Caught in the act: in vivo development of macrolide resistance to Campylobacter jejuni infection.

We report the first instance of macrolide resistance developing in vivo following appropriate antibiotic use in a healthy volunteer as a part of a controlled human infection with Campylobacter jejuni. In vivo development of macrolide resistance may be an important contributor to antibiotic resistance in C. jejuni.

Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers.

In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 x 10(8) or 4 x 10(9)cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 x 10(9)cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p<0.001) and 0.82 (p<0.001), respectively after challenge and 0.78 (p<0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p<0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on "CFA agar," which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 x 10(7) to 5 x 10(9) CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates.

Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2.

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic; interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because, surprisingly, the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms.

Determination of binding constants of diabodies directed against prostate-specific antigen using electrochemiluminescence-based immunoassays.

Two diabody molecules directed against the prostate-specific antigen (PSA) were generated from a combinatorial phage display library. The C-termini of diabodies incorporated the FLAG peptide epitope (P008D diabody) or the myc epitope (P001D). Both diabodies have the same antigen-binding site. Equilibrium-binding constants of these molecules were determined in two immunoassays using the ORIGEN detection system based on electrochemiluminescence. The binding of diabodies to biotinylated PSA was detected with either polyclonal antimouse Fab F(ab')2 or a monoclonal antibody directed against the FLAG epitope. Both detecting antibody preparations were covalently labeled with a ruthenium (II) tris (bipyridyl) moiety, Ru(bpy)3(2+), which allows quantification via an electrochemically triggered light reaction in an ORIGEN analyzer. The binding constants obtained by Scatchard analysis of non-linear curve fitting calculations from electrochemiluminescence immunoassays were compared with data derived from kinetic-binding studies using the BIAcore technology based on surface plasmon resonance. Depending on the detecting antibody, the dissociation constants KD determined at equilibrium with the ORIGEN technology are between 0.1 and 0.4 nM for both diabodies. From the kinetic constants kon and koff measured with the BIAcore instrument KD was calculated to be 0.2 nM for P001D and 0.6 nM for P008D. It is concluded that these two very different methods generate comparable affinity data for the diabodies.

Screening and kinetic analysis of recombinant anti-CEA antibody fragments.

Four different carcinoembryonic antigen (CEA)-binding antibody fragments were prepared using the genes of the variable regions of the T84 epitope-specific antibody 7F7 and phage display techniques. The genes were successfully cloned and expressed in the pCANTAB5 phage display vector to investigate the kinetic binding parameters of each synthesized construct. Single chain fragments, Fab fragments, and two diabodies were purified and compared in their CEA-binding properties with the parent IgG using surface plasmon resonance detection. The on-rates for all these molecules were in the same order of magnitude (about 1 x 10(5) M-1 s-1) whereas major differences were detected in the off-rates. IgG and diabodies had slow off-rates due to bivalent binding, while single chain and Fab fragments dissociated rather fast. We also present a method for the immobilization of large amounts of CEA on CM5 sensorchips. These high density surfaces can be used for observing mass transport limited binding of CEA-specific molecules and are convenient tools for screening and quality control.

Spectrophotometric quantitation of silver in X-ray film images and silver-stained electrophoresis gel bands.

This paper describes a novel spectrophotometric method for quantitating the silver in silver-stained electrophoresis gel bands and X-ray film images. Silver in excised areas is quantitated by (i) oxidation/solubilization of Ag0 to Ag+ with nitric acid, (ii) neutralization followed by extraction into an organic solution of dithizone (diphenylthiocarbazone), and (iii) visible absorbance measurement at 600 nm. Spectrophotometric quantitation is possible because dithizone undergoes a dramatic color change from blue-green to yellow upon binding Ag+ (delta epsilon 600 = 3.28 x 10(4) M-1 cm-1). Experiments with silver-stained gels and with X-ray film images obtained by chemiluminescence and autoradiography demonstrated that the color change relates to the amount of silver present and hence to the amount of material represented by the band or image. This method is a convenient, inexpensive alternative to the use of a densitometer to quantitate silver-containing images.

Aspects of antibody-catalyzed primary amide hydrolysis.

Because there are many known C-terminally amidated peptides of biological importance, there is great potential in medicine and organic synthesis for antibodies that catalyze primary amide bond hydrolysis or formation. We characterized a catalytic antibody, 13D11, raised to a phosphinate hapten, that hydrolyzed the primary amide of a dansyl-alkylated derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a kcat of 1.65 x 10(-7) s-1 (kcat/kuncat = 132) and a Km of 432 microM, and was stereospecifically hapten-inhibited (Ki = 14.0 microM). Control experiments indicated that the catalytic activity was not the result of a contaminating protease. In accordance with the hapten being a transition-state analog of base hydrolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity declined rapidly suggesting the antibody was inactivated during the long incubation period. This work demonstrates the feasibility of generating catalytic antibodies to hydrolyze unactivated amide bonds without cofactor assistance.

Conjugates of COL-1 monoclonal antibody and beta-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug beta-D-galactosyl-5-fluorouridine.

5'-O-beta-D-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme beta-D-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC50 of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500:1-1000:1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of beta-D-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).

Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization.

Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies. Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.

Cytotoxic T lymphocyte recognition of hybrid MHC class I molecules.

We have utilized a group of MHC class I genes produced by in vitro recombination between Dp and Dd to study recognition of MHC class I molecules by cytolytic T cells (CTLs). Both polyclonal allo-specific and H-2-restricted CTLs require that alpha 1 and alpha 2 of the target class I molecule be derived from the same haplotype for efficient killing. By using T-cell lines we showed that within the bulk population there must exist a fraction of T cells which can recognize epitopes in alpha 1 or alpha 2. Critical residues for T-cell recognition have been identified using these chimeric genes.

Interaction of an immunodominant epitope with Ia molecules in T-cell activation.

The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis of the T-cell proliferative response induced by synthetic peptides covering almost the entire HEL sequence. All the T-cell hybridomas from H-2d mice analyzed recognize the HEL sequence 107-116 in association with the I-Ed molecule. Correlating with the restriction of T-cell recognition, HEL-(105-120)-peptide binds to I-Ed but not to I-Ad molecules. Conservative or semiconservative substitutions at positions 113 (Asn----Lys), 114 (Arg----His), or 115 (Cys----Ala) abrogate the ability of HEL-(105-120) to activate T cells. Substitutions at residues 113 and 115 affect T-cell recognition but not the binding to I-Ed molecules, whereas, as shown by binding data and competition experiments, an Arg----His substitution at position 114 profoundly impairs the capacity of the peptide to interact with I-Ed molecules. In agreement with these results, [Lys113]HEL-(105-120)-peptide but not [His114]HEL-(105-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity.

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved.

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains.

Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.

Three distinct epitopes within the loop region of hen egg lysozyme defined with monoclonal antibodies.

Five monoclonal antibodies specific for the loop region of hen egg lysozyme were prepared by immunisation with a synthetic conjugate of a proteolytic fragment of lysozyme coupled to bovine serum albumin. Their fine specificities were investigated using a panel of variant lysozymes and peptide fragments of lysozyme in a quantitative radio-immunoassay procedure. Knowledge of the structure of hen lysozyme to high resolution and the use of computer graphics enables the localisation of the epitopes recognised by the antibodies with some precision. The antibodies were shown to define three distinct, overlapping epitopes within what was previously considered to be a single antigenic site. These results are discussed in relation to current ideas of the antigenic nature of proteins and other recent studies in which anti-protein antibodies have been elicited by immunisation with small peptides.

Nucleotide sequences of five anti-lysozyme monoclonal antibodies.

The nucleotide sequences of the heavy and light chain immunoglobulin mRNAs derived from five hybridomas (Gloop 1-5) secreting IgGs specific for the loop region of hen egg lysozyme were determined. These monoclonal antibodies recognise three distinct but overlapping epitopes within the loop region. The sequences of two pairs of antibodies with indistinguishable fine specificities were similar in both chains whereas the sequences of antibodies of non-identical specificities were very different. It is proposed that the D-segments expressed in two of the antibodies (Gloop3 and Gloop4) are the products of one, or perhaps two, previously unidentified germ line D-genes. Gloop1 and Gloop2 use a D-segment previously identified in antibodies specific for the hapten 2-phenyloxazolone; however it is recombined in a different reading frame in the anti-lysozyme antibodies, producing a different amino acid sequence.