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We describe a novel live oral vaccine type. Conceptually, this vaccine is based on a non-lytic, recombinant filamentous bacteriophage that displays an antigen of interest. To provide proof of concept we used the amino-terminal part of a conserved influenza A virus epitope, i.e. matrix protein 2 ectodomain (M2e) residues 2 to 16, as the antigen of interest. Rather than using the phages as purified virus-like particles as a vaccine, these phages were delivered to intestinal Peyer's patches as a live bacterium-phage combination that comprises Escherichia coli cells that conditionally express invasin derived from Yersinia pseudotuberculosis. Invasin-expressing E. coli cells were internalized by mammalian Hep-2 cells in vitro and adhered to mouse intestinal microfold (M) cells ex vivo. Invasin-expressing E. coli cells were permissive for recombinant filamentous bacteriophage f88 that displays M2e and became persistently infected. Oral administration of the live engineered E. coli-invasin-phage combination to mice induced M2e-specific serum IgG antibodies. Mice that had been immunized with invasin-expressing E. coli cells that carried M2e2-16 displaying fd phages seroconverted to M2e and showed partial protection against challenge with influenza A virus. Oral delivery of a live vaccine comprising a bacterial host that is targeted to Peyer's patches and is persistently infected with an antigen-displaying phage, can thus be exploited as an oral vaccine., (Copyright © 2017. Published by Elsevier B.V.)

In this work a combination therapy that acts upon the immune suppressive, the innate and specific arms of the immune system is proposed. This combination therapy, which consists of intratumoral interleukin-12 (IL-12) gene therapy, human tyrosinase (hTyr) DNA vaccination and metronomic cyclophosphamide (CPX), was evaluated in a B16-F10 mouse model. The following groups were compared: (1) no treatment, (2) control vector, (3) intratumoral IL-12 gene therapy, (4) intratumoral IL-12 gene therapy+metronomic CPX, (5) intratumoral IL-12 gene therapy+metronomic CPX+hTyr DNA vaccination. Next to clinical efficacy and safety, we characterized acute effects of IL-12 and anti-tumor immune response after a second tumor challenge. All treatment groups showed increased survival and higher cure rates than control groups. Survival of non-cured mice was increased when metronomic CPX was combined with IL-12 gene therapy. Furthermore, mice that received metronomic CPX had significantly lower percentages of regulatory T cells. Addition of the hTyr DNA vaccine increased cure rate and resulted in increased survival compared to other treatment groups. We also demonstrated that the manifest necrosis within days after IL-12 gene therapy is at least partly due to IL-12 mediated activation of NK cells. All cured mice were resistant to a second challenge. A humoral memory response against the tumor cells was observed in all groups that received IL-12 gene therapy, while a cellular memory response was observed only in the vaccinated mice. In conclusion, every component of this combination treatment contributed a unique immunologic trait with associated clinical benefits., (Copyright © 2014 Elsevier B.V. All rights reserved.)