Engineered Outer Membrane Vesicles Derived From Probiotic Escherichia Coli Nissle 1917 As Recobinant Subunit Antigen Carreirs For The Development Of Pathogen-Mimetic Vaccines
The greatest strides in vaccine delivery over the last decade have come primarily from a new class of nanoparticulate antigen carrier that focuses on reverse-engineering the pathogen-immune cell interaction on the molecular level. Such "pathogen-like particles", or PLPs, take an elegant approach to biomimicry, attempting to artificially isolate or recreate a pathogen's natural ability to stimulate a targeted immune response. In this work, we focused on the transformation of the probiotic E. coli strain Nissle 1917 into an outer membrane vesicle (OMV) platform for TH1-biasing delivery of a variety of recombinant antigens. We hypothesize that by harnessing the natural immunomodulation of the Nissle 1917 (EcN) bacterium, and pairing this immunomodulation with appropriate vaccine targets that require potent TH1-biasing vaccine responses, we can engineer a recombinant antigen delivery platform that uniquely enhances antigen-specific immunity through pathogen-mimetic vaccination. As bionanoparticulate PLPs often suffer from requiring multiple boosts and external adjuvants to achieve pathogen-mimetic memory responses, we further enhanced our EcN OMV platform with controlled release delivery using injectable polymeric microspheres as a transient OMV depot. From the immunological characterization of free and encapsulated EcN OMVs' vaccine capability, two vaccine targets were chosen to demonstrate the efficacy of the OMVs as a PLP platform for vaccine delivery. To test the capacity of the OMVs to functionally display and vaccinate against a heterologous antigen of viral origin, OMVs expressing a subunit of H1N1 hemagglutinin were produced and tested on BALB/c mice. Not only did the resulting immunological assays for vaccine response show great promise for a protective response, generating a 2.6-fold increase in IgG2a:IgG1 titers and a 8.1iii fold increase in IFN-[gamma]:IL-4 T-cell secretion versus a gold-standard control, but further analysis using hemagglutination-inhibition assays demonstrated >50-fold enhancement in cross-strain protection against H3N2. Secondly, to test EcN OMVs' capacity to direct unique immunomodulation to less standard vaccine targets, OMVs expressing the peanut allergen Arah2 were produced as both a prophylactic vaccine (for preventing peanut allergy) and an immunotherapy (for treating extent peanut allergy). Using a BALB/c mouse model for peanut allergy sensitization, a free EcN OMV vaccine dose was administered prior to sensitization, which following anaphylactic challenge post-sensitization resulted in protective survival of 100% of vaccinated mice. Encapsulated controlled release of lower doses of the Arah2-displaying EcN OMVs administered following sensitization were also successful at protecting >50% of mice from some level of anaphylaxis post-challenge while minimizing side-effects relative to traditional sublingual immunotherapy. The engineering and in vitro/in vivo testing of EcN OMVs as vaccine antigen carriers demonstrated promising efficacy as a pathogen-mimetic platform for protective immunomodulation. Successful testing with a variety of recombinant antigens provides the foundation upon which further development of the EcN OMV platform can lead to a promising host of PLP vaccines. iv
Outer membrane vesicle; Vaccine delivery; Pathogen-like particle
Putnam, David A.
Chang, Yung-Fu; Shuler, Michael Louis; Delisa, Matthew
Ph. D., Biomedical Engineering
Doctor of Philosophy
dissertation or thesis