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The use of immunogenic formulations targeting pattern recognition receptors towards modulation of immune responses is a promising strategy to develop better vaccines against infectious and malignant diseases. Molecules targeting TLR2 offer interesting properties that are relevant for vaccine development, including the possibility to covalently attach the lipidic ligands to the antigens. However, the type of immune response elicited by these formulations is still controversial. In this work, we used the model antigen ovalbumin (OVA) expressed in fusion with the bacterial lipoprotein OprI in order to characterize the immunomodulatory properties of this TLR ligand. Murine bone marrow-derived dendritic cells stimulated with OprI-OVA fusion lipoprotein produced high levels of the pro-inflammatory cytokines TNF-α and IL-6 and also IL-10, IL-12(p70) and IL-27, while TGF-β and IL-23 were not detected. Using OT-II and OT-I mice, an enhancement of MHC class II and class I antigen presentation was observed for the OVA antigen in fusion with OprI. Mice immunized by intraperitoneal route with this fusion lipoprotein in prime-boost protocols developed strong specific antibody responses including IgG1, IgG2c, IgG2b, IgG3 and IgE. These results, together with data obtained by restimulation of splenocytes from the immunized mice, point to an immune response profile that does not correspond to a strict Th1 or Th2 polarization. Finally, in a challenge experiment using a melanoma syngeneic mouse model (B16-OVA), prophylactic inoculation with OprI fused with the unrelated antigen eGFP increased the tumor growth, while the fusion with the tumor-associated antigen OVA delayed the tumor growth and increased mice survival., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity., (Copyright © 2011 Elsevier B.V. All rights reserved.)