Roles in innate immune receptors: Host : pathogen interactions and strengthening vaccines

Sammanfattning: During the past decades our knowledge of innate immunology has increased drastically. This hasimproved our understanding about how innate immune cells can distinguish self from non-self, commensal bacteria from pathogens, and how it affects later adaptive immune responses. This knowledge can lead to new approaches for treatment of diseases and autoimmunity, and the development of new vaccines. Vaccines are among the greatest inventions in medical history. However traditional vaccine approaches, such as live attenuated or inactivated viruses, have failed as vaccine candidates to address certain diseases including HIV/AIDS. Nonliving non-replicating DNA vaccinesrepresent an alternative approach, capable of inducing broad cell-mediated and humoral responses, while being safe and fast to produce. Still, despite its efficacy in animal models, DNA vaccines have not yet succeed to induce effective immune responses in human. To enhance the immunogenicity, a combination of more optimized vectors, delivery methods and adjuvants will be required. Skin electroporation (EP) is a promising method known to elicit robust humoral and CD8+ T cell responses. However, the data on CD4+T cell responses has been limited. In paper I we compare immunization by skin EP with intramuscular injection, and find that EP increases both the magnitude and the polyfunctionality of the CD4+ T cell responses to the HIV antigen Gag. In paper II we show that plasmid encoding a secreted flagellin (pFliC) adjuvant promotes both humoral and MHC Class I-dependent cellular immunity when delivered through different routes representing dermal, systemic, and mucosal tissues. Additionally, it enhances mucosal humoral and MHC Class II-dependent cellular immunity when delivered mucosally. With in vitro studies we could show that secreted pFliC has the ability to activate macrophages through Toll-like receptor 5 (TLR5) but also cytoplasmic Nod-like receptor C4 (NLRC4), leading to inflammasome dependent cell death (pyroptosis). In paper III we continue to study NLRC4 activation upon recognition of flagellin. We have established a system that allows for inducible expression of a NLRC4 agonist in a macrophage cell line, without additional stimuli. Using this system we have shown that NLRC4 induced caspase-1-dependent pyroptosis isindependent of LPS priming, reactive oxygen species, or classical mitochondrial involvement. Nevertheless, pyroptotic macrophages release the alarmin high mobility group box 1 (HMGB1). Importantly, the functional isoform of HMGB1 is affected by the priming event and unprimed pyroptotic cells release a chemotactic form of HMGB1. However, priming during pyroptosis causes oxidation of the protein thereby changing it to a TLR4-agonist. Combined, these studies will contribute to the understanding of the regulation of inflammasome activity, and how to deliver the next generation of DNA vaccines in combination with adjuvants.

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