Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis.

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.

Neutralisation of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera.

Envenoming by snakes results in severe systemic and local pathology. Intravenous administration of antivenom, prepared from IgG of venom immunised horses or sheep, is the only effective treatment of systemic envenoming. Conventional antivenoms, formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab')2 fragments, are however ineffective against the local venom effects because of their inability to penetrate the blood/tissue barrier. We have embarked on a new research program to examine (i) whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain IgG (V(H)H) can be exploited to neutralise the local effects of envenoming and (ii) whether a novel antivenom to treat both the systemic and local effects of envenoming can be formulated by combining anti-snake venom V(H)H and conventional F(ab')2. In this preliminary study, we demonstrate that camels and llamas respond to immunisation with Echis ocellatus venom with high antibody titres and broad antigen specificity. These encouraging immunological results were matched by the successful elimination of venom-induced haemorrhage by IgG from the venom-immunised camels and llamas. Unexpectedly, we report for the first time that camelid serum contains a non-IgG, highly potent inhibitor of venom-induced haemorrhage.

Emergence and evolution of functional heavy-chain antibodies in Camelidae.

Antibodies of jawed-vertebrates are composed of paired heavy (H) and light (L) polypeptide chains. Surprisingly, the sera of camelids, nurse shark and wobbegong shark, and possibly ratfish contain antibodies that lack L-chains. In camelids, these Heavy-chain antibodies (HCAbs) are gamma-isotypes, and are functional in antigen binding. In this review we focus on the dedicated immunoglobulin (Ig) genes that encode the HCAb in Camelidae (camels, dromedaries and llamas), about their origin, and how these camel immunoglobulins evolved and acquire a large and diverse repertoire of antigen binding sites in absence of the H-L combinatorial diversity.

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs.

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.

The topology of the S protein in the yeast-derived hepatitis B surface antigen particles.

Hepatitis B surface antigen particles are highly immunogenic and have been shown to provide a suitable support for the presentation of foreign epitopes. More information about the topology of their constitutive protein, the S (small envelope) protein, is a prerequisite to any rational attempt to replace region of this protein with foreign epitopes without modifying the assembly of the particle. The topology of the S protein within the lipid membrane was investigated here by combining extensive proteolysis of the external protein domains with proteinase K and (FTIR-ATR). The proteolytic hydrolysis of the S protein and the identification of the digestion products allowed characterization of the membrane-protected regions of the protein. FTIR spectra of the digested hepatitis B particles revealed that the peptides associated with the particles are rich in alpha-helix structure. The kinetic of 2H/H exchange provided evidence that a large fraction of the native S protein is poorly accessible to the aqueous medium.

Sequence and structure of the membrane-associated peptide of glycophorin A.

Glycophorin A (GPA) has been reconstituted into dimyristoylphosphatidylcholine vesicles and digested with proteinase K to identify the membrane domain and to characterize its structure and orientation. After digestion of the inner and outer domain of GPA by protease action restricted to the aqueous phase, a protected peptide migrates on an electrophoresis gel as a 7.5-kDa dimer (His66-Ile95). The secondary structure and orientation in a lipid bilayer of the 7.5-kDa dimer have been studied by Fourier transform infrared spectroscopy. Our proteolytic and spectroscopic data are in agreement with a topological model in which the His66-Glu72 peptide adopts a beta-sheet conformation and is oriented parallel to the lipid-water interface and the Ile73-Ile95 domain is helical and oriented parallel to the lipid acyl chains, in a transmembrane configuration. Digestion of the domain protruding to the outside of the liposome generates "head-head" and "head-tail" dimers of 16 and 38 kDa, respectively. This observation is discussed in terms of the specificity of the dimer formation process.

Topology of diphtheria toxin B fragment inserted in lipid vesicles.

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells in response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresis, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194-223, 265-375 and 429-528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly alpha-helices and beta-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.

Enzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes.

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions.

HLA typing and lymphocytotoxic crossmatches using conventional techniques or immunobeads. A comparative study.

In a prospectively designed comparative study, HLA class I and class II typing was performed after B/T lymphocyte separation by nylon wool (NIH technique) or using immunomagnetic beads (IMB technique). Samples from 70 healthy individuals (group A) and from 47 patients with potential typing problems (group B) were examined. Complete typing failures occurred more frequently with NIH than with IMB. Except for class I in group A, the rate of correct decisions for antigen assignment was significantly higher with IMB. For class II serum reactions, specificity, predictive value and efficiency were superior for IMB, while sensitivity appeared higher for NIH. This can possibly be explained by a prozone effect which was observed in 9 of 36 titration experiments only with the IMB technique. Correlation of both methods in 104 crossmatches prior to kidney transplantation was poor using peripheral blood lymphocytes, but excellent with spleen cells. In addition to the important advantages of the IMB technique for potential problem cases, especially for class II, typing time is reduced to one third, which is of particular clinical importance in kidney transplantation. However, lymphocytotoxic crossmatches prior to transplantation, if performed by IMB, should be carried out using different serum dilutions or with donor spleen cells instead of peripheral blood lymphocytes.

Alpha subunit of mitochondrial F1-ATPase from the fission yeast. Deduced sequence of the wild type and identification of a mutation that alters apparent negative cooperativity.

The nuclear gene atp1 encoding the mitochondrial ATP synthase alpha subunit of the fission yeast Schizosaccharomyces pombe was sequenced. It contains a 1,608-base pair-long open reading frame interrupted by two introns of 175 and 269 base pairs, located near the 5'-end of the gene. The initiation site of transcription AAAC was located 60 nucleotides upstream of the translation initiation codon. The deduced polypeptide sequence contains a 27-amino acid residue presequence, presumably involved in mitochondrial targeting, preceding a mature protein of 509 amino acid residues. The atp1 alleles from mutant A2313 (Bouty, M., and Goffeau, A. (1982) Eur. J. Biochem. 125, 471-477) and its related phenotypic revertant R351 (Falson, P., Di Pietro, A., Darbouret, D., Jault, J. M., Gautheron, D. C., Boutry, M., and Goffeau, A. (1987) Biochem. Biophys. Res. Commun. 148, 1182-1188) were also cloned and sequenced. A single nonsense mutation CAA-TAA (Gln173-stop) in mutant A2313 became a missense mutation TAA-TTA (stop-Leucine) in revertant R351. Glutamine 173 is located in the first putative element of the nucleotide binding site. Its substitution by a leucine residue appears responsible for the lower enzyme affinity toward ADP and for the loss of cooperativity of F1-ATPase activity.

Development of a hepatitis B vaccine from transformed yeast cells.

The production in yeast cells of the recombinant DNA hepatitis B vaccine of SmithKline Biologicals involves an optimized fermentation process followed by cell disruption and extraction, together with other soluble yeast components of the surface antigen of the hepatitis B virus. The subsequent purification process includes precipitation steps, ion exchange and gel permeation chromatography, and caesium chloride ultracentrifugation. The yeast-derived antigen occurs as spherical particles containing the non-glycosylated HBsAg polypeptide, lipid, and Tween 20. The purity of the polypeptide is above 95% and confirmed by the absence of an immune response to yeast-derived contaminants in vaccinees. Yeast DNA levels were less than 10 pg/vaccine dose. Various biochemical analyses showed that the recombinant polypeptide was faithfully expressed and did not undergo unwanted processing or degradation during fermentation or purification. These results indicate that the recombinant HBsAg can be effectively produced in yeast and processed to a high degree of purity to yield HBsAg particles displaying most of the characteristic properties of plasma-derived HBsAg.