Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Abeta peptide.

A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta[1-40], Abeta[1-42] and Abeta[1-43] has been developed. The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli. Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry. ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence. The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.

Panning and selection of proteins using ribosome display.

Eukaryotic ribosome complexes can be used as a means to display a library of proteins, and isolate specific binding reagents by screening against target molecules. Here we present, as an example, a method for the display of a library of immunoglobulin variable-like domains (VLDs) for the production of stable mRNA/ribosome/protein complexes. These complexes are produced by the addition of specific in vitro transcriptional promoter elements and translation control sequences to the template DNA. Furthermore, an appropriate spacer (anchor) domain is included for efficient folding of the nascent translated protein, which remains attached to the ribosome complex. Ribosome complexes are panned against hen egg lysozyme-conjugated magnetic beads and genes encoding specific, binding, V-like domains are recovered by RT-PCR and cloned into an Escherichia coli expression vector.

Protein affinity maturation in vivo using E. coli mutator cells.

This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E. coli mutator cells. The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences. Selection conditions can be modified for antibody fragments with increased production levels. Growth conditions in E. coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment.

Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics.

Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment. Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library. Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency. Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv). Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naïve repertoires (10(14)). Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles. These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers. We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems.

Oxidation of ammonia in osmium polypyridyl complexes.

The oxidations of cis- and trans-[OsIII(tpy)(Cl)2(NH3)](PF6), cis-[OsII(bpy)2(Cl)(NH3)](PF6), and [OsII(typ)(bpy)(NH3)](PF6)2 have been studied by cyclic voltammetry and by controlled-potential electrolysis. In acetonitrile or in acidic, aqueous solution, oxidation is metal-based and reversible, but as the pH is increased, oxidation and proton loss from coordinated ammonia occurs. cis- and trans-[OsIII(tpy)(Cl)2(NH3)](PF6) are oxidized by four electrons to give the corresponding OsVI nitrido complexes, [OSVI(typ)(Cl)2(N)]+. Oxidation of [Os(typ)(bpy)(NH3)](PF6)2 occurs by six electrons to give [Os(tpy)(bpy)(NO)](PF6)3. Oxidation of cis-[OsII(bpy)2(Cl)(NH3)](PF6) at pH 9.0 gives cis-[OsII(bpy)2(Cl)(NO)](PF6)2 and the mixed-valence form of the mu-N2 dimer [cis-[Os(bpy)2(Cl)2[mu-N2)](PF6)3. With NH4+ added to the electrolyte, cis-[OsII(bpy)2(Cl)(N2)](PF6) is a coproduct. The results of pH-dependent cyclic voltammetry measurements suggest OsIV as a common intermediate in the oxidation of coordinated ammonia. For cis- and trans-[OsIII(tpy)(Cl)2(NH3)]+, OsIV is a discernible intermediate. It undergoes further pH-dependent oxidation to [OsVI(tpy)(Cl)2(N)]+. For [OsII(tpy)(bpy)(NH3)]2+, oxidation to OsIV is followed by hydration at the nitrogen atom and further oxidation to nitrosyl. For cis-[OsII(bpy)2(Cl)-(NH3)]+, oxidation to OsIV is followed by N-N coupling and further oxidation to [cis-[Os(bpy)2(Cl)2(mu-N2)]3+. At pH 9, N-N coupling is competitive with capture of OsIV by OH- and further oxidation, yielding cis-[OsII(bpy)2(Cl)(NO)]2+.

Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B.

A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following production of the scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells were used as an inoculum for liquid cultures. After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered. Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels. Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.

Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents.

Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti-neuraminidase NC10 scFv-streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcore biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti-ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.

Agglutination-inhibition assay for the detection of recombinant proteins tagged with peptide epitopes.

We have demonstrated that the expression of recombinant proteins labeled with an immunoreactive epitope can be rapidly assessed and quantitated using a modified haemagglutination inhibition assay in enzyme-linked immunosorbent assay (ELISA) trays. The agglutination of erythrocytes from a droplet of whole blood provided a simple visual assay. The additional reagents required for the assay were a recombinant anti-human erythrocyte Fab fragment fused to a peptide epitope and a bivalent antibody with specificity to the same epitope. In this report, we found that a convenient and sensitive epitope was the octapeptide FLAG in conjunction with the M2 anti-FLAG antibody, which had affinity to FLAG incorporated either at the C-terminus or N-terminus of the recombinant protein. The agglutination-inhibition (AI) assay was configured to detect as little as 1 mg/L of soluble recombinant protein in a 30-min assay. Since the AI assay was substantially more rapid and convenient than dot-blot or Western blot analyses, our laboratory now uses this method routinely for the assay of FLAG-labeled recombinant products following protein expression and subsequent small- and large-scale purification procedures.

Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2.

The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described. In a whole-blood, erythrocyte agglutination assay format, recombinant scFv antibodies (expressed in Escherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 peptides, were shown to be able to detect efficiently natural antibodies against HIV in human serum. Performance in trials suggests that these single chain reagents have potential as alternatives to existing Fab-peptide chemical conjugates. We also report the construction of an inducible expression vector, pGC, which can be used both in laboratory experiments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as the Fab peptide conjugate in assays where whole (negative) blood was spiked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives. This deficiency was overcome by inclusion of the natural 1C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagent (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a result of this important modification, performance of the extended scFv reagent (for both HIV-1 and HIV-2) equalled that of the current commercial technology in limited trials.

Escherichia coli expression of a bifunctional Fab-peptide epitope reagent for the rapid diagnosis of HIV-1 and HIV-2.

BACKGROUND: The current format of a rapid whole-blood agglutination assay for HIV relies on a bifunctional molecule which comprises a 1C3 Fab fragment, with specificity for the human red blood cell surface marker (glycophorin A), chemically conjugated to a synthetic peptide that corresponds to a single immunodominant region of HIV envelope glycoprotein. In this assay erythrocyte agglutination occurs if the blood sample contains anti-HIV antibodies. OBJECTIVES: To establish whether a bacterially synthesised Fab fragment encoding several C-terminal immunodominant peptide tails can be produced in sufficient purity and yield to function in whole-blood agglutination assays. STUDY DESIGN: An E. coli dicistronic Fab expression cassette was constructed comprising of light and heavy chain gene fragments derived from a glycophorin specific monoclonal antibody (1C3), genetically linked with C-terminal immunoreactive peptide epitopes. Expression and purification procedures were established to enable the rapid production of 1C3 Fab-peptide epitope conjugates. RESULTS: A recombinant 1C3 Fab fragment was expressed with two different immunological epitope markers, Glu-Glu-Phe (EEF) and FLAG, at the C-terminus of the Fd heavy and kappa light chain, respectively. This model Fab-EEF/FLAG conjugate was detected in culture supernatant by SDS-PAGE gels and Western blots, and could be successfully used in erythrocyte agglutination assays. Furthermore, an HIV specific 1C3 Fab reagent, containing immunoreactive peptide epitopes from the surface glycoproteins of HIV-1 and HIV-2, was also expressed but at lower levels and with increased sensitivity to proteolytic degradation. Nevertheless, this recombinant Fab reagent with dual diagnostic specificity performed very effectively in whole-blood diagnosis of patients infected with either HIV-1 or HIV-2. CONCLUSION: A recombinant 1C3 Fab fragment terminated by immunoreactive peptide epitopes can be expressed in E. coli in a soluble, antigen-binding form, and it can successfully mimic the commercial Fab-HIV reagents in whole-blood agglutination assays.

Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression.

The present paper analyzes the influence of major histocompatibility complex (MHC) class II (Ir) genes on MHC class II-restricted T-cell responses to West Nile virus (WNV) and recombinant vaccinia virus-derived Kunjin virus antigens and identifies the immunodominant Kunjin virus antigens. Generally, mice were primed by intravenous infection with WNV or Kunjin virus, and their CD4+ T cells were stimulated in vitro 14 days later with WNV or Kunjin virus antigens to pulse macrophage or B-cell antigen-presenting cells (APC). WNV-specific in vitro T-cell responses from H-2b mice were higher than those from H-2d, H-2k, and H-2q mice. When recombinant vaccinia virus-derived Kunjin virus antigen preparations were tested in vitro, Kunjin virus-immune T cells of H-2b haplotype responded most strongly to structural (prM, C, E) and membrane-associated nonstructural (NS1) proteins encoded by VKV 1031 and showed weaker responses to cytosolic nonstructural protein NS5 (VKV 1022), whereas the responders of H-2k haplotype responded most strongly to the antigens encoded by VKV 1022 and gave lesser responses to VKV 1031. H-2d T cells gave weaker responses than either H-2b or H-2k cells, with responses to VKV 1031 generally being higher than those to VKV 1022. Responses to VKV 1023 or VKV 1024 encoding all of the NS3 to NS5 gene sequence or to VKV 1023 encoding all of NS3 were weak or absent. Within a given inbred strain, B cells and macrophages differed in their abilities to present recombinant vaccinia virus-derived Kunjin virus antigens, both in terms of magnitude of T-cell responses induced and the particular Kunjin virus protein presented. T cells from different non-MHC genetic backgrounds varied in their requirements of macrophage numbers as APC for maximum reactivity, suggesting that the concentration of class II MHC antigens and other molecules affecting APC-T-cell interaction varied in mice with different genetic backgrounds. Regardless of MHC haplotype, responses to VKV 1024, which encompasses VKV 1023 and VKV 1022, were either absent or lower than those to VKV 1022, possibly reflecting differences in the processing requirements of these two proteins. When mice were primed intravenously with recombinant vaccinia virus and when their CD4+ T cells were stimulated in vitro with native Kunjin virus antigens, VKV 1031 primed more efficiently than Kunjin virus and VKV 1022 primed similarly to Kunjin virus.

Broad cross-reactivity with marked fine specificity in the cytotoxic T cell response to flaviviruses.

Cytotoxic T (Tc) cells were generated in mice of five H-2 haplotypes against the flaviviruses Kunjin and West Nile (WNV). A panel of recombinant vaccinia viruses which between them expressed cDNA of the entire Kunjin virus genome were used to infect targets. Anti-Kunjin virus responses to determinants derived from non-structural proteins, especially NS3, NS4A and NS4B, were dominant in most mouse strains; usually only one class I major histocompatibility complex (MHC) restriction element was involved. WNV-immune Tc cells showed similar but not identical patterns of antigen recognition to Kunjin virus-immune Tc cells. The extent to which WNV-immune Tc cells recognized Kunjin virus-encoded determinants varied considerably between mice of different MHC haplotypes.

Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin.

Kunjin virus-infected cells were lysed and the cytoplasmic extract was subjected to sedimentation analysis. After centrifugation at 16,000 x g for 10 min about 70% of the original RNA-dependent RNA polymerase (RDRP) was recovered in the pellet; most of this enzymic activity was recovered in the soluble fraction after treatment with NP40 detergent. Membrane fractions were prepared from cytoplasmic extracts by centrifugation in discontinuous density gradients comprising w/w or w/v sucrose solutions, either for 3 h (top-loaded on 4 ml 20-60% sucrose) or for 19 h (centre-loaded in 37 ml 0-60% sucrose). Similar separations of bands of light membranes were obtained in all gradients. Multi-layered heavy membrane bands obtained with w/w sucrose gradients were resolved into two well-separated bands (F4 and F5) using w/v sucrose gradients. Thin-section electron microscopy of embedded membrane fractions, gel analysis of intracellular RNA, and RDRP assays showed that the w/w centre loading method and the w/v top-loading (short spin) method produced similar recoveries and distributions of smooth and rough membranes, intact virus particles and RDRP activity. The distribution of intracellular viral RNA and proteins was coincident with the RDRP, all being located in the F4 and F5 bands which contained the characteristic membrane structures induced during flavivirus infection. Significant advantages of the preferred method (w/v sucrose, top loading and short spin) were its rapidity, good preservation of membranes and RDRP, and the concentrations of RDRP achieved in the small volume fractions collected from a total of 4.5 ml.

Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression.

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins.

A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5'-C.GP20.E.GP44.P19.P10.P71.(?).P21.P98-3' YF 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala decreases where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg decreases Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN greater than Murray Valley greater than Japanese encephalitis greater than St Louis encephalitis viruses within this closely related serological complex. A non-coding 5' sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3' sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.

Gene mapping and positive identification of the non-structural proteins NS2A, NS2B, NS3, NS4B and NS5 of the flavivirus Kunjin and their cleavage sites.

Partial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5'...GP44.P19.P10.P71.(?).P21.P98-3', YF 5'...NS1.ns2a.ns2b.NS3.ns4a.ns4b.NS5 -3'. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg decreased Gly, similar in form to the sequence Arg-Arg decreased Ser defined at the cleavage sites of YF NS3 and NS5. A new consensus cleavage site for NS1, NS2A and NS4B in the form Val-X-Ala decreased, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flavivirus sequences (a total of 18 sites). NS1 and NS4B, but not NS2A, were preceded by a putative signal sequence.

Particulate antigens may be reprocessed after initial phagocytosis for presentation to T cells in vivo.

Macrophages were pulsed with Listeria monocytogenes antigens by intraperitoneal injection prior to harvesting and thoroughly washing the cells. The pulsed macrophages were injected into the feet of Listeria-immune or naive mice to elicit a delayed hypersensitivity reaction. Where soluble Listeria antigen was used, presentation by donor macrophages to host T cells required identity within the I region of the H-2 complex. However, presentation of alcohol-killed Listeria organisms or of a living, temperature-sensitive mutant of Listeria was apparently not H-2 restricted. When T cells enriched in vitro for Listeria reactivity were injected into the feet of naive mice, they reacted in an H-2 restricted manner to antigen presented to them either by the pulsed macrophages or host cells apparently acquiring antigen from the original pulsed macrophages.