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

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing

A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers

Summary: The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalent ones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the α9-helix and an intersubunit β sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development. : Rutten et al. describe a universal repair and stabilize approach that corrects rare mutations and stabilizes refolding regions to obtain high-quality HIV Envs with high yields. The crystal structure shows how the optimization of the trimer interface between α9, α6, and the intersubunit β-sheet stabilizes the membrane-proximal base. Keywords: envelope protein, chronic, ConC_base, HIV, SOSIP, stabilization, transmitted/founder, vaccine, X-ray structure, hybrid shee

Directing HIV-1 for degradation by non-target cells, using bi-specific single-chain llama antibodies

While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing

Summary of llama immunizations.

<p>Each llama received the indicated gp140 immunogen as described in the materials and methods and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#ppat.1004552-McCoy2" target="_blank">[26]</a>.</p><p>Summary of llama immunizations.</p

VHH mutation alters affinity, potency and breadth of neutralization.

<p>(A) VHH binding to clade B gp120 Bal.26 in ELISA detected by their C-terminal Myc tag. (B) VHH neutralization of Bal.26 HIV pseudovirus in the TZM-bl assay. Mutant VHH were generated by site-directed mutagenesis as detailed in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#s4" target="_blank">Materials and Methods</a>. (C) The fold change in IC50 µg/ml values for B9 S54W relative to B9, indicating the increase potency of the mutant are plotted on the Y-axis. The IC50 µg/ml values for B9 against each virus in the legend are plotted on the X-axis, indicating the baseline potency of B9. All assays were carried out in duplicate and error bars represent standard deviation. These data are representative of at least three independent experiments.</p

A14, B9, B21 binding to HIV ENV.

<p>VHH binding to immunogens (A) clade B gp140 R2, (B) clade C gp140 96ZM651.02, (C) clade A 92UG037 gp140, (D) CRF BC CN54 gp140, (E) YU2 WT and D368R gp120, and (F) RSC3 mutant and RSC3 delta mutant gp120, was assessed by ELISA as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#s4" target="_blank">Materials and Methods</a>. The positive control for gp140 binding was J3 (McCoy et al. 2012) that for gp41 binding 2H10 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004552#ppat.1004552-LutjeHulsik1" target="_blank">[32]</a>. The positive control for both RSC3 gp120 proteins was D47 a non-neutralizing RSC3-specific VHH (L McCoy unpublished data). All binding assays were carried out in duplicate and error bars represent standard deviation. These data are representative of at least three independent experiments.</p