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PorM is a membrane protein that is involved in the assembly of the type IX secretion system (T9SS) inPorphyromonas gingivalis, a major bacterial pathogen that is responsible for periodontal disease in humans. In the context of structural studies of PorM to better understand T9SS assembly, four camelid nanobodies were selected, produced and purified, and their specific interaction with the N-terminal or C-terminal part of the periplasmic domain of PorM was investigated. Diffracting crystals were also obtained, and the structures of the four nanobodies were solved by molecular replacement. Furthermore, two nanobodies were used as crystallization chaperones and turned out to be valuable tools in the structure-determination process of the periplasmic domain of PorM.

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly ( K d = 0.26 μM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

International audience; Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission.

International audience; Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as VHH and CHH, respectively, and which differ from conventional VH and CH counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both VHH and VH genes followed by a unique DH-JH cluster and C region genes, which include both CHH and CH genes. Although this general gene organization greatly resembles that of other typical mammalian Vn-Dn-Jn-Cn translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM+ stage, similar to the case for conventional IgG.

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.

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., Author Summary Due to the absence of an effective vaccine or cure for acquired immunodeficiency syndrome (AIDS), HIV-1 infections still result in high mortality. Two antibodies, 2F5 and 4E10, previously isolated from HIV-1 infected patients, prevent infections by binding to the MPER of gp41, a part of the virus that is difficult to access and only transiently exposed. Here, we immunized llamas with a gp41-based immunogen and subsequently isolated a small antibody fragment (VHH) that can easily access and recognize the MPER. We showed that a unit of two VHH, named bi-2H10, was indeed capable of preventing HIV-1 from infecting cells. We determined the three dimensional structure of the VHH and mapped its interaction site to an MPER region that overlaps with the 2F5 epitope. The 2H10 VHH displays a membrane binding component important for neutralization that resembles that of 2F5. In conclusion, we have developed an immunogen and a small antibody that may have great potential for development of novel anti-HIV/AIDS vaccines and treatments.