Enhancement of toxin- and virus-neutralizing capacity of single-domain antibody fragments by <Emphasis Type="Italic">N</Emphasis>-glycosylation

Single-domain antibody fragments (VHHs) have several beneficial properties as compared to conventional antibody fragments. However, their small size complicates their toxin- and virus-neutralizing capacity. We isolated 27 VHHs binding Escherichia coli heat-labile toxin and expressed these in Saccharomyces cerevisiae. The most potent neutralizing VHH (LT109) was N-glycosylated, resulting in a large increase in molecular mass. This suggests that N-glycosylation of LT109 improves its neutralizing capacity. Indeed, deglycosylation of LT109 decreased its neutralizing capacity three- to fivefold. We also studied the effect of glycosylation of two previously isolated VHHs on their ability to neutralize foot-and-mouth disease virus. For this purpose, these VHHs that lacked potential N-glycosylation sites were genetically fused to another VHH that was known to be glycosylated. The resulting fusion proteins were also N-glycosylated. They neutralized the virus at at least fourfold-lower VHH concentrations as compared to the single, non-glycosylated VHHs and at at least 50-fold-lower VHH concentrations as compared to their deglycosylated counterparts. Thus, we have shown that N-glycosylation of VHHs contributes to toxin- and virus-neutralizing capacity.     

VHH characterization. Comparison of recombinant with chemically synthesized anti‐HER2 VHH

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its “antigen.” Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the “antigen” HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates.     

Glycosylated F4 (K88) Fimbrial Adhesin FaeG Expressed in Barley Endosperm Induces ETEC-neutralizing Antibodies in Mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4⁺ ETEC to the F4 receptors present on porcine intestinal enterocytes.     

Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk

Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 10⁴ colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable.     

Stimulation of chymosin secretion by simultaneous expression with chymosin-binding llama single-domain antibody fragments in yeast

We studied the effect of coexpression of chymosin and chymosin-binding llama single-domain antibody fragments (VHHs) on the secretion of chymosin by Saccharomyces cerevisiae cells. A VHH expression library containing chymosin-specific VHHs was obtained by immunization of a llama and coexpressed with chymosin in yeast. From this library, we obtained two VHH clones that stimulated chymosin secretion by screening colonies for the level of chymosin secreted. These VHHs bound biotinylated chymosin in an immunoblot procedure but failed to bind chymosin in ELISA, suggesting that their interaction with chymosin was of low affinity. In a second approach, chymosin-specific VHHs were first selected using phage display and then coexpressed with chymosin in yeast cells. Screening yeast cells for higher levels of chymosin secretion resulted in 11 VHHs. Sequence analysis revealed that these 11 VHHs formed four sets of related VHHs that were different from the previously isolated two VHHs. Although binding of VHHs to chymosin could not be demonstrated in ELISA using soluble VHHs, it could be unambiguously demonstrated for clones isolated by phage display, using phage-displayed VHHs. Finally, quantitative Western blot analysis of chymosin amounts demonstrated that coexpression with VHH domains can stimulate the level of secreted chymosin 1.5- to 6-fold.     

The effect of mutation at M307 in <Emphasis Type="Italic">FUT1</Emphasis> gene on susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.     

Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.     

Comparison between Enterotoxigenic Escherichia coli Strains Expressing “F42,” F41 and K99 Colonization Factors

Two enterotoxigenic Escherichia coli (ETEC) strains (coded 567/7 and 103) isolated from piglets with neonatal diarrhea were described as producers of a new adhesin (F42). With the use of molecular biology and immunology techniques such as DNA hybridization with probes for F41 and K99 genes and Western-blotting of the superficial proteins of these strains and standard E. coli strains carrying genes for F41 and K99 adhesins, it was demonstrated that this new adhesin either shares extensive genetic and immunological determinants with F41 adhesin or they are the same fimbriae.     

Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.     

Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo

As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.     

Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen

The importance of the lymphocyte source to generate hybridomas or to construct antibody gene libraries from which to identify potent monoclonal antibodies is understudied. However, the few comparative studies that exist seem to favor the lymph node tissue as a B-cell source. Here the peripheral blood and lymph node lymphocytes of a dromedary immunized with prostate-specific antigen (PSA) have been employed to clone two independent gene banks of the variable domains of heavy-chain antibodies (i.e. the VHHs). Several PSA-specific VHHs were retrieved after panning of these phage-displayed VHH libraries. Some of them were derived from the same B-cell lineage, possibly reflecting the restricted primary repertoire of heavy-chain antibodies. Other binders originated from different B-cell lineages and apparently converged toward a striking homologous amino acid sequence motif in their CDR3. This illustrates the strong somatic hypermutation and stringent antigen-driven selection ongoing in these animals. Although the various antigen binders exhibit a broad range of kinetic rate constants for their interaction with the PSA, leading to equilibrium constants from 70 pM to 100 nM, no significant difference existed between the binders from the two B-cell sources. The VHHs of both libraries were categorized in three groups based on nonoverlapping epitopes. Some of these VHHs could inhibit and others could enhance the proteolytic activity of the antigen. Remarkably, VHHs seem to sense or induce conformational changes on different PSA isoforms, a feature that might be exploited to study the PSA conformational flexibility and to discriminate the stages of prostate cancer.     

Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies

Antigen specific llama VHH antibody fragments were compared to antigen specific mouse monoclonal antibodies with respect to specificity, affinity and stability. The llama VHH antibody fragments and the mouse monoclonal antibodies investigated were shown to be highly specific for the protein antigen hCG or the hapten antigen RR-6. The affinity of the interaction between monovalent llama VHH antibody fragments and their antigen is close to the nanomolar range, similar to the bivalent mouse monoclonal antibodies studied. Llama VHH antibody fragments are similar to mouse monoclonal antibodies with respect to antigen binding in the presence of ammonium thiocyanate and ethanol. The results show that relative to antigen specific mouse monoclonal antibodies, antigen specific llama VHH fragments are extremely temperature stable. Two out of six llama VHHs are able to bind antigen specifically at temperatures as high as 90 degrees C, whereas four out of four mouse monoclonal antibodies are not functional at this temperature. Together with the finding that llama VHH fragments can be produced at high yield in Saccharomyces cerevisiae, these findings indicate that in the near future antigen specific llama VHH fragments can be used in for antibodies unexpected products and processes.     

Kinetics of PKCε activating and inhibiting llama single chain antibodies and their effect on PKCε translocation in HeLa cells

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.     

Comparison of VHH‐Fc antibody production in Arabidopsis thaliana,Nicotiana benthamiana and Pichia pastoris

VHHs or nanobodies are widely acknowledged as interesting diagnostic and therapeutic tools. However, for some applications, multivalent antibody formats, such as the dimeric VHH‐Fc format, are desired to increase the functional affinity. The scope of this study was to compare transient expression of diagnostic VHH‐Fc antibodies in Nicotiana benthamiana leaves with their stable expression in Arabidopsis thaliana seeds and Pichia pastoris. To this end, VHH‐Fc antibodies targeting green fluorescent protein or the A. thaliana seed storage proteins (albumin and globulin) were produced in the three platforms. Differences were mainly observed in the accumulation levels and glycosylation patterns. Interestingly, although in plants oligomannosidic N‐glycans were expected for KDEL‐tagged VHH‐Fcs, several VHH‐Fcs with an intact KDEL‐tag carried complex‐type N‐glycans, suggesting a dysfunctional retention in the endoplasmic reticulum. All VHH‐Fcs were equally functional across expression platforms and several outperformed their corresponding VHH in terms of sensitivity in ELISA.     

Dual beneficial effect of interloop disulfide bond for single domain antibody fragments

The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.     

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Summary Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.     

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.     

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis

Background

Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis.

Methodology/Principal Findings

Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10⁻¹⁰ M.

Conclusions/Significance

A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。