repetitive boosting can overcome some of the constraints for immunogenicity of the 2F5 epitope of the MPER, but strategies for the induction of the polyreactive antibodies needed for neutral- izing activity have to be developed. In this regard, Matyas et al have shown that liposomes containing the MPER and lipid A induced polyreactive IgM but not IgG antibodies that simulta- neously bind gp41 and phosphatidylinositol-4-phosphate and neutralized HIV-1 in a peripheral blood mononuclear cells neutralization assay [51]. That the prime boost regimen reported here and constrained scaffolds can induce 2F5 core 664DKW epitope raises the question of the utility of immunization with a gp41-inter protein constructs. The prototype gp41 intermediate construct also contained the post-fusion 6 helix bundle [14,32], and immune serum from guinea pigs immunized with this construct did not focus antibodies on 664DKW residues (Alam, M, Chen, B. et al. unpublished data). Studies are underway with a GCN4 stabilized gp41 pre-hairpin intermediate construct that does not contain the post-fusion six- helix bundle. Finally, these results indicate that mounting of responses to 2F5 core epitope as it exists in the fusion intermediate conformation of gp41-inter alone is insufficient for HIV-1 neutralization. We have previously shown that mutation in the 2F5 CDR H3 that abrogate 2F5 binding to lipids but maintain binding to the pre-hairpin gp41 intermediate conformation are unable to neutralize HIV-1 (2). Thus, it is likely that the induced DKW-specific antibodies described in this study also lack a hydrophobic CDR H3 loop that renders them incapable of reacting with viral membrane lipids and are thus unable to neutralize. This is consistent with the results that 2F5 CDR H3 mutations greatly reduced the binding of recombinant 2F5 to MPER peptide liposomes (Figure S8-A). 11F10 mAb, which lacks a hydrophobic CDRH3 [27], also gave weak binding to MPER peptide liposomes (Figure S8-B). We had proposed that the lack of lipid binding of 2F5 CDR H3 mutants would prevent them from pre-concentrating on the viral membrane and such antibodies would fail to capture the transient fusion intermediate state of gp41 or to neutralize. Thus, while IgGs from immunized rhesus macaques showed some binding to MPER peptide-liposomes (Figure S8 C–F), the lack of lipid binding would prevent the rhesus IgGs to maintain a kinetic head start required for HIV-1 neutralization. Materials and Methods Ethics Statement The guinea pig studies were carried out following a protocol (A334-09-11) approved by the Animal Use and Care Committee of the Duke University IACUC, which ensures that all animals in experimental research are used appropriately and are treated with the highest standards of humane care. Colony bred Indian-origin rhesus monkeys used in the immunization studies were housed and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution in accordance with the principles of the National Institute of Health. All studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in BIOQUAL (Rockville, MD). BIOQUAL is fully accredited by AAALAC and through OLAW, Assurance Number A-3086. The animal protocol used in this study was approved by the BIOQUAL IACUC (#08-3449-71). All physical procedures associated with this work were done under anesthesia to minimize pain and distress in accordance with the recommen- dations of the Weatherall report, ‘‘The use of non-human primates in research’’. Teklad 5038 Primate Diet was provided once daily Induction of MPER Antibodies in Rhesus by animal size and weight. The diet was supplemented with fresh fruit and vegetables. Fresh water was given ad libitum. Proteins, Peptides, mAbs, Phospholipids and Adjuvants Recombinant JRFL gp140CF (C = gp1202gp41 cleavage site deleted; F = fusion domain-deleted, Figure S6) was produced and purified using methods described earlier [52]. MPER peptides containing the 2F5 epitope (QQEKNEQELLELDKWASLWN) or the epitopes of both 2F5 and 4E10 mAbs (NEQELLELDK- WASLWNWFNITNWLWYIK) were synthesized with a C- terminal hydrophobic membrane anchor tag (YKRWIILGLN- KIVRMYS) and purified by reverse phase HPLC. The purity of the custom made (CPC Scientific) MPER peptides were assessed by HPLC to be greater than 95% and confirmed by mass spectrometric analysis. The gp41 inter proteins (92UG gp41-inter and GCN4-gp41 inter, Figure S7) having gp41 sequence of clade A HIV-1 isolate 92UG037.8 were produced as described earlier [32]. Chloroform stocks of phospholipids 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-gly- cero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-gly- cero-3-phosphoserine (POPS), 1,2-dimyristoyl-sn-glycero-3-phos- phate (DMPA), cholesterol (CH), monophosphoryl Lipid A (MPL- A) and bovine heart cardiolipin were obtained from Avanti Polar Lipids and were used without further purification. R848 was purchased from Axxora LLC. The custom made oCpG with 59 TCGTCGTTGTCGTTTTGTCGTT 39 (used in guinea pigs immunization) and 59-(P = S)TCGTCGTTTTTCGGTCGTT- TT-39 (used in rhesus macaques immunization) sequences was obtained from The Midland Certified Reagent Co. Emulsigen was purchased from MVP Laboratories. 2F5, 4E10 mAbs were obtained from Polymun and Synagis was obtained from Medimmune. 13H11 mAb and Fabs of 2F5 and 13H11 were produced as described earlier [28,52]. Recombinant gp41 MN was obtained from Immunodiagnostics. The recombinant 2F5 mAb and the mutants were produced in 293T cells as described earlier [29]. The epitope scaffold proteins ES2, ES4 and ES5 and the 11F10 mAb were made as described earlier [27]. For antibody preparation, 250 mg of light chain plasmid DNA and 250 mg of heavy chain plasmid DNA, or for ES2 and ES4 scaffolds, 500 mg plasmid DNA, were mixed with 1 ml of 293fectin (Invitrogen, Carlsbad, CA) for 20 minutes before the DNA-293fectin complex was added into 850 ml of FreeStyle 293F cells (1.46106 cells/ml) in a 2-L shaking flask. The transfected cells were returned to suspension incubation for 2 days, and then, the culture was fed with 50 ml of protein expression enriched medium CellBoost-5 (HyClone, Logan, UT) and protein expression enhancer Sodium Butyrate at final concentration of 2 mM (SIGMA, St. Louis, MO). After 6 days post transfection, supernatants were harvested by centrifugation, filtered through 0.22 mm filter. The 11F10 antibody was purified by running the supernatant over a protein A column (Pierce Protein A Plus Agarose, Thermo, Rockford, IL), followed by elution at low pH. Purified antibody was dialyzed against PBS, analyzed SDS-PAGE and stored at 280uC. The ES2 and ES4 scaffolds were purified by running the supernatant over a 2F5 antibody affinity column, followed by elution at low pH. Bacterial expression and subsequent purification of epitope scaffold ES5 was performed as previously described [27]. Briefly, purification from inclusion bodies was undertaken under dena- turing conditions of 8 M urea, 20 mM Tris-Cl, pH 8.0, 1 mM beta-mercaptoethanol or DTT, followed by nickel chelating chromatography in binding buffer comprised of 8 M urea, 500 mM NaCl, Tris-Cl, pH 8.0, imidazole 10 mM. Washes and elutions were undertaken in binding buffer supplemented with PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27824