30 mM and 500 mM imidazole, respectively. Refolding was performed at 4uC by dilution (1:100) into refolding buffer comprised of 50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 500 mM L-Arginine, 0.1 mM glutathione reduced, 0.01 mM glutathione oxidized, 0.03% N-laurylsarcosine and 0.1 mM ZnCl2. Construction of adjuvant-containing MPER liposomes The procedure used to prepare MPER peptide liposomes earlier [19] was employed to construct adjuvant containing MPER liposomes. Briefly, chloroform stocks of POPC, POPE, DMPA and CH were mixed in glass tubes at a molar ratio of 45:25:20:1.33 respectively. The MPER peptide stock (made in chloroform-methanol 7:3 v/v) solution was added to this mixture to give a final peptide to total lipid ratio of 1:420. Appropriate volumes of stock solutions of adjuvants MPL-A (in chloroform) and R848 (in methanol) needed to yield desired dose of adjuvants were added to the above lipid mixture. The lipids-peptide-adjuvant mixture was dried under the stream of nitrogen to remove all visible traces of chloroform and methanol. Any residual amount of chloroform and methanol was removed by placing the dried film under vacuum for overnight. The dried film was hydrated by adding appropriate volume of PBS buffer (pH 7.4) and incubating at 37uC for 45 minutes. The hydrated mixture was then sonicated in a bath sonicator (Misonix 3000) and extruded through 0.4 mm and 0.2 mm polycarbonate membranes using either a mini- extruder obtained from Avanti Polar Lipids or a Lipex model thermo barrel (Northern Lipids) extruder that uses high pressure nitrogen gas for extrusion. The extruded liposomes were quality controlled by checking for the binding of 2F5 or both 2F5 and 4E10 depending on the sequence of the MPER peptide as described earlier [19]. The functionally active nature of liposome incorporated MPL-A and R848 was confirmed by the ability of MPER-adjuvant liposomal constructs to induce cytokine release in human PBMC. Immunizations Guinea pigs were purchased from Charles River and housed in the Vivarium at the Duke Human Vaccine Institute Animal Research Facility. Some initial studies in guinea pigs (Figure 1B, 2B and 2E) involved immunization through intramuscular, intranasal and sublingual routes with total dose of 100 mg of immunogen distributed in a 16:8:1 ratio respectively. In all other studies, guinea pigs (4 animals per immunogen group) were immunized every three weeks intramuscularly. At each immuni- zation, the JRFL gp140CF group of guinea pigs were administered intramuscularly with 400 ml (in two sites 200 ml/site) of recombi- nant JRFL gp140CF formulated in 15% emulsigen and oCpG to give a dose of 100 mg per animal of recombinant JRFL gp140 and 50 mg per animal of oCpG. The MPER liposome group of guinea pigs received MPER/MPL-A liposomes formulated in 15% emulsigen and oCpG. The MPER/MPL-A liposome immuniza- tion involved intramuscular administration of guinea pigs at two sites (200 ml per site). Each animal received a dose of 100 mg MPER peptide, 200 mg of MPL-A and 50 mg of oCpG respectively. Serum samples were collected 10 days after each immunization and stored at 280uC until use. Two groups of rhesus macaques were immunized intramuscu- larly; one with JRFL gp140CF (100 mg per animal) adjuvanted with AS01B (GSK Bio) and the second with MPER liposomes (1 mg MPER peptide per animal) formulated in emulsigen and oCPG (1 mg per animal) at weeks 0, 4, 8, 12 and 16. The ASO1B adjuvant is a GSK-liposome adjuvant system containing MPL-A and QS21. Serum samples were collected at weeks 2, 6, 10, 14 and 18. The JRFL gp140CF immunized rhesus macaques were Induction of MPER Antibodies in Rhesus boosted with MPER/MPL-A/R848 liposomes (1 mg each of MPER peptide, MPL-A and R848 per animal) at weeks 48, 51 and 55. Serum samples were collected at weeks 48, 49, 50, 51, 55 and 56 and were stored at 280uC until use. Surface Plasmon Resonance assays for screening of serum samples for MPER specific binding and epitope mapping The MPER specific binding responses present in immunized animal serum samples were measured using a BIAcore 3000 instrument and data analyses were performed with BIAevaluation 4.1 software (Biacore). Biotinylated 2F5 nominal epitope peptide (Biotin-SP62, QQEKNEQELLELDKWASLWN), 4E10 nominal epitope peptide (Biotin-4E10p, SLWNWFNITNWLWYIK) and a bi-epitope MPER peptide (Biotin-MPER656, NEQELLELDK- WASLWNWFNITNWLW) were immobilized (150 RU) on difference flow channels of a Streptavidin chip (Biacore). The activity of immobilized MPER peptides was confirmed by checking the binding of 2F5 or 4E10 mAbs at 5 or 10 mg/ml concentration. The serum samples after 50 fold dilution in PBS buffer (pH 7.4) were injected over the MPER peptide surfaces for 5 minutes at a 30 ml/minute flow rate. The responses were continued to be monitored for 5 minutes after the injection of diluted serum samples. The peptide surfaces were regenerated by flowing over Glycine-HCl pH 2.0. A biotinylated scrambled MPER peptide (NKEQDQAEESLQLWEKLNWL) immobilized on the remaining flow channel served as a negative control surface to subtract out the non-specific interactions of serum sample with the chip. The dissociation phase of the MPER peptide specific binding of sera was biphasic and could be resolved into a faster and slower component. The dissociation rates shown in the figures were derived from the initial faster phase. The gp41 inter proteins (92UG gp41-inter and GCN4-gp41 inter) specific binding responses in rhesus macaques sera were measured by flowing 50 fold diluted rhesus macaques sera over a CM5 chip immobilized with ,500 RU of recombinant HIV-1 gp41 MN, gp41-inter proteins and HIV-1 p66 protein surfaces. The binding responses on HIV-1 p66 protein surface were used to subtract out responses due to non-specific interactions. The IgGs purified from rhesus macaques sera using Protein G spin columns and desalting columns (Thermo Scientific) following manufacturer’s recom- mended protocol were flowed at a 100 mg/ml concentration over the gp41-inter proteins surfaces. The rhesus macaque serum IgGs (150 mg/ml) binding to 2F5 epitope scaffolds was performed using a CM5 chip immobilized with ,1500 RU of ES proteins. Epitope mapping of MPER specific responses found in immunized animal sera was carried out using a BIAcore A100 instrument and Streptavidin chip (Biacore). The biotinylated 2F5 nominal epitope peptide (Biotin-SP62) and seven of the biotiny- lated alanine scanning mutant MPER peptides were immobilized in duplicate on 16 different spots on four flow channels of the chip. The median spot on each flow channel served as a blank surface to subtract out non-specific interactions. The 50 fold diluted serum samples were injected over the native and mutant MPER peptide and reference surfaces for 5 minutes at a 30 ml/minute flow rate. The dissociation phase was monitored for 10 minutes and the surfaces were regenerated using Glycine-HCl pH 2.0 between cycles. The responses at the end of the dissociation phase (450– 500 seconds after the injection of serum samples was over) were averaged and used to calculate normalized binding response which is defined as the ratio between the average binding response to a mutant peptide and the corresponding binding response to the native peptide. PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27824