Vaccination with mRNA-encoded membrane-bound HIV Envelope trimer induces neutralizing antibodies in animal models
Parham Ramezani-RadChristopher A. CottrellEster Marina-ZárateAlessia LiguoriElise LandaisJonathan L. TorresAmber MyersJeong Hyun LeeSabyasachi BabooClaudia T. FlynnKatherine McKenneyEugenia SalcedoXiaoya ZhouOleksandr KalyuzhniyErik GeorgesonNicole PhelpsDanny LuSaman EskandarzadehSergey MenisMichael KubitzBettina GröschelNushin AlaviAbigail M. JacksonWen-Hsin LeeAndy S TranElana Ben‐AkivaKatarzyna Kaczmarek MichaelsJolene K. DiedrichChiamaka A. EnemuoVanessa LewisArpan PradhanSudhir Pai KasturiTorben SchiffnerJon M. SteichenDiane G. CarnathanSunny HimansuJohn R. YatesJames C. PaulsonGabriel OzorowskiDarrell J. IrvineGuido SilvestriDevin SokAndrew B. WardShane CrottyWilliam R. Schief
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A protective vaccine against HIV will likely need to induce broadly neutralizing antibodies (bnAbs) that engage relatively conserved epitopes on the HIV envelope glycoprotein (Env) trimer. Nearly all vaccine strategies to induce bnAbs require the use of relatively complex immunization regimens involving a series of different immunogens, most of which are Env trimers. Producing protein-based clinical material to evaluate such relatively complex regimens in humans presents major challenges in cost and time. Furthermore, immunization with HIV trimers as soluble proteins induces strong non-neutralizing responses to the trimer base, which is normally occluded on the virion. These base responses could potentially detract from the induction of nAbs and the eventual induction of bnAbs. mRNA vaccine platforms offer potential advantages over protein delivery for HIV vaccine development, including increased production speed, reduced cost, and the ability to deliver membrane-bound trimers that might facilitate improved immuno-focusing to non-base epitopes. We report the design of mRNA-delivered soluble and membrane-bound forms of a stabilized native-like Env trimer (BG505 MD39.3), initial immunogenicity evaluation in rabbits that triggered clinical evaluation, and more comprehensive evaluation of B cell, T cell, and antibody responses in non-human primates. mRNA-encoded membrane-bound Env immunization elicited reduced off-target base-directed Env responses and stronger neutralizing antibody responses, compared with mRNA-encoded soluble Env. Overall, mRNA delivery of membrane-bound Env appears promising for enhancing B cell responses to subdominant epitopes and facilitating rapid translation to clinical testing, which should assist HIV vaccine development. HIV envelope trimer mRNA enables membrane-bound expression and represents a functional immunogen in pre-clinical mammalian models.Keywords:
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During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block. During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block. Human immunodeficiency virus (HIV) 1The abbreviations used are: HIV, human immunodeficiency virus; Ab, antibody; Env, viral envelope glycoprotein; BPTI, bovine pancreatic trypsin inhibitor; Ub, human ubiquitin; Mb, sperm whale myoglobin; GFP, green fluorescent protein; MBP, maltose-binding protein; SPR, surface plasmon resonance. entry is mediated by the viral envelope (Env) glycoprotein. Env is initially produced as gp160, which is proteolytically cleaved into non-covalently associated transmembrane (gp41) and surface (gp120) subunits. gp120 is primarily involved in recognition of cellular receptors, whereas gp41 is anchored in the viral membrane and mediates membrane fusion. The gp41 ectodomain contains two helical heptad repeat sequences (N- and C-peptide regions) (1.Lu M. Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1995; 2: 1075-1082Crossref PubMed Scopus (679) Google Scholar, 2.Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9770-9774Crossref PubMed Scopus (891) Google Scholar). Peptides corresponding to these helical regions (N- and C-peptides) are dominant-negative inhibitors of HIV membrane fusion (2.Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9770-9774Crossref PubMed Scopus (891) Google Scholar, 3.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar). Isolated N- and C-peptides form a six-helix bundle (trimer-of-hairpins) when mixed in solution (4.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (519) Google Scholar, 5.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1480) Google Scholar, 6.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1861) Google Scholar). In this structure, three N-peptides form a central parallel trimeric coiled coil (N-trimer) surrounded by three anti-parallel C-peptides that nestle between neighboring N-peptides. Based largely on these inhibitory and structural data, a working model of HIV-1 membrane fusion has been proposed (Fig. 1) (3.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar, 5.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1480) Google Scholar). Initial interaction of Env with its target cell occurs via gp120 binding to CD4 and a coreceptor (typically CCR5 or CXCR4). This binding induces a series of large conformational changes in gp120 that are propagated to gp41 via the gp41-gp120 interface. At this stage, gp41 transiently adopts an extended "prehairpin intermediate" conformation that bridges both the viral and cellular membranes. This state is believed to persist for at least 15 min (3.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar, 7.Munoz-Barroso I. Durell S. Sakaguchi K. Appella E. Blumenthal R. J. Cell Biol. 1998; 140: 315-323Crossref PubMed Scopus (269) Google Scholar, 8.Melikyan G.B. Markosyan R.M. Hemmati H. Delmedico M.K. Lambert D.M. Cohen F.S. J. Cell Biol. 2000; 151: 413-423Crossref PubMed Scopus (491) Google Scholar) but eventually collapses into a trimer-of-hairpins structure that pulls both membranes into tight apposition and induces membrane fusion (Fig. 1). In this model, the prehairpin intermediate exposes the isolated N-trimer, whereas the C-peptide region exists in an unknown and possibly unstructured conformation remote from the N-trimer (3.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar). At this stage, the prehairpin intermediate is vulnerable to binding of exogenous N- and C-peptides. Binding of these peptide inhibitors denies access of the endogenous N- or C-peptide regions to their appropriate intramolecular partners, thwarting hairpin formation and membrane fusion. This model predicts that any molecule that binds to the prehairpin intermediate and disrupts association of the N- and C-peptides will inhibit membrane fusion and has been successfully applied to the development of several potent entry inhibitors (9.Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (388) Google Scholar, 10.Eckert D.M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11187-11192Crossref PubMed Scopus (255) Google Scholar, 11.Eckert D.M. Malashkevich V.N. Hong L.H. Carr P.A. Kim P.S. Cell. 1999; 99: 103-115Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Additionally, the gp41 prehairpin intermediate has several promising features as an inhibitory target (12.Eckert D.M. Kim P.S. Annu. Rev. Biochem. 2001; 70: 777-810Crossref PubMed Scopus (1158) Google Scholar). Peptide mimics of the N-trimer region have been structurally characterized at high resolution (4.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (519) Google Scholar, 5.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1480) Google Scholar, 6.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1861) Google Scholar). The interface between the N- and C-peptides is highly conserved among diverse HIV strains of both laboratory-adapted and clinical isolates (9.Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (388) Google Scholar). The N-trimer also presents a long (>100 Å) deep groove with an extensive binding surface (4.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (519) Google Scholar, 5.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1480) Google Scholar, 6.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1861) Google Scholar). These special properties have led many groups to search for Abs that can disrupt this interface (reviewed in Ref. 13.Burton D.R. Desrosiers R.C. Doms R.W. Koff W.C. Kwong P.D. Moore J.P. Nabel G.J. Sodroski J. Wilson I.A. Wyatt R.T. Nat. Immunol. 2004; 5: 233-236Crossref PubMed Scopus (686) Google Scholar). C-peptide Inhibitors—Several peptide fusion inhibitors derived from the N- and C-peptide regions of gp41 have been described (2.Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9770-9774Crossref PubMed Scopus (891) Google Scholar, 3.Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar, 12.Eckert D.M. Kim P.S. Annu. Rev. Biochem. 2001; 70: 777-810Crossref PubMed Scopus (1158) Google Scholar, 14.Wild C. Oas T. McDanal C. Bolognesi D. Matthews T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10537-10541Crossref PubMed Scopus (484) Google Scholar, 15.Wild C. Greenwell T. Matthews T. AIDS Res. Hum. Retroviruses. 1993; 9: 1051-1053Crossref PubMed Scopus (378) Google Scholar, 16.Jiang S. Lin K. Strick N. Neurath A.R. Nature. 1993; 365: 113Crossref PubMed Scopus (487) Google Scholar). The most potent are peptides derived from the C-peptide region (e.g. C34, DP178/T20, T1249), which have low nm IC50s against viral entry in cell-cell fusion (syncytia formation), and viral infectivity assays (reviewed in Ref. 17.Weiss C.D. AIDS Rev. 2003; 5: 214-221PubMed Google Scholar). Several mutations leading to T-20 resistance have been mapped to the N-peptide region of gp41 (18.Rimsky L.T. Shugars D.C. Matthews T.J. J. Virol. 1998; 72: 986-993Crossref PubMed Google Scholar), providing strong support that the N-trimer is the primary target of C-peptide inhibitors. gp41 N-trimer as a Vaccine Target—As demonstrated by the efficacy of C-peptide inhibitors, the N-trimer region of gp41 is a very attractive candidate for vaccine efforts. Many such efforts have been undertaken using various peptide mimics of the N-trimer region (e.g. N-peptide, 5-helix, IZN36, and N35CCG-N13) (17.Weiss C.D. AIDS Rev. 2003; 5: 214-221PubMed Google Scholar, 19.Golding H. Zaitseva M. de Rosny E. King L.R. Manischewitz J. Sidorov I. Gorny M.K. Zolla-Pazner S. Dimitrov D.S. Weiss C.D. J. Virol. 2002; 76: 6780-6790Crossref PubMed Scopus (117) Google Scholar, 20.Opalka D. Pessi A. Bianchi E. Ciliberto G. Schleif W. McElhaugh M. Danzeisen R. Geleziunas R. Miller M. Eckert D.M. Bramhill D. Joyce J. Cook J. Magilton W. Shiver J. Emini E. Esser M.T. J. Immunol. Methods. 2004; 287: 49-65Crossref PubMed Scopus (32) Google Scholar, 21.Louis J.M. Nesheiwat I. Chang L. Clore G.M. Bewley C.A. J. Biol. Chem. 2003; 278: 20278-20285Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These efforts have produced a large number of Abs with specific and high affinity binding to their targets but weak and/or narrow neutralizing activity in standard viral entry and spread assays. Interestingly, some of these anti-N-trimer Abs can inhibit fusion if bound to a temperature-arrested intermediate fusion state (19.Golding H. Zaitseva M. de Rosny E. King L.R. Manischewitz J. Sidorov I. Gorny M.K. Zolla-Pazner S. Dimitrov D.S. Weiss C.D. J. Virol. 2002; 76: 6780-6790Crossref PubMed Scopus (117) Google Scholar) or in the presence of soluble CD4 (21.Louis J.M. Nesheiwat I. Chang L. Clore G.M. Bewley C.A. J. Biol. Chem. 2003; 278: 20278-20285Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Currently, there are only two reported anti-gp41 Abs that exhibit potent and broadly neutralizing activity, 2F5 and 4E10, which bind just outside the C-terminal border of the C-peptide region, an area with uncertain structure (reviewed in Ref. 22.McGaughey G.B. Barbato G. Bianchi E. Freidinger R.M. Garsky V.M. Hurni W.M. Joyce J.G. Liang X. Miller M.D. Pessi A. Shiver J.W. Bogusky M.J. Curr. HIV Res. 2004; 2: 193-204Crossref PubMed Scopus (47) Google Scholar). In this study, we tested the hypothesis that the N-trimer of gp41 is sterically restricted in the prehairpin intermediate, which may explain the current dearth of broadly neutralizing Abs against this target (Fig. 1). All of the known fusion inhibitors that target this structure (e.g. C34, T-20, T-1249, D-peptides) are small (<40 residue) peptides and could circumvent such a steric block. We have constructed fusions of a well characterized C-peptide inhibitor (C34) to a series of protein cargoes of varying sizes to determine whether such a steric block exists and, if so, to define its size cutoff. Our results demonstrate that C-peptide fusion proteins lose inhibitory potency with increasing size and that the N-trimer region of gp41 is likely to be poorly accessible to proteins as large as Abs. These results have important implications for gp41 vaccine design as well as for the production of second-generation C-peptide entry inhibitors. This steric restriction also helps to better define the conformation of the prehairpin intermediate. Reagents—Plasmids were obtained from the following sources: pET vectors (Novagen), pMAL-c2G (New England Biolabs), pEBB-HXB2 and pEBB-JRFL (gifts from B. Chen) (23.Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar). Reverse phase HPLC was performed using a C18 column (Vydac). All Ni affinity purifications used His-Select HC nickel affinity gel (Sigma) or His-Select HC nickel magnetic resin (Sigma). The National Institutes of Health AIDS Research and Reference Reagent Program provided the following reagents: pNL4–3.Luc.R-E-(N. Landau), HeLa-CD4-LTR-β-galactosidase cells (M. Emerman), HOS-CD4-fusin/CCR5 cells (N. Landau). Protein Expression, Purification, and Characterization—C37-H6 (C37), derived from the HXB2 Env sequence, was expressed and purified as previously described (9.Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (388) Google Scholar). Proteins used in this study were bovine pancreatic trypsin inhibitor (BPTI), human ubiquitin (Ub), sperm whale myoglobin (Mb), enhanced green fluorescent protein (GFP; Clontech), and Escherichia coli maltose-binding protein (MBP; New England Biolabs). Linker sequences were Ser4Gly2 for BPTI-C37, Ub-C37, and GFP-C37 and Ser5Gly2 for Mb-C37 and MBP-C37 (linker sequences are slightly different for cloning reasons). The extended linker constructs had the following linker sequences: SSS(GGGS)3-SSSGG (MBP1-C37) and SSS(GGGS)3S(GGGS)3SSSGG (MBP2-C37). The DNA encoding each protein was cloned into the following plasmids: pET9a (for BPTI-C37, Ub-C37, Mb-C37, and GFP-C37), pET20b (for BPTI-H6, Ub-H6, Mb-H6, and GFP-H6); pMAL-c2G (for MBP-H6, MBP-C37, MBP1-C37, and MBP2-C37). Proteins were expressed in BL21(DE3)pLysS (Novagen) for pET9a and pET20b vectors and XL1-Blue (Stratagene) for pMal-c2G vectors. All proteins have C-terminal His tags (His6) and were purified using Ni affinity chromatography. BPTI required refolding after expression for correct formation of disulfide bonds. Briefly, after Ni affinity purification, BPTI-C37-H6 and BPTI-H6 were reduced with 100 mm β-mercaptoethanol at pH 8 and dialyzed into 5% acetic acid. The proteins were air oxidized in the presence of a 1:10 ratio of oxidized:reduced glutathione at pH 8, 4 °C for 24 h. The correctly folded proteins were isolated using reverse phase HPLC and were confirmed by near-UV circular dichroism (Aviv 62DS) and measurement of trypsin inhibiting activity as previously described (24.Coplen L.J. Frieden R.W. Goldenberg D.P. Proteins. 1990; 7: 16-31Crossref PubMed Scopus (34) Google Scholar). Cys-Gly-Gly-Asp-IZN36 (10.Eckert D.M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11187-11192Crossref PubMed Scopus (255) Google Scholar) was cloned into pET14b and expressed in BL21(DE3)pLysS. IZN36 was purified from inclusion bodies (solubilized in 6 m GuHCl) using Ni affinity chromatography. The protein was then dialyzed into 5% acetic acid and purified by reverse phase HPLC. This material was reduced with TCEP (Pierce) and biotinylated at its unique Cys residue using Biotin-HPDP (Pierce). After biotinylation, the His tag was removed by thrombin cleavage (Novagen), and the cleaved product was purified by reverse phase HPLC. The sequence of the final product is GSHMCGGDIKKEIEAIKKEQEAIKKKIEAIEKEISGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL. All protein masses were confirmed by matrix-assisted laser desorption ionization or electrospray mass spectrometry (University of Utah Core Facility). All proteins were judged >98% pure by SDS-PAGE. Protein concentrations were measured by UV absorbance at 280 nm (25.Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (3044) Google Scholar). Surface Plasmon Resonance (SPR) Analysis—Binding experiments were performed using a Biacore 2000 optical biosensor (University of Utah Protein Interaction Core Facility) equipped with research-grade CM5 sensor chips (Biacore). A standard coupling protocol was employed to immobilize streptavidin (SA; Pierce) (26.Johnsson B. Lofas S. Lindquist G. Anal. Biochem. 1991; 198: 268-277Crossref PubMed Scopus (1213) Google Scholar). Biotinylated IZN36 was captured on a SA surface, and free SA surfaces served as references. Binding analysis of C37 and C37 fusion proteins was performed at 25 °C with a data collection rate of 2.5 Hz. The binding buffer (phosphate-buffered saline; Invitrogen) + 0.005% P20 detergent (Biacore) + 1 mg/ml bovine serum albumin (fraction V; Fisher)) was prepared, vacuum filtered, and degassed immediately prior to use. Stock solutions of C37, C37 fusion proteins, and corresponding control proteins (without C37) were prepared in binding buffer at 100 nm. Protein binding was analyzed by injecting samples for 1 min over the IZN36 and reference surfaces using KINJECT at a flow rate of 50–100 μl/min. The dissociations were monitored for 3 min. The IZN36 surfaces were completely regenerated using one 3-s pulse of 6 m guanidine-HCl or three 6-s pulses of 0.1% SDS. Data from the reference flow cells were subtracted to remove systematic artifacts that occurred in all flow cells (27.Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (661) Google Scholar). The data were normalized to the highest point in the response curve to facilitate comparison. Binding at one concentration was analyzed using a 1:1 binding model in CLAMP (28.Myszka D.G. Morton T.A. Trends Biochem. Sci. 1998; 23: 149-150Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), assuming enough information from the curvature of the responses to determine the approximate kinetic parameters for the reactions (29.Canziani G.A. Klakamp S. Myszka D.G. Anal. Biochem. 2004; 325: 301-307Crossref PubMed Scopus (118) Google Scholar). Cell-cell Fusion and Viral Infectivity Assay—Cell-cell fusion was monitored as previously described (30.Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (485) Google Scholar). Briefly, HXB2 Env-expressing Chinese hamster ovary cells (gift from M. Krieger (31.Kozarsky K. Penman M. Basiripour L. Haseltine W. Sodroski J. Krieger M. J. Acquir. Immune Defic. Syndr. 1989; 2: 163-169PubMed Google Scholar)) were mixed with HeLa-CD4-LTR-β-galactosidase cells in the presence of inhibitors for 20 h at 37 °C. Syncytia were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) (Invitrogen) and counted. Viral infectivity was measured as previously described (9.Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (388) Google Scholar). Briefly, pseudotyped viruses were produced by co-transfecting 293T cells using FuGENE (Roche Applied Science) with pNL4–3.Luc.R-E- and either pEBB-HXB2 or pEBB-JRFL. After 36–48 h, viral supernatants were collected and sterile filtered. HXB2 or JRFL pseudotyped virus was added to HOS-CD4-fusin or HOS-CD4-CCR5 cells, respectively, in the presence of inhibitors. HXB2 assays included 20 μg/ml DEAE-dextran (23.Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar). After 12 h, virus and inhibitor were removed and replaced with fresh media. Cells were lysed 40–44 h after infection using Glo lysis buffer (Promega), and luciferase activity was measured using Bright-Glo (Promega). IC50 values for both assays were calculated by fitting data to the equation, y = k/(1+ [inhibitor]/IC50), where y is the normalized number of syncytia or luciferase activity and k is the scaling constant (k = 1 for syncytia assay and is floated for viral infectivity assay, see Fig. 2B legend). Assays for Inhibitor Proteolysis and Precipitation—C37 fusion inhibitors were incubated in tissue culture medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum; Invitrogen) at 37 °C for 20 h. Proteins were purified from the medium by a 1-h incubation at room temperature with magnetic Ni affinity beads. The resin was washed 3× with phosphate-buffered saline, and proteins were eluted by boiling in LDS sample buffer (Invitrogen). Eluted samples were separated by SDS-PAGE and visualized with SimplyBlue stain (Invitrogen). Unpurified media samples were analyzed before and after centrifugation (10 min. at 18,000 × g) by Western blot using polyclonal rabbit anti-His tag Ab (Abcam) and SuperSignal West Pico substrate (Pierce), as well as visually analyzed for precipitate. Production of Fusion Proteins—To test for steric constraints in accessing the gp41 N-trimer region, we constructed a series of inhibitors containing a C-peptide attached to cargo proteins of various sizes (Fig. 1). The cargo partners used in this study were selected for the following properties: monomeric, soluble, globular, stable, tolerant to C-terminal additions, and free of nonspecific peptide binding. Cargo proteins meeting these inclusion criteria and used in this study range from 6 to 41 kDa (Table I). For these studies, C37 (9.Root M.J. Kay M.S. Kim P.S. Science. 2001; 291: 884-888Crossref PubMed Scopus (388) Google Scholar), the recombinant His-tagged version of the previously characterized synthetic peptide C34 (30.Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (485) Google Scholar, 32.Malashkevich V.N. Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9134-9139Crossref PubMed Scopus (191) Google Scholar), was used as the reference inhibitor. In each fusion protein, C37 is connected at its N terminus to the C terminus of the cargo by a flexible 6- or 7-residue Ser/Gly linker. This linker was designed to be long enough to allow the proper orientation of C37 as it binds to the N-trimer but short enough for the attached cargo to prevent access to an occluded binding site. The N terminus of C37 was chosen for attachment of cargo because this attachment site points away from the membrane (whereas the C terminus of C37 is expected to be near the viral membrane and, therefore, less accessible; see Fig. 1). For each fusion protein, a matching control protein lacking C37 was also produced.Table IIC50 (in nm) of fusion proteins in cell-cell fusion and viral infectivity assaysProteinFusion partner molecular massCell-cell fusionIC50 ratio (cell-cell fusion)Viral infectivity HXB2IC50 ratio HXB2Viral infectivity JRFLIC50 ratio JRFLkDaC3700.851.02.81.08.21.0BPTI-C376.51.51.83.11.14.80.6Ub-C378.64.75.56.82.537.74.6Mb-C371730.836.258.021.041450.5GFP-C372728.934.011842.853365.0MBP-C374119222520674.81874228MBP1-C374175.188.488.932.264078.0MBP2-C374131.236.779.228.751662.9IC50 S.E. is <25% for both assays. IC50 ratios are relative to C37. Open table in a new tab IC50 S.E. is <25% for both assays. IC50 ratios are relative to C37. Size and Inhibitory Potency Are Inversely Correlated—The inhibitory potency of each inhibitor was tested using a cell-cell fusion (syncytia) assay utilizing HXB2 Env and two viral infectivity assays utilizing either HXB2 (X4) or JRFL (R5) Envs (Table I, Fig. 2). C37 shows high potency inhibition in all assays (IC50 = 0.85–8.2 nm). Inhibition is slightly weaker than seen with C34 (30.Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (485) Google Scholar), as expected from the loss of helix-stabilizing synthetic blocking groups found in C34. For reference, the anti-gp41 Abs 2F5 and 4E10 have reported IC50 values of ∼0.2–7 nm against HXB2/IIIB laboratory strains in cell-cell and viral infectivity assays similar to those used in this study (33.Zwick M.B. Komori H.K. Stanfield R.L. Church S. Wang M. Parren P.W. Kunert R. Katinger H. Wilson I.A. Burton D.R. J. Virol. 2004; 78: 3155-3161Crossref PubMed Scopus (109) Google Scholar, 34.Stiegler G. Kunert R. Purtscher M. Wolbank S. Voglauer R. Steindl F. Katinger H. AIDS Res. Hum. Retroviruses. 2001; 17: 1757-1765Crossref PubMed Scopus (445) Google Scholar). The smallest fusion protein, BPTI-C37, also displays high potency in both assays, very similar to C37, demonstrating that our C37-cargo linker does not interfere with inhibitory activity. Ub-C37 is a slightly weaker (2.5–5.5-fold) inhibitor than C37, whereas Mb-C37 and GFP-C37 both show more substantial (21–65-fold) reductions in potency in both assays. MBP-C37 shows the most dramatic change with a 75–228-fold drop in potency. None of the control proteins (cargo without C37 peptide) inhibits at up to 1 μm (10 μm for MBP with JRFL Env) in either assay (data not shown). In general, the cell-cell fusion and viral infectivity assays show similar losses of activity with increasing size of the inhibitors, with a slightly more pronounced effect on cell-cell fusion and JRFL-mediated viral entry. For HXB2 Env we observed up to a 4-fold greater potency in cell-cell fusion versus viral infectivity as seen in studies of other fusion inhibitors (2.Wild C.T. Shugars D.C. Greenwell T.K. McDanal C.B. Matthews T.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9770-9774Crossref PubMed Scopus (891) Google Scholar, 11.Eckert D.M. Malashkevich V.N. Hong L.H. Carr P.A. Kim P.S. Cell. 1999; 99: 103-115Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 30.Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (485) Google Scholar, 35.Sia S.K. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9756-9761Crossref PubMed Scopus (62) Google Scholar). As expected, inhibitors were less potent against the primary isolate JRFL in the viral infectivity assay. For most of the inhibitors, the viral infectivity data show a reproducible increase in infectivity (above the uninhibited values) at low inhibitor concentrations (see the legend to Fig. 2B). This "overshoot" has also occasionally been seen in other studies of fusion inhibitors (36.Wei X. Decker J.M. Liu H. Zhang Z. Arani R.B. Kilby J.M. Saag M.S. Wu X. Shaw G.M. Kappes J.C. Antimicrob. Agents Chemother. 2002; 46: 1896-1905Crossref PubMed Scopus (1382) Google Scholar, 37.Reeves J.D. Gallo S.A. Ahmad N. Miamidian J.L. Harvey P.E. Sharron M. Pohlmann S. Sfakianos J.N. Derdeyn C.A. Blumenthal R. Hunter E. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16249-16254Crossref PubMed Scopus (382) Google Scholar, 38.Labrijn A.F. Poignard P. Raja A. Zwick M.B. Delgado K. Franti M. Binley J. Vivona V. Grundner C. Huang C.C. Venturi M. Petropoulos C.J. Wrin T. Dimitrov D.S. Robinson J. Kwong P.D. Wyatt R.T. Sodroski J. Burton D.R. J. Virol. 2003; 77: 10557-10565Crossref PubMed Scopus (328) Google Scholar) but has not been explained. The C-peptide Remains Accessible when Linked to Fusion Partners—To ensure that linkage of C37-H6 to each of the partner proteins did not affect the accessibility of C37 for binding to a sterically open target, the fusion proteins and C37 were assayed for binding to IZN36, a soluble mimic of the N-trimer (10.Eckert D.M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11187-11192Crossref PubMed Scopus (255) Google Scholar), using SPR. Each fusion protein was flowed over the control and IZN36 surfaces. C37 reversibly bound to IZN36 with a low nmKD (Fig. 3). The calculated KD for the fusion proteins are clustered in a narrow range around the C37 value (2-fold lower to 2-fold higher). The est
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The effect of trimer water cluster on conduction phenomena was investigated using three kinds of liquid dielectrics: di-octhyle-phthalate (DOP), including dimer; acctonitrile (ACT), including dimer and much trimer; and a mixture of ACT and DOP. It is shown that, when the concentration of DOP in the mixture increases, the amount of trimer decreases and the cluster becomes smaller. In the conduction test, it is evident that the conduction current is electron current. It is also shown that, as the amount of trimer decreases and the cluster becomes smaller, the electron current decreases. This conduction phenomena appears to be due to trimer bridges.< >
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The trimer form of the human immunodeficiency virus type a (HIV-1) envelope protein gp41 plays pivotal role in HIV-1 entry. The gp41 is composed with trimers of the N-terminal and C-terminal helical region (NHR and CHR), which form a six-helical bundle (6HB) in the membrane fusion step. A trimeric peptide was synthesized using a C34 derived from CHR using a newly designed template with three equivalent linkers. The C34 trimer induced neutralizing antibodies which can recognize the structure of the C34 trimer. Besides, the C34 trimer showed 100-fold unexpectedly higher anti-HIV-1 activity than the C34 monomer, indicating that a trimeric form is critical for its inhibitory activity.
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The possible effect of trimer water cluster on conduction phenomena was investigated in three kinds of liquid dielectrics: di-octhyle-phtalate (DOP), including dimer; acctonitrile, including dimer and much trimer; and mixture of ACT and DOP. It is shown that the greater the concentration of DOP in the mixture is, the less trimer there will be and the smaller the cluster will be. For the conduction test, it is evident that the conduction current is electron current. Moreover, the less trimer there is and the smaller the cluster is, the smaller the electron current will be. It appears that the electronic conduction current is caused by the trimer conductive bridge. It is likely that if a large amount of trimer exists in the liquid material, the conduction current is large.
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