Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.     

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

Maraviroc (MVC) is a CCR5 antagonist that inhibits HIV-1 entry by binding to the coreceptor and inducing structural alterations in the extracellular loops. In this study, we isolated MVC-resistant variants from an HIV-1 primary isolate that arose after 21 weeks of tissue culture passage in the presence of inhibitor. gp120 sequences from passage control and MVC-resistant cultures were cloned into NL4-3 via yeast-based recombination followed by sequencing and drug susceptibility testing. Using 140 clones, three mutations were linked to MVC resistance, but none appeared in the V3 loop as was the case with previous HIV-1 strains resistant to CCR5 antagonists. Rather, resistance was dependent upon a single mutation in the C4 region of gp120. Chimeric clones bearing this N425K mutation replicated at high MVC concentrations and displayed significant shifts in 50% inhibitory concentrations (IC₅₀s), characteristic of resistance to all other antiretroviral drugs but not typical of MVC resistance. Previous reports on MVC resistance describe an ability to use a drug-bound form of the receptor, leading to reduction in maximal drug inhibition. In contrast, our structural models on K425 gp120 suggest that this resistant mutation impacts CD4 interactions and highlights a novel pathway for MVC resistance.     

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.     

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.     

HIV-1 Envelope Glycoprotein Trimers Display Open Quaternary Conformation When Bound to the gp41 Membrane-Proximal External-Region-Directed Broadly Neutralizing Antibody Z13e1

We describe cryo-electron microscopic studies of the interaction between the ectodomain of the trimeric HIV-1 envelope glycoprotein (Env) and Z13e1, a broadly neutralizing antibody that targets the membrane-proximal external region (MPER) of the gp41 subunit. We show that Z13e1-bound Env displays an open quaternary conformation similar to the CD4-bound conformation. Our results support the idea that MPER-directed antibodies, such as Z13e1, block viral entry by interacting with Env at a step after CD4 activation.     

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.     

Binding of HIV-1 gp41-Directed Neutralizing and Non-Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.     

Structural and Biochemical Characterization of Cysteinylation in Broadly Neutralizing Antibodies to HIV-1

Antibodies with exceptional breadth and potency have been elicited in some individuals during natural HIV-1 infection. Elicitation and affinity maturation of broadly neutralizing antibodies (bnAbs) is therefore the central goal of HIV-1 vaccine development. The functional properties of bnAbs also make them attractive as immunotherapeutic agents, which has led to their production and optimization for passive immunotherapy. This process requires in vitro manufacturing and monitoring of any heterogeneous expression, especially when subpopulations of antibodies are produced with varying levels of biological activity. Post-translational modification (PTM) of antibodies can contribute to heterogeneity and is the focus of this study. Specifically, we have investigated cysteinylation in a bnAb lineage (PCDN family) targeting the N332-glycan supersite on the surface envelope glycoprotein (Env) of HIV-1. This PTM is defined by capping of unpaired cysteine residues with molecular cysteine. Through chromatography and mass spectrometry, we were able to characterize subpopulations of cysteinylated and non-cysteinylated antibodies when expressed in mammalian cells. The crystal structures of two PCDN antibodies represent the first structures of a cysteinylated antibody and reveal that the cysteinylation in this case is located in CDRH3. Biophysical studies indicate that cysteinylation of these HIV-1 antibodies does not interfere with antigen binding, which has been reported to occur in other cysteinylated antibodies. As such, these studies highlight the need for further investigation of cysteinylation in anti-HIV and other bnAbs.     

VSV-Displayed HIV-1 Envelope Identifies Broadly Neutralizing Antibodies Class-Switched to IgG and IgA

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Enhanced Exposure of the CD4-Binding Site to Neutralizing Antibodies by Structural Design of a Membrane-Anchored Human Immunodeficiency Virus Type 1 gp120 Domain

The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the β20-β21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant [OD(GSL)(Δβ20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.The human immunodeficiency virus type 1 (HIV-1) envelope is composed of surface gp120 and transmembrane gp41. Initial attempts to develop HIV vaccines through the induction of antibodies focused on recombinant gp120 glycoproteins. Two phase III clinical trials conducted in the United States and Thailand showed no protection from a gp120-based subunit vaccine against HIV infection, nor did the vaccine delay HIV-1 disease progression (11, 25). In addition, a phase II trial completed in Thailand with a live recombinant HIV-1 canarypox vaccine (vCP1452) in combination with a gp120 subunit protein did not stimulate a markedly improved immune response (28). The lack of efficacy was likely to be related to its failure to elicit broadly neutralizing antibodies (4, 10, 33).Several broadly neutralizing human monoclonal antibodies (MAbs) have been derived from infected individuals, including immunoglobulin G1 (IgG1) b12, 2G12, 2F5 and 4E10, which are directed against CD4-binding-site (BS), carbohydrate, and membrane-proximal regions of HIV Env (reviewed in reference 9). Among the most potent, the b12 antibody occludes the site of CD4 binding on gp120 and prevents virus attachment to CD4 on target cells (39). Other CD4-BS antibodies recognize epitopes on monomeric gp120 that overlap with b12 but lack the ability of b12 to neutralize primary HIV-1 isolates (5). An understanding of the specificity of b12 binding, neutralization, and protection should aid in the development of immunogens that induce neutralizing antibodies of a similar specificity.The structure of the b12-gp120 complex (39) shows that b12 binds to a conformationally conserved surface, which is centered around the CD4-binding loop on the outer domain of gp120. In the CD4-bound conformation of gp120, the CD4-binding loop or β15-strand makes antiparallel intermolecular hydrogen bonds to the C″ strand of CD4 (14). Overall, the outer domain of gp120 comprises 82% of the gp120 contact surface with b12, while most of the contacts outside of the outer domain have marginal importance (39). One exception, however, are contacts to the loop connecting the outer domain with the α5-helix of the inner domain (39), which appear to be important.Because it represents the smallest structural unit containing the b12 epitope, and therefore maximizes the b12-immunogenic surface relative to the overall surface, an outer domain-only immunogen with high b12 affinity represents an attractive immunogen. An outer domain construct (named OD1) was previously derived from HIV-1 strain YU2 gp120 and found to bind 2G12 and a number of anti-V3 antibodies (36); however, b12 binding to this construct was difficult to detect by enzyme-linked immunosorbent assay, probably due to an enhanced off rate (36, 39). A large, relatively flat interface exists between the inner and outer domains of gp120 in both CD4-bound and b12-bound conformations. We reasoned that the removal of the inner domain might partially destabilize it and decided to replace the inner domain with another polar surface, the cell membrane. We expressed outer domain proteins (ODs) in various membrane-anchored forms and tested their abilities to bind b12. An HIV-1 clade B R5 and X4 dual-tropic virus, R3A, was selected as a prototype (20). Laboratory-adapted virus strain R3A TA1 contains a truncated V1/V2 and a truncated V3 (named 9,9), maintains CCR5 tropism, and is highly sensitive to b12 neutralization (15, 23). We used available atomic-level structures to model an R3A gp120 core and to design truncations of flexible, potentially immunodominant structures, which emanate from OD, including the β20-β21 hairpin and the V3 loop. Thus, by using structure-based design to modify the OD form of R3A TA1, we attempted to remove strain-specific determinants, to enhance cell-surface expression, and to increase specific b12 binding compared to other native forms.     

Drift of the HIV-1 Envelope Glycoprotein gp120 toward Increased Neutralization Resistance over the Course of the Epidemic: a Comprehensive Study Using the Most Potent and Broadly Neutralizing Monoclonal Antibodies

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.     

Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual

Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.     

Design of a Non-glycosylated Outer Domain-derived HIV-1 gp120 Immunogen That Binds to CD4 and Induces Neutralizing Antibodies

The outer domain (OD) of the HIV-1 envelope glycoprotein gp120 is an important target for vaccine design as it contains a number of conserved epitopes, including a large fraction of the CD4 binding site. Attempts to design OD-based immunogens in the past have met with little success. We report the design and characterization of an Escherichia coli-expressed OD-based immunogen (OD_EC), based on the sequence of the HxBc2 strain. The OD_EC-designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gp120. Biophysical studies showed that OD_EC is folded and protease-resistant, whereas OD_EC lacking the designed mutations is highly aggregation-prone. In contrast to previously characterized OD constructs, OD_EC bound CD4 and the broadly neutralizing antibody b12 but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, OD_EC was highly immunogenic, and the sera showed measurable neutralization for four subtype B and one subtype C virus including two b12-resistant viruses. In contrast, sera from rabbits immunized with gp120 did not neutralize any of the viruses. OD_EC is the first example of a gp120 fragment-based immunogen that yields significant neutralizing antibodies.     

Requirements for the Induction of Broadly Neutralizing Antibodies against HIV-1 by Vaccination

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime–boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.