CONICET | Buscador de Institutos y Recursos Humanos

Camelids (camels, dromedaries, llamas) produce unusual antibodies composed only of heavy chains. The antigen combining site of these antibodies is formed solely by the heavy-chain variable domain (VHH). These VHH domains are easily produced as recombinant proteins and show similar antigen binding affinity than their parent antibodies. Their CDR3s form long finger-like extensions that can protrude into cavities on antigens, e.g. the active site crevice of enzymes. Thus VHHs are a suitable fragment for the development of enzyme inhibitors. Salmonella enterica are intracellular bacteria that are pathogenic for humans and animals, causing gastroenteritis and typhoid fever. These bacteria express an enzyme called SpvB, which is essential for Salmonella virulence. SpvB catalyzes the transference of the ADP-ribose moiety from NAD to actin, causing the depolymerization of actin filaments and cell death. To generate VHHs with inhibitory properties against SpvB, we have isolated cDNA from lymphocytes of llamas immunized with SpvB, obtaining a VHH library of 4x107 transformants. Phage display allowed an enrichment of binders through consecutive rounds of panning. Clones recognizing the antigen were sequenced and classified according to the length and variability of their CDR3s. Five independent clones were isolated, and the VHH proteins codified by them were expressed and purified. All VHHs were able to inhibit SpvB in enzymatic tests using radioactive probes. We quantitatively analyzed the inhibition of SpvB activity by fluorescence using pyrene-labelled actin as substrate. For each test, actin-pyrene, cold NAD, and both pure SpvB and the four VHHs were used. The test showed strong inhibition for the clones named VHH5 and VHH6. Furthermore, VHH5 was subcloned in a vector for eukaryotic expression. As such, eukaryotic cells were transfected and cells expressing the VHHs were infected with Salmonella enterica to test their effect on virulence. Fluorescence microscopy analyses clearly show that VHH5 is able to inhibit in vivo the ADP-ribosylation of actin by SpvB. This result constitutes a proof of principle of the use of single domain antibodies for the specific intracellular inhibition of normal or pathogenic enzymatic functions.