Production, purification, and quality assessment of borrelial proteins CspZ from Borrelia burgdorferi and FhbA from Borrelia hermsii.

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH15-20 and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1.

The functionality of a therapeutic antibody candidate restored by a single mutation from proline to threonine in the variable region.

mAbs play an essential role in the therapeutic arsenal. Our laboratory has patented the Rendomab-B49 mAb targeting the endothelin B receptor (ETB). This G protein-coupled receptor plays a driving role in the progression of numerous cancers. We chimerized our mAb (xiRB49) to evaluate its preclinical therapeutic efficacy in different ETB+ tumor models with an antibody drug conjugate approach. As previously reported, the chimerization process of an antibody can alter its functionality. In this article, we present the chimerization of RB49. xiRB49 purified by Protein A remained perfectly soluble and did not aggregate, but it lost all its ability to recognize ETB. A detailed analysis of its variable region using IMGT tools allowed us to identify an unusual proline at position 125. In silico mAb modeling and in vitro experiments were performed for a better understanding of xiRB49 structure-function relationships. Our results show that the proline in position 125 on the heavy chain alters the xiRB49 CDR3 light chain conformation and its mutation to threonine allows complete functional recovery.

Comparative Study of Single-stranded Oligonucleotides Secondary Structure Prediction Tools.

BACKGROUND: Single-stranded nucleic acids (ssNAs) have important biological roles and a high biotechnological potential linked to their ability to bind to numerous molecular targets. This depends on the different spatial conformations they can assume. The first level of ssNAs spatial organisation corresponds to their base pairs pattern, i.e. their secondary structure. Many computational tools have been developed to predict the ssNAs secondary structures, making the choice of the appropriate tool difficult, and an up-to-date guide on the limits and applicability of current secondary structure prediction tools is missing. Therefore, we performed a comparative study of the performances of 9 freely available tools (mfold, RNAfold, CentroidFold, CONTRAfold, MC-Fold, LinearFold, UFold, SPOT-RNA, and MXfold2) on a dataset of 538 ssNAs with known experimental secondary structure. RESULTS: The minimum free energy-based tools, namely mfold and RNAfold, and some tools based on artificial intelligence, namely CONTRAfold and MXfold2, provided the best results, with [Formula: see text] of exact predictions, whilst MC-fold seemed to be the worst performing tool, with only [Formula: see text] of exact predictions. In addition, UFold and SPOT-RNA are the only options for pseudoknots prediction. Including in the analysis of mfold and RNAfold results 5-10 suboptimal solutions further improved the performances of these tools. Nevertheless, we could observe issues in predicting particular motifs, such as multiple-ways junctions and mini-dumbbells, or the ssNAs whose structure has been determined in complex with a protein. In addition, our benchmark shows that some effort has to be paid for ssDNA secondary structure predictions. CONCLUSIONS: In general, Mfold, RNAfold, and MXfold2 seem to currently be the best choice for the ssNAs secondary structure prediction, although they still show some limits linked to specific structural motifs. Nevertheless, actual trends suggest that artificial intelligence has a high potential to overcome these remaining issues, for example the recently developed UFold and SPOT-RNA have a high success rate in predicting pseudoknots.

Lyme borreliosis diagnosis: state of the art of improvements and innovations.

With almost 700 000 estimated cases each year in the United States and Europe, Lyme borreliosis (LB), also called Lyme disease, is the most common tick-borne illness in the world. Transmitted by ticks of the genus Ixodes and caused by bacteria Borrelia burgdorferi sensu lato, LB occurs with various symptoms, such as erythema migrans, which is characteristic, whereas others involve blurred clinical features such as fatigue, headaches, arthralgia, and myalgia. The diagnosis of Lyme borreliosis, based on a standard two-tiered serology, is the subject of many debates and controversies, since it relies on an indirect approach which suffers from a low sensitivity depending on the stage of the disease. Above all, early detection of the disease raises some issues. Inappropriate diagnosis of Lyme borreliosis leads to therapeutic wandering, inducing potential chronic infection with a strong antibody response that fails to clear the infection. Early and proper detection of Lyme disease is essential to propose an adequate treatment to patients and avoid the persistence of the pathogen. This review presents the available tests, with an emphasis on the improvements of the current diagnosis, the innovative methods and ideas which, ultimately, will allow more precise detection of LB.

Genetic background and immunological status influence B cell repertoire diversity in mice.

The relationship between the immune repertoire and the physiopathological status of individuals is essential to apprehend the genesis and the evolution of numerous pathologies. Nevertheless, the methodological approaches to understand these complex interactions are challenging. We performed a study evaluating the diversity harbored by different immune repertoires as a function of their physiopathological status. In this study, we base our analysis on a murine scFv library previously described and representing four different immune repertoires: i) healthy and naïve, ii) healthy and immunized, iii) autoimmune prone and naïve, and iv) autoimmune prone and immunized. This library, 2.6 × 109 in size, is submitted to high throughput sequencing (Next Generation Sequencing, NGS) in order to analyze the gene subgroups encoding for immunoglobulins. A comparative study of the distribution of immunoglobulin gene subgroups present in the four libraries has revealed shifts in the B cell repertoire originating from differences in genetic background and immunological status of mice.

Multitarget selection of catalytic antibodies with ß-lactamase activity using phage display.

ß-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of ß-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 109 with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM ß-lactamase as targets for selection of catalytic antibodies with ß-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the ß-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with ß-lactamase activity, and (b) the plasticity of the ß-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.

Generation of Catalytic Antibodies Is an Intrinsic Property of an Individual's Immune System: A Study on a Large Cohort of Renal Transplant Patients.

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the current study, we have followed the evolution of the levels of catalytic IgG in a large cohort of renal transplant patients over a 2-y period. Our results demonstrate that, prior to transplant, patients with renal failure present with heterogeneous levels of IgG hydrolyzing the generic proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA) substrate. PFR-MCA hydrolysis was greater for patients' IgG than for a therapeutic preparation of pooled IgG from healthy donors. Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 mo followed by a steady increase during the next 21 mo. Patients who displayed high levels of catalytic IgG pretransplant recovered high levels of catalytic Abs 2 y posttransplant. Interestingly, IgG-mediated hydrolysis of a model protein substrate, procoagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, whereas it did 12 mo posttransplant. Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual's immune system and that recovery of pretransplant levels of catalytic IgG is accompanied by changes in the repertoire of target Ags.

Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library.

Catalytic antibodies and their applications in biotechnology: state of the art.

Catalytic antibodies are immunoglobulins endowed with enzymatic properties. Discovered in the second part of the 1980s, the enthusiasm they initially aroused was counterbalanced by the difficulty of their production and their low catalytic rates. Nevertheless, improvements in expression systems and engineering technologies, combined with various studies suggesting that catalytic antibodies play a role in the immune system, have opened the way to new applications for these proteins. Herein we review catalytic antibodies from a biotechnological point of view, focusing our study on the different production methods, expression systems and their potential clinical applications dedicated to these proteins.

IVIg treatment reduces catalytic antibody titers of renal transplanted patients.

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.

Efficient refolding of a recombinant abzyme : structural and catalytic characterizations.

Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure-function relationships is of great interest. Recombinant scFv fragments are smaller and easier to genetically manipulate than whole antibodies, making them well suited for this kind of study. Nevertheless they are often described as proteins being laborious to produce. This paper describes a highly efficient method to produce large quantities of refolded soluble catalytic scFv. For the first time, the functionality of a refolded catalytic scFv displaying a ß-lactamase activity has been validated by three approaches: (1) use of circular dichroism to ensure that the refolded had secondary structure consistent with a native scFv fold, (2) development of enzyme-linked immunosorbant assay and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (3) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate.

A novel molecular analysis of genes encoding catalytic antibodies.

Among the numerous questions remaining opened about catalytic antibodies (abzymes), the understanding of the origin of the genes encoding them is of vital significance. An original statistical analysis of genes encoding abzymes is described in the present report. Results suggested that these genes display a high conservation degree with their germline counterpart and a limited number of amino acid changes. Hence, on the contrary with high-affinity antibodies, maturation process by accumulation of somatic hypermutations is not required for the catalytic function. We demonstrated that despite a weak somatic mutation rate, the physicochemical properties of mutated amino acid (AA) are predominantly dissimilar with that of the germline AA. Further, we developed a novel approach in order to analyze the nature of genes encoding catalytic antibodies. For the first time, an unexpected and significant high level expression of rare gene subgroups was noticed and emphasized. The data described in this paper would lay the foundation for future studies about origin of genes encoding catalytic antibodies.

Do proteolytic antibodies complete the panoply of the autoimmune response in acquired haemophilia A?

Acquired haemophilia A (AHA) is a rare bleeding disorder characterized by the sudden generation of autoantibodies against factor VIII (FVIII) in individuals with no previous history of abnormal haemostasis. Understanding the pathogenesis of this disease has been hampered by the rarity of the patients and the difficulty in obtaining biological material from untreated patients. Still, progress has been made recently in understanding the pathogenesis of AHA. In particular, the importance of CD4(+) T cells in AHA development has been documented and the epitopes targeted by T cells on FVIII have been delineated. Accordingly, a polymorphism in the cytotoxic T-lymphocyte-associated protein 4 gene (CTLA4), known to participate in the regulation of CD4(+) T-cell responses, and a preferential usage of certain human leukocyte antigen class II haplotypes, have been associated with the disease. Recent findings have documented the presence of immunoglobulin G (IgG) with proteolytic activity against FVIII and factor IX (FIX) in patients with AHA. While FVIII-hydrolysing IgG has been shown to inactivate FVIII, FIX-hydrolysing IgG from AHA patients activate FIX in vitro. Here, we describe the latest findings on the immuno-pathogenesis of AHA, with a special focus on the potential role played by antibodies endowed with proteolytic properties.

Novel peptide inhibiting both TEM-1 ß-lactamase and penicillin-binding proteins.

9G4H9, a catalytic antibody displaying ß-lactamase-like activity, has been developed by the anti-idiotypic approach using ß-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of ß-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 ß-lactamase (K(i) = 333 µm) and several penicillin-binding proteins (IC50 values ranging from 6-62 µm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 ß-lactamase.

Peptide mass-assisted antibody cloning strategy for accurate characterization of potential therapeutic monoclonal antibodies against neurodegenerative diseases.

The development of therapeutic recombinant antibodies involves accurate characterization of immunoglobulin variable light (VL) and heavy (VH) chains. However, it has been reported that the use of subgroup or isotype-specific primers for the amplification of monoclonal antibody (mAb) variable domains introduces heterogeneities within the variable domains, or amplifies aberrant productive Ig domains. To address these issues, we have combined the rapid amplification of cDNA ends PCR (RACE-PCR) for the full-length VL and VH amplification, with peptide mass fingerprinting of the corresponding Ig chain. Using this strategy, we amplified full-length cDNA chains of SAF34 and SAF32, two potential therapeutic mAbs against neurodegenerative diseases directed to the prion protein (PrP). We report an unambiguous correlation between hybridoma cDNA sequences and protein fingerprints of the variable domains of each mAb, indicating the discrimination between mutated, pseudo-genes and functional Ig genes. As a proof of principle for this dual strategy of full-length PCR amplification of variable domains and their characterization by MALDI-TOF, we show that the corresponding scFvs recognize the native PrP and retain full capacity to bind to human PrP, as does the parental mAb. This finding addresses the need for reliable light and heavy chain characterization, a key factor for humanization of mouse antibodies and for its use in passive immunotherapy applications.

Mutational and inhibitory analysis of a catalytic antibody. Implication for drug discovery.

Data on catalytic antibodies (abzymes) having critical roles in pathologies, for example in some auto-immune diseases accumulate at overwhelming pace. Nevertheless, the misunderstanding of how antibodies can mimic a catalytic activity may hamper the development of therapeutic tools. We thus investigated the structure function roles of residues of a catalytic antibody endowed with a beta-lactamase activity. The catalytic antibody 9G4H9 was generated using the internal image properties of anti-idiotypic antibodies. The single-chain fragment was cloned and produced in Escherichia coli. Based on the structure function knowledge of beta-lactamases pattern and on the tridimensional model of the scFv, five residues were selected for mutagenic analysis to learn about the contribution of putative residues in catalysis. Light chain mutants R24A, S26A, S28A, and E98A lost catalytic activity significantly. Mutant K27A retained catalytic activity but the estimated K(M) was affected. Kinetic outcomes support the argument that S26 and S28 function as nucleophile and E98 as general acid/base catalyst. We have selected a peptide Pep90 able to inhibit 9G4H9 catalytic activity. We also demonstrate the tryptophan and proline residues of Pep90 (YHFLWGP) are responsible for binding and inhibitory properties of Pep90 on 9G4H9. A three-dimensional model docked with Pep90 is therefore built in which critical residues of Pep90 are buried at the interface of CDR-L1 and CDR-L3 loops whereas other are exposed to the solvent.

Expression and detection strategies for an scFv fragment retaining the same high affinity than Fab and whole antibody: Implications for therapeutic use in prion diseases.

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.

Expression of a functional scFv fragment of an anti-idiotypic antibody with a beta-lactam hydrolytic activity.

The single chain variable fragment (scFv) of an anti-idiotypic catalytic monoclonal antibody, 9G4H9, displaying a beta-lactamase-like activity was cloned. The recombinant protein was expressed through the periplasm in Escherichia coli in the presence or in the absence of FkpA, a chaperone-like enzyme and tested for its hydrolytic activity. The results show that the catalytic parameters for hydrolysis of ampicillin by scFv9G4H9 are clearly influenced by the presence of FkpA, indicating that the correct folding of the fragment represents a crucial step for catalysis.