Immunobiological role of llama heavy-chain antibodies against a bacterial beta-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.
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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 · 1.78 Impact Factor
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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
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ABSTRACT: Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.Veterinary Immunology and Immunopathology 08/2008; 126(1-2):64-73. DOI:10.1016/j.vetimm.2008.06.015 · 1.75 Impact Factor