N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies

The Human immunodeficiency virus type 1(HIV-1) gp41 membrane proximal external region(MPER) is targeted by broadly neutralizing antibodies(e.g. 2F5, 4E10, Z13 e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However,these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope(amino acids(aa) 662–667 in the MPER)but also several other residues(aa 652–655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.     

Targeting antibody responses to the membrane proximal external region of the envelope glycoprotein of human immunodeficiency virus

Although human immunodeficiency type 1 (HIV-1) infection induces strong antibody responses to the viral envelope glycoprotein (Env) only a few of these antibodies possess the capacity to neutralize a broad range of strains. The induction of such antibodies represents an important goal in the development of a preventive vaccine against the infection. Among the broadly neutralizing monoclonal antibodies discovered so far, three (2F5, Z13 and 4E10) target the short and hidden membrane proximal external region (MPER) of the gp41 transmembrane protein. Antibody responses to MPER are rarely observed in HIV-infected individuals or after immunization with Env immunogens. To initiate antibody responses to MPER in its membrane-embedded native conformation, we generated expression plasmids encoding the membrane-anchored ectodomain of gp41 with N-terminal deletions of various sizes. Following transfection of these plasmids, the MPER domains are displayed on the cell surface and incorporated into HIV virus like particles (VLP). Transfected cells displaying MPER mutants bound as efficiently to both 2F5 and 4E10 as cells transfected with a plasmid encoding full-length Env. Mice immunized with VLPs containing the MPER mutants produced MPER-specific antibodies, the levels of which could be increased by the trimerization of the displayed proteins as well as by a DNA prime-VLP boost immunization strategy. Although 2F5 competed for binding to MPER with antibodies in sera of some of the immunized mice, neutralizing activity could not be detected. Whether this is due to inefficient binding of the induced antibodies to MPER in the context of wild type Env or whether the overall MPER-specific antibody response induced by the MPER display mutants is too low to reveal neutralizing activity, remains to be determined.     

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1(JR-FL), designated HIV-1(JR2), and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LELDKWANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (NWFDISNWLW). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.     

Synthesis and analysis of the membrane proximal external region epitopes of HIV‐1

The membrane proximal external region (MPER) of gp41 abuts the viral membrane at the base of HIV‐1 envelope glycoprotein spikes. The MPER is highly conserved and is rich in Trp and other lipophilic residues. The MPER is also required for the infection of host cells by HIV‐1 and is the target of the broadly neutralizing antibodies, 4E10, 2F5, and Z13e1. These neutralizing antibodies are valuable tools for understanding relevant conformations of the MPER and for studying HIV‐1 neutralization, but multiple approaches used to elicit MPER binding antibodies with similar neutralization properties have failed. Here we report our efforts to mimic the MPER using linear as well as constrained peptides. Unnatural amino acids were also introduced into the core epitope of 4E10 to probe requirements of antibody binding. Peptide analogs with C‐terminal Api or Aib residues designed to be helical transmembrane (TM) domain surrogates exhibit enhanced binding to the 4E10 and Z13e1 antibodies. However, we find that placement of constrained amino acids at nonbinding sites within the core epitope significantly reduce binding. These results are relevant to an understanding of native MPER structure on HIV‐1, and form a basis for a chemical synthesis approach to mimic MPER stricture and to construct an MPER‐based vaccine. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody

The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the K(d) values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH(2) could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope.     

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.     

HIV-1 antibodies and vaccine antigen selectively interact with lipid domains

The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody–membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies.     

Structure-guided Alterations of the gp41-directed HIV-1 Broadly Neutralizing Antibody 2F5 Reveal New Properties Regarding its Neutralizing Function

The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER). The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab). However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.     

HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.     

A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.     

A Fusion Intermediate gp41 Immunogen Elicits Neutralizing Antibodies to HIV-1

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41_int-Cys) and show that it folds into an elongated ∼12-nm-long extended structure based on small angle x-ray scattering data. Gp41_int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41_int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140_CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated 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 gp140_CA018 was higher than that induced by gp41_int-Cys, the majority of animals immunized with gp41_int-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.     

Dramatic Potentiation of the Antiviral Activity of HIV Antibodies by Cholesterol Conjugation

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10–100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.     

Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope

The membrane-proximal region of the ectodomain of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the target of three of the five broadly neutralizing anti-HIV-1 antibodies thus far isolated. We have determined crystal structures of the antigen-binding fragment for one of these antibodies, 2F5, in complex with 7-mer, 11-mer, and 17-mer peptides of the gp41 membrane-proximal region, at 2.0-, 2.1-, and 2.2-A resolutions, respectively. The structures reveal an extended gp41 conformation, which stretches over 30 A in length. Contacts are made with five complementarity-determining regions of the antibody as well as with nonpolymorphic regions. Only one exclusive charged face of the gp41 epitope is bound by 2F5, while the nonbound face, which is hydrophobic, may be hidden due to occlusion by other portions of the ectodomain. The structures reveal that the 2F5 antibody is uniquely built to bind to an epitope that is proximal to a membrane surface and in a manner mostly unaffected by large-scale steric hindrance. Biochemical studies with proteoliposomes confirm the importance of lipid membrane and hydrophobic context in the binding of 2F5 as well as in the binding of 4E10, another broadly neutralizing antibody that recognizes the membrane-proximal region of gp41. Based on these structural and biochemical results, immunization strategies for eliciting 2F5- and 4E10-like broadly neutralizing anti-HIV-1 antibodies are proposed.     

The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent

The peptide of HIV-1 envelope gp41 (a.a 628-683), referred to herein as P5, contains P1, a conserved galactose-specific lectin domain for binding the mucosal HIV-1-receptor, galactosyl ceramide (GalCer), as shown earlier, and a potential calcium-binding site (a.a 628-648). P1 contains contiguous epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, Z13. However, similar neutralizing antibodies could not be raised in animal model using immunogens based on these epitopes. We now show that the structure of both P5 and P1 peptides, as measured by circular dichroism, differs according to their environment: aqueous or lipidic, and as a function of calcium concentration. P5, but not P1, binds to calcium with a low binding affinity constant in the order of 2.5x10(4). Calcium binding results in a conformational change of P5, leading in turn to a decrease in affinity for GalCer. Hence, the affinity of the gp41-lectin site for the galactose harbored by the mucosal HIV-1 receptor GalCer is modulated by the peptide secondary and tertiary structure and the local environment. Therefore, definition of the conformation of this novel extended gp41 membrane proximal region, containing the conserved peptide P1 and the Ca(2+) binding site, could help designing an immunogen efficient at inducing neutralizing anti-HIV-1 antibodies.     

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.     

Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants

We previously reported that soluble, stable YU2 gp140 trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein immunogens could elicit improved breadth of neutralization against HIV-1 isolates compared to monomeric YU2 gp120 proteins. In this guinea pig immunization study, we sought to extend these data and determine if adjuvant could quantitatively or qualitatively alter the neutralizing response elicited by trimeric or monomeric immunogens. Consistent with our earlier studies, the YU2 gp140 immunogens elicited higher-titer neutralizing antibodies against homologous and heterologous isolates than those elicited by monomeric YU2 gp120. Additionally, the GlaxoSmithKline family of adjuvants AS01B, AS02A, and AS03 induced higher levels of neutralizing antibodies compared to emulsification of the same immunogens in Ribi adjuvant. Further analysis of vaccine sera indicated that homologous virus neutralization was not mediated by antibodies to the V3 loop, although V3 loop-directed neutralization could be detected for some heterologous isolates. In most gp120-inoculated animals, the homologous YU2 neutralization activity was inhibited by a peptide derived from the YU2 V1 loop, whereas the neutralizing activity elicited by YU2 gp140 trimers was much less sensitive to V1 peptide inhibition. Consistent with a less V1-focused antibody response, sera from the gp140-immunized animals more efficiently neutralized heterologous HIV-1 isolates, as determined by two distinct neutralization formats. Thus, there appear to be qualitative differences in the neutralizing antibody response elicited by YU2 gp140 compared to YU2 monomeric gp120. Further mapping analysis of more conserved regions of gp120/gp41 may be required to determine the neutralizing specificity elicited by the trimeric immunogens.     

Structure and Immunogenicity of a Peptide Vaccine,Including the Complete HIV-1 gp41 2F5 Epitope: IMPLICATIONS FOR ANTIBODY RECOGNITION MECHANISM AND IMMUNOGEN DESIGN*

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund''s adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.     

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.     

Synthetic peptides containing T and B cell epitopes from human immunodeficiency virus envelope gp120 induce anti-HIV proliferative responses and high titers of neutralizing antibodies in rhesus monkeys.

We have previously described a synthetic peptide (T1-SP10) derived from two noncontiguous regions of HTLVIIIB envelope gp120 (T1, amino acids 428-443; SP10, amino acids 303-321) that induced type-specific anti-HIV neutralizing antibodies and T cell proliferative responses against native HIV gp120 when used as a carrier-free immunogen in goats. In this study, HTLVIIIB T1-SP10 synthetic peptides were used to immunize rhesus monkeys to determine if the peptides were capable of eliciting HIV-specific neutralizing antibody and proliferative responses in primates. Four compounds (alum, polyA:polyU, threonyl-muramyldipeptide (MDP) and IFA) were also compared for efficacy as adjuvants in this system. Rhesus monkeys immunized with T1-SP10 peptides generated high titers of antibodies against the immunogens and also against HTLVIIIB gp120. Sera from all four animals given T1-SP10 in IFA or threonyl-MDP neutralized infection by HTLVIIIB and blocked virus-dependent cell fusion events. A peak neutralization titer of 1:940 was seen in one animal given IFA (19600) and a titer of 1:900 was seen in one of the monkeys (17371) given threonyl-MDP. Proliferative responses of immune rhesus PBMC to T1-SP10 appeared after the first injection. After eight immunizations, two of eight monkeys (one injected with peptides in threonyl-MDP and one given peptides in IFA) had PBMC proliferative responses to native HTLVIIIB gp120. These data demonstrate that the carrier-free T1-SP10 synthetic peptide construct can induce high titers of neutralizing anti-HIV antibody responses and PBMC proliferative responses to HIV in primates.     

Efficiency of neutralizing antibodies targeting the CD4-binding site: influence of conformational masking by the V2 loop in R5-tropic clade C simian-human immunodeficiency virus

In R5-tropic clade C simian-human immunodeficiency viruses (SHIV-Cs), we identified a 3-asparagine (3N) deletion mutation in the V2 loop stem of gp120 as the major determinant of neutralization escape of the anti-CD4-binding site (anti-CD4-bs) neutralizing monoclonal antibody (nMAb) b12. However, the more potent anti-CD4-bs nMAbs VRC01 and VRC03 were not sensitive to this mutation. Using isogenic tier 1 or tier 2 proviruses differing only in the 3N mutation, we showed that this mutation might result in selective conformational b12 epitope masking. Therefore, human immunodeficiency virus (HIV) Env immunogens targeting the CD4-bs and designed to neutralize tier 2 viruses should take conformational masking by the V2 loop into account.