Jean-Claude Mani

Functional Specific Binding of Testosterone to Schistosoma haematobium 28-Kilodalton Glutathione S-Transferase

ABSTRACT During parasitic disease such as schistosomiasis, sex hormones have an important influence on the age- and gender-dependent level of infection. Since mammal glutathione S-transferase (GST) has the ability to bind hormones and particularly sexual steroids to influence their transport, metabolism, and physiological action, we have evaluated the capacity of testosterone to bind the 28-kDa GST of the Schistosoma haematobium parasite (Sh28GST). For the first time, we have demonstrated a specific binding of testosterone to parasite GST protein with high affinity (Kd = 2.57 × 10−7 M). In addition, we have assessed the effect of this binding on Sh28GST enzymatic activity, a mechanism closely associated with the reduction of Schistosoma fecundity. We showed that testosterone has the functional ability to inhibit the Sh28GST enzymatic activity in a dose-dependent manner, suggesting that this hormone could be directly involved in an antifecundity mechanism. This effect seemed to be related to the binding of testosterone to one peptide involved in the enzymatic site (i.e., amino acids 24 to 43). During human infection, binding of sexual hormones to Schistosoma Sh28GST could play a key role in parasite metabolism, especially the decrease of fecundity, and could be involved in the sex-dependent immune response to Sh28GST that we have previously observed in infected adults.

Human Anti-Thyroid Peroxidase Single-Chain Fragment Variable of Ig Isolated from a Combinatorial Library Assembled In-Cell: Insights into the In Vivo Situation

In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves’ disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vλ1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves’ disease patients tested were able to strongly inhibit (60–100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.

Monoclonal Antibody 667 Recognizes the Variable Region A Motif of the Ecotropic Retrovirus CasBrE Envelope Glycoprotein and Inhibits Env Binding to the Viral Receptor

ABSTRACT Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.