F., Majumdar S., Ofek G., Yang Y., Kwong P. the MPER region, exhibited competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140CA018 was higher than that induced by gp41int-Cys, the majority of animals immunized with gp41int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols. (35). Aleglitazar Here we structurally characterized the intermediate conformation of gp41 (gp41int) and coupled it covalently to liposomes, which were then administered alone or in combination with soluble gp140 in guinea pigs. Immunization was performed with a mixture of two adjuvants, Carbopol-971P and MF59, that preserved the liposomal structure prior to immunization. Analyses of the postimmune sera exhibited strong gp41-specific IgG responses, the presence of antibodies targeting MPER, and neutralizing activity against a panel of tier 1 and tier 2 HIV-1 viruses. Our study thus indicates the benefit of a membrane environment in the induction of neutralizing antibodies by gp41int. EXPERIMENTAL PROCEDURES Ethics Statement The animal study was carried out in strict accordance with the United Kingdom Animals (Scientific Procedure) Act 1986, and the protocol was approved by the local Ethical and Welfare Committee of the University of Cambridge and the United Kingdom Home Office (Project license number 80/2238). Recombinant Gp140 Purification HIV-1 gp140CA018 is an A/G recombinant subtype Env derived from a Cameroon patient. The gp140 glycoprotein was purified using a published protocol with agarose affinity column followed by diethylaminoethyl-Sepharose and ceramic hydroxyapatite columns to remove all contaminants (89). The purified glycoprotein was concentrated using an Amicon YM-30 (30-kDa-cutoff) ultrafiltration disc (Millipore) and stored at ?80 C until use. Gp41 Protein Expression and Purification The gp41 constructs are based on the HXB2 group M subtype B sequence. Gp41int-Cys and gp41int-fd contain gp41 residues 584C684. A part of gp41 heptad repeat region 1 (HR1) is usually N-terminally fused in-frame with the GCN4 trimerization domain name (90). Gp41int-fd contains a C-terminal fold-on trimerization domain name rendering gp41int-fd similar to the reported GCN-gp41-inter construct (91). Both cysteines at positions 598 and 604 have INCENP been mutated to serine. Gp41int-fd has the following sequence: MAQIEDKIEEILSKIYHIENEIARIKKLIGEAstrain Rosetta 2 (DE3) (Novagen). Cells were grown to an culture, indicating that production can be scaled up and is suitable for good manufacturing practice production for further immunization trials. SAXS Analysis of Gp41int-Cys X-ray scattering data were collected on beam line BM-29 (European Synchrotron Radiation Facility, Grenoble, France) at 20 C, a wavelength of 0.9919 ?, and a sample-to-detector (PILATUS 1M, DECTRIS) distance of 2.849 m. The scattering intensities of the gp41int-Cys were measured at concentrations of 0.75 and 3 mg/ml in the gel filtration buffer. The data were normalized to the intensity of the incident beam, the scattering of the buffer was subtracted, and the resulting intensities were scaled for concentration. Data processing and analysis were performed using the ATSAS package (92), and molecular weights were estimated based on the method Aleglitazar of Putnam (93). The final merged scattering data were further evaluated using PRIMUS (94). The isotropic scattering intensity modeling of the SAXS data, 10 sets of independent models were calculated using Dammin (96). Circular Dichroism Spectroscopy All spectra were recorded on a Jasco J-810 spectropolarimeter at 20 C. Prior to analysis, proteins were dialyzed in 10 mm potassium phosphate, pH 8.0, and the secondary structure content was calculated with the Jasco Spectra Manager 2 software package. Surface Plasmon Resonance Surface plasmon resonance analysis was performed with a Biacore X3000 (GE Healthcare). As a flow buffer, 10 mm HEPES, 150 mm NaCl, pH 7.4 with 0.005% P-20 was used. Gp41int-Cys was immobilized to 1000 response models Aleglitazar using 50 g/ml protein in flow buffer on an activated CM-5 sensor chip (GE Healthcare, Aleglitazar BR-1000-50) according to the manufacturer’s instructions. Specific binding to the target protein was corrected for nonspecific binding to the deactivated control channel. The flow rate was 50 l/min. Regeneration of the sensor chip was achieved with 100 mm glycine, pH 3.3, 20 mm NaOH for 60 s at 50 l/min. mAb 2F5 was injected at concentrations of 50, 100, and 250 nm, and bnAb 10E8 was injected at concentrations of 1 1.3 and 6 m. Data were analyzed with BIAevalution software version 4.1. The curves.

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