Immunobiological role of llama heavy-chain antibodies against a bacterial β-lactamase
ABSTRACT In 1993, a fraction of antibodies (Abs) devoid of L chain was found naturally occurring in the Camelidae. They were found to lack L chains, as well as the first constant heavy-chain domain (CH(1)) and therefore they were named "heavy-chain Abs" (HCAbs). Subsequent studies focused on the functional, structural and biochemical properties of recombinant variable fragments (rVHHs) of HCAbs. It was stated that rVHHs have an augmented capacity to interact with "partially hidden" epitopes, like enzymes active sites, and have an increased stability to thermal and chemical aggression. It has been suggested that these unconventional Abs could represent an evolutionary advantage, being more efficient than conventional Abs to inhibit microbial enzymes, and thus exerting a more protective immune response against pathogens. The present work focuses on the immunobiological role of HCAbs, in their capacity to inhibit microbial enzymes. Two animal models were selected, comprising a model for common vertebrates without HCAbs (rabbits), and a model for vertebrates with both conventional and unconventional Abs (Lama glama). A recombinant bacterial beta-lactamase (CTX-M-2) was selected as the microbial enzymatic antigen. After conventional immunization schedules, neither serum titers nor serum inhibitory capacity showed significant differences when rabbits and llamas were compared. These results indicate that the a priori assumption that the adaptive immune system of camelids could be better "prepared" to respond to bacterial enzymes because of the presence of HCAbs, is not always accurate. Furthermore, when the different llama antibody isotypes and subclasses were purified, it was demonstrated that the inhibitory capacity of total serum was due exclusively to IgG(1). HCAbs not only failed to inhibit CTX-M-2, but instead they activated its enzymatic activity. Altogether, these results indicate that the hypotheses extrapolated from the rVHHs properties need to be revised; the real role of HCAbs in vivo remains unknown, as well as their evolutionary cause.
- SourceAvailable from: Nicolás Caggiano
07/2014; 8(2). DOI:10.5209/rev_RCCV.2014.v8.n2.46537
- "Muchos autores han descripto que los dominios VHH de los HCAbs tienen importantes ventajas en comparación con las Igs convencionales, sin embargo las propiedades fisiológicas de la molécula de HCAbs en su totalidad no parece tener alguna ventaja cuando se compara con las Igs convencionales, (Ferrari et al., 2007). Además nosotros no fuimos capaces de demostrar funciones efectoras como fijación de complemento por parte de los HCAbs, cuando las Igs convencionales tienen sitios de fijación para C1q, (Saccodossi et al., 2012). "
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- "IgG 1 and IgG 3 were purified from the selected serum samples as previously described (De Simone et al., 2005; Ferrari et al., 2007), using a HiTrap-protein G column (GE Healthcare, Piscataway, NJ, USA) and low pH elution buffers (pH 3.5 for IgG 3 and pH 2.7 for IgG 1 ). All Ab fractions were neutralized with Tris 1 M, dialyzed against PBS and protein purity was assessed by 10% SDS-PAGE. "
ABSTRACT: Since they were first described in 1993, it was found that recombinant variable fragments (rVHHs) of heavy-chain antibodies (HCAbs) from Camelidae have unusual biophysical properties, as well as a special ability to interact with epitopes that are cryptic for conventional Abs. It has been assumed that in vivo raised polyclonal HCAbs (pHCAbs) should behave in a similar manner than rVHHs; however, this assumption has not been tested sufficiently. Furthermore, our own preliminary work on a single serum sample from a llama immunized with a β-lactamase, has suggested that pHCAbs have no special ability to down-modulate catalytic activity. In this work, we further explored the interaction of pHCAbs from four llamas raised against two microbial enzymes and analyzed it within a short and a long immunization plan. The relative contribution of pHCAbs to serum titer was found to be low compared with that of the most abundant conventional subisotype (IgG(1)), during the whole immunization schedule. Furthermore, pHCAbs not only failed to inhibit the enzymes, but also activated one of them. Altogether, these results suggest that raising high titer inhibitory HCAbs is not a straightforward strategy - neither as a biotechnological strategy nor in the biological context of an immune response against infection - as raising inhibitory rVHHs.animal 03/2012; 6(3):510-7. DOI:10.1017/S1751731111001789 · 1.84 Impact Factor
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- "Previous studies have shown that HcAbs can act as effective high affinity binding ligands for foreign target molecules (Hamers-Casterman et al., 1993; Huang et al., 2005; Maass et al., 2007). However, other studies have found no added benefit from these unusual molecules; one group failed to detect any specific HAb binding following immunisation (Lange et al., 2001), while another found HcAb ineffective as an enzyme inhibitor (Ferrari et al., 2007). To ascertain the situation that prevails for a llama immunised against botulinum neurotoxin (BoNT) and to better evaluate HcAb capabilities found in the natural immune repertoire, we purified and tested the llama IgG subclasses obtained from a llama that had been immunised for BoNT. Figure 1 Schematic of llama IgG subclass structures. "
ABSTRACT: Lama immunoglobulins consist of conventional antibody (IgG1) and unique forms that lack light chains, called heavy chain antibodies (IgG2 and IgG3). These unusual antibodies possess unique properties ideal for diagnostics and therapeutics. To evaluate the IgG from a llama immunised with botulinum complex toxoids A through F each IgG subclass was tested as capture and recognition ligand in xMAP fluid array immunoassays. The optimal combination, IgG3 capture and IgG2 tracer, detected as low as 64 pg/ml of BoNT/A complex toxoid. Also, heavy chain antibodies were shown to bind BoNT as effectively as conventional IgG1, while possessing much greater thermal stability. [Received 15 October; Accepted 28 December 2007]The Botulinum J 01/2008; 1(1). DOI:10.1504/TBJ.2008.018953