Infection of mice with the S. Typhimurium SL1344 and S. Typhimurium (pHCM1) strains.

Survival rate (A), body weight evolution (B), disease index (C) and proinflammatory cytokine expression in spleen, Il-6 (D), Il-1β (E) and Tnf-α (F). Groups treated with ampicillin (Amp) are indicated with +Amp. In panel (A), survival rate of control animals and those infected with the S. Typhimurium SL1344 (WT) strain and treated with Amp, are 100%. Animals infected with the S. Typhimurium (pHCM1) strain have the same survival rate independently of the treatment with Amp. Results are expressed as mean ± SEM (n= 6-8 animals). Means without a common letter differ, P<0.05.

Effect of immunization with the RSP protein on body weight evolution (A) and immunoglobulin concentration (B-I).

Open bars represent non-immunized (No-Imm) mice; solid bars represent immunized (Imm) mice. Results are expressed as mean ± SEM (n= 6-8 animals). IgA, immunoglobulin A; IgG, immunoglobulin G; nd, non-detected. Means without a common letter differ, P<0.05.

Survival rate (A), body weight change (B) and clinical signs (C) after challenge with the SL1344 (pHCM1) strain.

Open bars/plots represent non-immunized (No-Imm) mice; solid bars/plots represent immunized (Imm) mice. In panel B, results are expressed as mean ± SEM (n= 12-14 animals). Means without a common letter differ, P<0.05.

Immunoglobulin concentration and cytokine expression in the gastrointestinal tract after RSP immunization and challenge with Salmonella.

Total IgA concentration in jejunum content (A), in colon content (B) and in feces (C). RSP-specific IgA titers in jejunum content (D), in colon content (E) and in feces (F). Il-1β (G), Il-6 (H) and Tnf-α (I) expression in colon mucosa. Open bars represent non-immunized (Non-Imm) mice; solid bars represent immunized (Imm) mice. Results are expressed as means ± SEMs (n= 10–12 animals). Means without a common letter differ, P<0.05. IgA, immunoglobulin A; IgG, immunoglobulin G; Il, interleukin; Int, interaction between both factors; Tnf-α, tumor necrosis factor alpha.

Immunoglobulin concentration, spleen weight, Salmonella counts in spleen tissue and cytokine expression in spleen tissue after immunization with the RSP protein and challenge with the SL1344 (pHCM1) strain.

Total IgG concentration (A) and RSP-specific IgG titers (B) in plasma. Spleen weight (C) and Salmonella counts in spleen tissue (D). Il-1β (E), Il-6 (F) and Tnf-α (G) expression in spleen tissue. Results are expressed as means ± SEMs (n= 12-14 animals). Means without a common letter differ, P<0.05. IgA, immunoglobulin A; IgG, immunoglobulin G; Il, interleukin; nd, non-detected; Tnf-α, tumor necrosis factor alpha.

Selection of RSP-binding Nbs from immune libraries displayed on the surface of E. coli.

(A) Flow cytometry analysis of the enrichment of bacterial population displaying Nb libraries generated after the immunization of dromedaries with the C-terminal domain of the RSP protein. 3 rounds of selection with the RSP protein by magnetic cell sorting (MACS) and one round of fluorescence activated cell sorting (FACS) were performed, in which bacteria were incubated with 50 nM of biotin-labeled RSP (or 100 nM of fibrinogen used as a specificity control) and stained with Streptavidin-APC. (B) Flow cytometry of the individual bacterial clones selected from the immune library. The bacterial surface display of the corresponding nanobody was detected using an anti-c-myc monoclonal antibody. The binding of biotin-labeled antigens to Nbs displayed on bacteria was performed with the incubation of bacterial cells with 50 nM of biotin-labeled RSP.

Analysis of the interaction of the VHH-RSP#3 isolated in the immune library with the native RSP protein at a macro and microscopic level.

(A) Assay of the ability of E. coli DH10BT1R cells expressing nanobodies on its surface to agglutinate cells of the S. Typhimurium SL1344 (R27) strain. Blue arrow marks the aggregation of bacteria after the interaction of E. coli cells producing nanobodies directed against the RSP protein (VHH-RSP #3) and Salmonella cells harboring the R27 plasmid. (B) Transmission electron microscopy imaging mixtures of E. coli DH10BT1R cells expressing nanobodies against the RSP protein on its surface and cells of the S. Typhimurium SL1344 strain, harboring or not the R27 plasmid. The studies were performed by labeling the nanobodies producing bacteria (E. coli DH10BT1R) with a mouse antibody anti c-myc tag and goat anti-mouse IgG conjugated to 12 nm gold particles. Blue bars represent 1 μm while black bars represent 2 μm.

Characterization of the purified VHH-RSP#3 as a specific binder to the RSP protein.

(A) Purification of Nb3-Fc by affinity column. Coomassie staining of the SDS-PAGE (10%) showing the purified Nb-RSP #3-Fc from transfected mammalian cell culture supernatants. Molecular weight markers are indicated on the left. (B) ELISA showing the binding capacity of the purified VHH-RSP#3-Fc to the RSP or BSA protein. The plot represents the OD values at 490 nm obtained with the indicated concentrations of the purified Nb3-Fc after 1 hour of interaction with the corresponding antigen.

Interference of E. coli DH10BT1R cells expressing nanobodies on its surface with the conjugative transfer of the R27 plasmid in Salmonella.

R27 plasmid was conjugated from a donor strain (SL1344) to a recipient strain (SL1344 ibpA::lacZ-Kmr) with the absence (labelled as “none”) or presence of the interferent strains E. coli DH10BT1R, plasmid-free clone; E. coli DH10BT1R (pNVFib1), clone expressing a fibrinogen-specific VHH; E. coli DH10BT1R (pNeae2 VHH-RSP #3), clone expressing RSP-specific VHH. Means without a common letter differ, P<0.05.