Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics

Structure-Guided Redesign Improves NFL HIV Env Trimer Integrity and Identifies an Inter-Protomer Disulfide Permitting Post-Expression Cleavage

Soluble HIV-1 envelope glycoprotein (Env) trimers are under active investigation as vaccine candidates in relevant pre-clinical models. Like SOSIPs, the cleavage-independent native flexibly linked (NFL) trimers are faithful mimics of the Env spike. Here, we analyzed multiple new designs to explore alternative modifications, informing tertiary interactions, while maintaining NFL trimer homogeneity and integrity. Accordingly, we performed a proline (P) substitution screen in the gp41 heptad repeat 1 region, identifying other trimer-enhancing Ps, including L555P. This P improved trimer integrity compared to I559P in selected properties. Next, we screened 15 structure-guided potential cysteine pairs in gp140 and found that A501C-L663C (“CC2”) forms an inter-protomer disulfide bond that demonstrably increased NFL trimer thermostability. We combined these two approaches with trimer-derived substitutions, coupled with glycine substitutions at helix-to-coil transitions, developed by our group. To increase the exposure of the fusion peptide (FP) N-terminus, we engineered an enterokinase (EK) cleavage site upstream of the FP for controlled post-expression cleavage. In combination, the redesigns resulted in highly stable and homogeneous NFL mimics derived from different clades. Following recombinant EK cleavage, the NFL trimers retained covalent linkage, maintaining a native-like structure while displaying enhanced stability and favorable antigenic features. These trimers also displayed increased exposure of neutralizing epitopes in the FP and gp120/gp41 interface, while retaining other neutralizing epitopes and occluding non-neutralizing elements. This array of Env-structure-guided designs reveals additional interactive regions in the prefusion state of the HIV Env spike, affording the development of novel antigens and immunogens

Fusion of the molecular adjuvant C3d to cleavage-independent native-like HIV-1 Env trimers improves the elicited antibody response

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30–60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV

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

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

<div><p>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.</p> </div

Antibody binding to CL:B2G1.

<p>Bars represent ELISA optical density readings at 450 nm wavelength corresponding to the binding of antibodies (2F5 in black, 4E10 in green, 11F10 in gray and the CDRH3-altered variants in blue, violet, yellow, brown and orange) to CL (top panel), CL:B2G1 complex (middle) and B2G1 (bottom). Light blue color designates CDRH3 altered antibodies with no W substitutions, violet with one W substitution, yellow with two W substitutions, brown with three W substitutions, and orange with one W and one or two Y substitutions. The red and dark blue bars represent the positive and negative binding levels of control IgG included in the assay. The dotted line indicates an arbitrary “cut-off” between the negative and positive values.</p

Schematic representation of the 2F5 wt and loop-shortened CDRH3 and approximate location of the MPER.

<p>Left, the wt 2F5 CDRH3 apex is shown as a schematic representation based upon the mAb structure. Residues P₉₈ to A_100G are shown with each residue represented by a colored cylinder. On the right, a schematic of the length-altered version, where residues T₉₉,T₁₀₀, P_100E and I_100F , represented by the green cylinders with the discontinuous border, were deleted. The red residues, L_100A, F_100B, G_100C and V_100D, were maintained for the length alterations as described in the text. The W and Y substitutions are noted next to the original residue in the variant schematic depiction (Rosetta modeling of a variant CDRH3 was performed, but clashes with peptide binding were assessed to not be compatible with the predicted conformations). The letters a, b and c represent the locations of the second W substitutions to generate antibody variants r4W2a, b and c, respectively.</p

CD analysis and binding kinetics of the CDRH3 length-altered antibodies to selected MPER probes.

<p>(A) CD spectra of 10 µM wt MPER peptide and linked peptide in H₂O. (B) Shown on the vertical axis are the affinity constants (K_D), of the wt 2F5 Mab and the CDRH3 length-altered variants (r2, r4, r6, e2 and e4) for the selected MPER probes. Gray colored bars indicate antibody interactions with the MPER peptide analyte, white bars represent interactions with the linked peptide, and black bars indicate interactions with the ES2 2F5-epitope scaffold. The dotted line indicates an arbitrary cut off which segregates low affinity interactions (K_D>100 nM) from high affinity interactions (K_D<100 nM). For a complete description of binding kinetic constants see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002806#ppat-1002806-t001" target="_blank">Table 1</a>. (C) The Octet binding curves of wt 2F5 Mab to the three probes in solution are shown for the wt MPER peptide, the linked peptide, and the ES2 2F5-epitope scaffold. The experimental binding curves are shown in blue and the applied Languir 1∶1 model fitting of the curves are shown in red. Representative binding curves of the CDRH3-length altered 2F5 variants r4 and e4 are shown below. For the binding curves of all variants, see supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002806#ppat-1002806-g001" target="_blank">figures 1S</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002806#ppat-1002806-g002" target="_blank">2S</a>.</p

MPER Binding and HIV neutralization of mutant antibodies after W/Y substitutions.

<p>(A) Antibody affinity constants corresponding to antibody binding to MPER peptide before and after the W/Y substitutions, in white and shaded columns, respectively. (B) Statistical analysis comparing the means of the binding measurements to the MPER peptide from the antibodies before and after the W substitutions. The affinity constant (K_D), the on-rate (k_on) and the off-rate (k_off) are shown on the vertical axis with each dot representing antibody binding to the MPER peptide. Closed circles represent antibodies with no Ws in the CDRH3 while open circles represent antibodies with W substitutions. Arrows indicate paired antibodies before and after the substitutions. The p values were obtained by subjecting the data to a paired t test statistical analysis. (C) Correlation between antibody K_D to MPER peptide, left panel, or K_D to ES2 scaffold, right panel, and HIV neutralization. Antibody IC₅₀ values are shown on the vertical axis and the affinity constant (K_D) to the MPER peptide in the horizontal axis. Red circles represent antibody IC₅₀ values against the sensitive viruses (HXBc2, MN and 7312a-C3) and blue squares are IC₅₀ against the more resistant isolates. Above the plots, in the gray boxes, are the names of the antibodies whose measurements are found directly underneath. The data were subjected to a Pearson correlation test, two-tailed resulting in a statistically significant p-value (p = 0.0052) and a positive correlation r-value (r = 0.695 ) for the wt MPER peptide and a non-significant p-value for the ES2 scaffold (p = 0.669 and r = 0.125).</p

HIV neutralization values of the CDRH3 length-altered antibodies.

<p>The top and middle panels display the CDRH3 sequences of the length-altered CDRH3 2F5 variants and their HIV neutralization activities. Residues colored in magenta (LFGV) were conserved while flanking residues colored in green (PTT and PIA) were removed sequentially to produce the shorter loop antibodies. In brown are the glycine (G) residues inserted to elongate the CDRH3 length. The W and Y substitutions in the altered-CDRH3 are colored in blue and orange, respectively. The bottom panel shows neutralization values achieved by selected CDRH3-altered 2F5 variants against a larger set of HIV isolates.</p