Studies on the synergistic protective activity of cancer antigens associated to engineered bacterial Outer Membrane Vesicles

Introduction Bacterial Outer Membrane Vesicles (OMVs) are naturally produced by all Gram-negative bacteria and are emerging as an attractive vaccine platform. The exploitation of OMVs in cancer immunotherapy was tested by decorating them with cancer epitopes and by following their capacity to elicit protective immune responses, alone or in combination, using cancer mouse models. Furthermore, we tested whether cancer cell-derived exosomes (TEXs) and OMVs can form complexes and whether such complexes, which carry TEX- associated tumor antigens and OMV-associated immune-stimulatory molecules, elicit anti-tumor immune responses in vivo. Results Immunization with OMVs engineered with two B cell epitopes (EGFRvIII and D8-mFAT1) induced tumor growth inhibition after mouse challenge with cell lines expressing the corresponding epitopes. Furthermore, mice immunized with engineered OMVs carrying two cancer epitopes, the EGFRvIII B cell epitope and the M30 CD4+ T cell epitope, were completely protected from EGFRvIIIB16F10 cell line expressing both epitopes, indicating the importance of multi-antigen immunization in cancer immunotherapy. Furthermore TEXs- OMVs immunization induced antibody responses against exosome antigens with a Th1-type profile. Finally, the combination of CT26-derived TEXs and MBP-D8-mFAT1 OMVs elicited synergistic protective activity against mouse challenge with CT26 cell line. Conclusions OMVs decorated with tumor antigens elicit antigen-specific, protective anti- tumor responses in mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs, either by direct OMV engineering or by TEX-OMV combination, demonstrates the attractiveness of the OMV platform in cancer immunotherapy

Flow cytometry as an integrative method for the evaluation of vaccine immunogenicity: A validation approach

The applied bioanalytical assays used for the evaluation of human immune responses from samples collected during clinical trials must be well characterized, fully validated and properly documented to provide reliable results. Even though recommendations for the standardization of flow cytometry instrumentation and assay validation for its clinical application have been published by several organizations, definitive guidelines are not available yet. The aim of the present paper is to provide a validation approach for flow cytometry, examining parameters such as linearity, relative accuracy, repeatability, intermediate precision, range and detection limits and specificity, in order to demonstrate and document its applicability for clinical research purposes and its possible use as one of the methods for the evaluation of vaccine immunogenicity

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides