Plasma profiles obtained for the standard drug and test compounds used in a <i>T. evansi</i> experimental goat model of infection.

<p>Drug plasma concentrations of a) diminazene at 5 mg/kg (purple), b) DB 1192 at 2.5 mg/kg (orange) and 1.25 mg/kg (green), c) DB 75 at 2.5 mg/kg (orange) and 1.25 mg/kg (green) and d) DB 867 at 2.5 mg/kg (orange) and 1.25 mg/kg (green) are depicted. Horizontal red line shows ten-times the IC₅₀ for that respective compound.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using the polymerase chain reaction (PCR), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>The results are depicted as negative (−) or positive (+) for the presence of parasite DNA in goat blood samples, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using a card agglutination test (CATT/<i>T. evansi</i>), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>Results are depicted as strongly positive (+++), intermediate (++), weak (+) or negative (−) for agglutination, when goat plasma was mixed with an antigenic reagent, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of <i>E. coli</i> Cells with Two β-Domains of Opposite Topologies

<div><p>Screening of antibody (Ab) libraries by direct display on the surface of <i>E. coli</i> cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic <i>E. coli</i> O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or V_HH) on the surface of <i>E. coli</i> K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM_EHEC). We demonstrated that both systems displayed functional sdAbs on the surface of <i>E. coli</i> cells with little proteolysis and cellular toxicity, although <i>E. coli</i> cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both <i>E. coli</i> display libraries were screened for TirM_EHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirM_EHEC was demonstrated by flow cytometry of <i>E. coli</i> cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the <i>E. coli</i> cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.</p> </div

Pharmacokinetic parameters determined for a treatment schedule of four applications of diminazene (at 5 mg/kg), DB 75, DB 867 and DB 1192 (at 2.5 mg/kg and 1.25 mg/kg), based on average plasma concentration levels taken from <i>T. evansi</i> experimentally infected goats.

<p>Pharmacokinetic parameters determined for a treatment schedule of four applications of diminazene (at 5 mg/kg), DB 75, DB 867 and DB 1192 (at 2.5 mg/kg and 1.25 mg/kg), based on average plasma concentration levels taken from <i>T. evansi</i> experimentally infected goats.</p

Signs of acute toxicity investigated after the i.m. application of DB 75, DB 867 and DB 1192 in a multiple compound application (4×1 mg/kg) toxicity trial.

<p>The presence or absence of each sign is depicted as a (✓) or as a (x), respectively.</p><p>*<b>Signs of acute toxicity:</b> Hypo: <i>Hypotension</i>; Hyper: <i>Hypertension</i>; (L): <i>Lacrimation</i>; (S): <i>Salivation</i> (excess); (Tr): <i>Tremors</i>; (Ir): <i>Irritation</i>; (U): <i>Urination</i> (excess); (D): <i>Diarrhoea</i>.</p

A five month follow up of an efficacy study for a <i>T. evansi</i> experimentally infected goat model, using the haematocrit centrifugation technique (HCT), after treatment with Diminazene, DB 75, DB 867 or DB 1192 at various compound doses.

<p>The results are depicted as negative (−) or positive (+) for motile trypanosomes observed in goat blood samples, along with goats, which died (<i>D</i>) or were removed from the project (<i>Rem</i>) during the study period.</p

<i>E. coli</i> cell surface display of VHH sdAbs with EhaA and Intimin β-domains.

<p>(<b>A</b>) Scheme of EhaA autotransporter and VHHA fusions (left), showing N-terminal SP, secreted passenger or VHH domain, and C-terminal β-domain. Model of VHHA fusion in the OM (right), with N-terminal VHH domain exposed to the extracellular milieu and with C-EhaA β-barrel inserted in the OM. These domains are connected with the E-tag epitope and the internal α-helical linker of the β-barrel. (<b>B</b>) Scheme of Intimin and NVHH fusions (left), showing N-terminal SP, LysM and β-domains, and secreted D0-D3 Ig-like and lectin-like domains, or VHH domain replacing D1-D3 in NVHH fusions. Model of NVHH fusion in the OM (right), with N-terminal LysM domain in the periplasm, β-barrel with linker in the OM, and connecting with C-terminal D0 and VHH domains exposed to the extracellular milieu. The E-tag and myc-tag epitopes flanking the VHH domain are indicated. (<b>C</b>) and (<b>D</b>) Western blots of whole-cell protein extracts from induced <i>E. coli</i> UT5600 harbouring pVgfpA (C) or pNVgfp (D). Intact <i>E. coli</i> cells were incubated with (+) or without (-) the indicated protease, Trypsin or Proteinase-K (ProtK), before lysis. Protein extracts were prepared in SDS (C) or SDS-urea (D) sample buffers and boiled (+) or not boiled (-) before SDS-PAGE. Western blots were developed with anti-E or anti-myc mAb, as indicated. The positions of full-length VgfpA and NVgfp fusions are labeled with arrows. Asterisks indicate protein bands detected in protease-treated samples. The mass of protein markers (in kDa) is shown on the left.</p

<i>E. coli</i> cell surface display and antigen binding activity of VgfpA and NVgfp fusions.

<p>Fluorescent flow cytometry analysis of induced <i>E. coli</i> UT5600 cells bearing the indicated plasmids: pAK-Not (control), pVgfpA, and pNVgfp. Histograms show the fluorescence intensity of bacteria stained with anti-E or anti-myc mAbs (as indicated) and secondary anti-mouse IgG-Alexa 488 (left panels) or incubated with biotinylated antigens (GFP or BSA, as labeled) and secondary Streptavidin-phycoerythrin (PE) (right panels).</p

Binding to TirM_EHEC by <i>E. coli</i> cells displaying selected clones from VHHA and NVHH libraries.

<p>Fluorescent flow cytometry analysis of induced <i>E. coli</i> EcM1 cells bearing the indicated plasmids selected from (<b>A</b>) the VHHA library: pVTIR1A, pVTIR2A, pVTIR3A; and (<b>B</b>) from the NVHH library: pNVTIR1, pNVTIR4, pNVTIR5. Histograms show the fluorescence intensity of bacteria incubated with biotinylated antigens (TirM_EHEC or BSA, as labeled) and secondary Streptavidin-PE.</p