).
- Koff, W. C. HIV vaccine development: challenges and opportunities towards solving the HIV vaccine-neutralizing antibody problem. Vaccine. 30, 4310-4315 (2012).
- Mascola, J. R., et al. Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation. 173, 340-348 (1996).
- Rerks-Ngarm, S., et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N. Engl. J. Med. 361, 2209-2220 (2009).
- Roederer, M., et al. Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV. Nature. 505, 502-508 (2014).
- Fennie, C., Lasky, L. A. Model for intracellular folding of the human immunodeficiency virus type 1 gp120. J. Virol. 63, 639-646 (1989).
- Willey, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A., Klausner, R. D. Biosynthesis cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc. Natl. Acad. Sci. U.S.A. 85, 9580-9584 (1988).
- Bosch, V., Pawlita, M. Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site. J. Virol. 64, 2337-2344 (1990).
- Hallenberger, S., et al. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature. 360, 358-361 (1038).
- Li, Y., Luo, L., Thomas, D. Y., Kang, C. Y. The HIV-1 Env protein signal sequence retards its cleavage and down-regulates the glycoprotein folding. Virology. 272, 417-428 (2000).
- Leonard, C. K., et al. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J. Biol. Chem. 265, 10373-10382 (1990).
- Wang, W., et al. A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization. Retrovirology. 10 (14), (2013).
- Helseth, E., Olshevsky, U., Furman, C., Sodroski, J. Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein. J. Virol. 65, 2119-2123 (1991).
- Haim, H., Salas, I., Sodroski, J. Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility. J. Virol. 87, 1884-1889 (2013).
- Chen, L., et al. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science. 326, 1123-1127 (2009).
- Kwong, P. D., et al. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature. 420, 678-682 (2002).
- Sakai, K., Takiguchi, M. Toward an effective AIDS vaccine development. Eur. J. Immunol. 43, 3087-3089 (2013).
- Veillette, M., et al. Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity. J. Virol. 88, 2633-2644 (2014).
- Wibmer, C. K., et al. Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes. PLoS Pathogens. 9 (e1003738), (2013).
- Kovacs, J. M., et al. HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120. Proc. Natl. Acad. Sci. U.S.A. 109, 12111-12116 (2012).
- Yuan, W., Bazick, J., Sodroski, J. Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker. J. Virol. 80, 6725-6737 (2006).
- Guttman, M., Lee, K. K. A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140. J. Virol. 87, 11462-11475 (2013).
- Ringe, R. P., et al. Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation. Proc. Natl. Acad. Sci. U.S.A. 110, 18256-18261 (2013).
- Desormeaux, A., et al. The highly conserved layer-3 component of the HIV-1 gp120 inner domain is critical for CD4-required conformational transitions. J. Virol. 87, 2549-2562 (2013).
- Haim, H., et al. Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state. PLoS Pathogens. 5 (e1000360), (2009).
- Haim, H., et al. Contribution of intrinsic reactivity of the HIV-1 envelope glycoproteins to CD4-independent infection and global inhibitor sensitivity. PLoS Pathogens. 7 (e1002101), (2011).
- Thali, M., et al. Effects of changes in gp120-CD4 binding affinity on human immunodeficiency virus type 1 envelope glycoprotein function and soluble CD4 sensitivity. J. Virol. 65, 5007-5012 (1991).
- Medjahed, H., Pacheco, B., Desormeaux, A., Sodroski, J., Finzi, A. The HIV-1 gp120 major variable regions modulate cold inactivation. J. Virol. 87, 4103-4111 (2013).
- Thali, M., et al. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J. Virol. 67, 3978-3988 (1993).
- Finzi, A., et al. Topological layers in the HIV-1 gp120 inner domain regulate gp41 interaction and CD4-triggered conformational transitions. Mol. Cell. 37, 656-667 (2010).
- Kassa, A., et al. Transitions to and from the CD4-bound conformation are modulated by a single-residue change in the human immunodeficiency virus type 1 gp120 inner domain. J. Virol. 83, 8364-8378 (2009).
- Xiang, S. H., et al. Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein. J. Virol. 76, 9888-9899 (2002).
- Mouquet, H., et al. Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies. Proc. Natl. Acad. Sci. U.S.A. 109, 3268-3277 (2012).
- Julien, J. P., et al. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS Pathogens. 9 (e1003342), (2013).
- Walker, L. M., et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 477, 466-470 (2011).
- Moore, J. P., Willey, R. L., Lewis, G. K., Robinson, J., Sodroski, J. Immunological evidence for interactions between the first, second, and fifth conserved domains of the gp120 surface glycoprotein of human immunodeficiency virus type 1. J. Virol. 68, 6836-6847 (1994).
- Robinson, J. E., Yoshiyama, H., Holton, D., Elliott, S., Ho, D. D. Distinct antigenic sites on HIV gp120 identified by a panel of human monoclonal antibodies. J. Cell. Biochem. Suppl. 16E (Q449), (1992).
- Bonsignori, M., et al. Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family. J. Virol. 86, 11521-11532 (2012).
- Brand, D., Srinivasan, K., Sodroski, J. Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein. J. Virol. 69, 166-171 (1995).
