scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.

Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer.

Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the VH and VL domains with 10-residue (Gly4Ser)2 and five-residue (Gly4Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the VH domain to the N-terminus of the VL domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M(r) 52000). These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M(r) 70000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M(r) 212000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface. However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.

Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase.

We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.

The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK).

Calves were vaccinated with cloned Moraxella bovis pili of serogroup C (experiment 1) or B (experiment 2) either as a monovalent formulation or as part of a multivalent preparation with pili of six other serogroups. Within 4 weeks of the second vaccine dose vaccinated calves and non-vaccinated controls were challenged via the ocular route with either virulent M. bovis strain Dal2d (serogroup C) or M. bovis strain 3WO7 (serogroup B) in experiments 1 and 2, respectively. Calves vaccinated with multivalent vaccines had significantly lower antibody titres than those vaccinated with monovalent preparations. Nevertheless, the levels of protection against infectious bovine keratoconjunctivitis (IBK) achieved with multivalent vaccines were 72% and 83% for the groups challenged with M. bovis strains of serogroups B and C, respectively. The serogroup C monovalent vaccine gave 100% protection against experimentally induced IBK and M. bovis isolates cultured from the eyes 6 days post-challenge were identified as belonging solely to serogroup C. Unexpectedly, only 25% protection was achieved against homologous strain challenge of calves that received the monovalent serogroup B vaccine. Furthermore, the majority of M. bovis isolates recovered from calves in this group belonged to serogroup C, as did half of those isolates cultured from the multivalent vaccinates. The remaining bacterial isolates from the latter group, together with all isolates from the non-vaccinated controls, belonged to serogroup B. Results are consistent with the hypothesis that derivatives of the serogroup B challenge inoculum had expressed serogroup C pilus antigen within 6 days of the challenge, possibly as a result of pilus gene inversion occurring in response to the presence of specific antibody in eye tissues and tears.

Characterization of pilin genes from seven serologically defined prototype strains of Moraxella bovis.

Numerous field isolates of Moraxella bovis have previously been classified by serological techniques into seven serogroups, each defined by homologous cross-reaction with antisera prepared against purified pili of a single prototype strain. The gene encoding pilin from each of the prototype strains has been characterized by nucleotide sequence determination. The coding sequences show extensive homology (70 to 80%) while the proximal downstream sequences show a dichotomy into nonhomologous sets. The pilin genes of three more strains were also characterized. The presence of an additional, partial pilin gene in each prototype strain was confirmed by Southern blot analysis, and the partial pilin genes from two strains of one serogroup were characterized by sequence determination. Features of the pilin gene sequences are considered in relation to pilin gene inversion and the serological variants of strains which may arise from gene inversion events.

A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis.

Pili (fimbriae) were prepared from Moraxella bovis strain Dalton 2d (Dal2d) and from a derivative of Pseudomonas aeruginosa K/2PfS that contained a plasmid-borne Dal2d pilin gene and produced pili having serogroup-specific identity to Dal2d. Nine calves were vaccinated with two doses each of 30 micrograms authentic M. bovis Dal2d pili in oil adjuvant and 10 calves were vaccinated with a similar dose of P. aeruginosa-derived Dal2d pili in the same formulation. All 19 calves and 10 non-vaccinated controls were challenged by instillation of 1 x 10(9) virulent M. bovis Dal2d cells into both conjunctival sacs 19 days after the second vaccine dose. The serological response to vaccination and the degree of protection against experimentally induced infectious bovine keratoconjunctivitis (IBK) were assessed. None of the nine calves vaccinated with authentic M. bovis Dal2d pili developed IBK while two of those vaccinated with P. aeruginosa-derived Dal2d pili developed lesions which accounted for a mean group lesion score of 0.3. In contrast, 9 of the 10 non-vaccinated calves developed IBK lesions, the majority of which were progressive, required early treatment and accounted for a mean group lesion score of 1.5. These results demonstrate the potential of a relatively low dose of pili produced by recombinant DNA technology for development of an effective vaccine against IBK.

The protective efficacy of pili from different strains of Moraxella bovis within the same serogroup against infectious bovine keratoconjunctivitis.

Three groups of ten calves were each immunised with a total of 400 micrograms pili prepared from three separate strains of Moraxella bovis in Alhydrogel-oil adjuvant as two divided, equal doses 21 days apart. Groups 1 and 2 each received a monovalent vaccine made from strain 4L and S276R respectively, which belonged to pili serogroup A. Group 3 received vaccine made from pili of strain Maff1, belonging to serogroup F. A further group of ten calves served as non-vaccinated controls. Calves in groups 1 and 2 had developed serogroup A-specific antibody and those in group 3 developed serogroup F-specific antibody, and some evidence of cross-reacting antibody was also detected when measured by an agglutination test using formalin-killed piliated cells of serogroup A strain 4L. Although antibody titres measured against purified pili by ELISA were highest with homologous serogroup antigens, cross-reactive titres to shared epitopes of M. bovis pili were also detected by this method. Ocular challenge of the 40 calves with virulent M. bovis of serogroup A strain S276R was carried out 14 days after the second vaccine dose. All non-vaccinated calves developed infectious bovine keratoconjunctivitis (IBK). The percentage protection in groups 1 (strain 4L) and 2 (strain S276R) was 60% and 80% respectively (P less than 0.05), with mean lesion scores of 0.7 and 0.3 out of a possible 6.0. The percentage protection of calves in group 3 (strain Maff1) was only 30%, with a mean lesion score of 1.4 compared with 2.2 for non-vaccinated controls. The present findings, together with other evidence indicating that immunity to IBK is serogroup-specific, suggest that inclusion of pili from one representative strain from each of the seven Australian and British serogroups in a polyvalent, subunit vaccine should effectively protect the majority of cattle against IBK caused by most field strains of M. bovis encountered in Australia and the United Kingdom.

Immunoglobulin gamma chains of a monotreme mammal, the echidna (Tachyglossus aculeatus): amino acid composition and partial amino acid sequence.

The echidna represents the lowest stage in phylogeny at which molecules clearly homologous to IgG antibodies appear to occur. We provide evidence that a fraction of gamma chains possess an unblocked N terminal sequence comparable to the VHIII sub-group of human gamma chains and that glycine is the C-terminal residue. Statistical comparison of amino acid composition of the component chains with other immunoglobulin heavy chains suggests that echidna gamma chains are more closely related to eutherian gamma chains than to the 7S Ig heavy chains from amphibia or aves. The results are consistent with our view that true gamma-type heavy chains did not appear in evolution until after the mammalian line diverged from the stem reptiles.

Plasma membrane of a murine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins.

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase. T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells.

Isolation and partial characterization of lymphocyte surface immunoglobulins.

Immunoglobulins were isolated from the surfaces of lymphocytes from a variety of lymphocyte populations including murine and human thymus lymphocytes and murine spleen and thoracic duct lymphocytes. Cell surface proteins were labeled with iodide-(125)I by lactoperoxidase-catalyzed iodination, and recovered in solution either by solubilization in dissociating solvents or active metabolic release. Immunoglobulins were identified and isolated by immunological coprecipitation. The polypeptide chain structure of immunoglobulins isolated from lymphocyte surfaces was analyzed by polyacrylamide gel electrophoresis of reduced, alkylated samples in acid urea. Human and murine thymus lymphocytes possessed only IgM immunoglobulin on their surfaces. This protein contained light chains and micro-type heavy chains and was characterized by a molecular weight of approximately 200,000. Murine splenic lymphocytes from CBA x C57 animals and congenitally athymic (nu/nu) mice possessed both IgM and IgG on their surfaces. The ratio of micro-chain to gamma-chain was about 3/1. The presence of IgM on thymus lymphocytes probably does not reflect trace contamination by B lymphocytes because comparable quantities of IgM were isolated from both cell populations. Metabolic turnover data suggest that this immunoglobulin is synthesized by the cell population studied. These results provide direct evidence for the presence of immunoglobulins composed of light and heavy polypeptide chains on the surfaces of lymphocytes of all classes.