Activation of Innate Immunity, Inflammation, and Potentiation of DNA Vaccination through Mammalian Expression of the TLR5 Agonist Flagellin

Abstract

Improving DNA vaccination remains a fundamental goal in vaccine research. Theoretically, this could be achieved by molecules encoded by DNA capable of activating TLRs to mimic inflammatory responses generated by infection. Therefore, we constructed an expression vector that allows mammalian cells to express the TLR5 agonist flagellin (FliC) at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection. These results demonstrate that expression of DNA-encoded TLR agonists by mammalian cells greatly enhance and broaden immune responses, imposing new possibilities on DNA vaccination to infectious agents and cancer.

Blocking of the TLR5 Activation Domain Hampers Protective Potential of Flagellin DNA Vaccine

“…The activation of TLR5 by flagellin has an adjuvant-like effect that enhances the immunogenicity of epitopes expressed on the flagellin molecule (13,20). However, Abs generated against the TLR5 activation domains expressed by WT flagellin hamper the host's ability to mount a protective innate immune response against flagellated pathogens.…”

Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy

“…This PAMP has recently been reported to function as an adjuvant when expressed by a plasmid. 25 Our results suggest that it may also be an interesting adjuvant for a tumor-associated antigen vaccine when expressed from an Ad vector. Further studies are needed to dissect the properties of Ad-PAMPs as vaccination adjuvants.…”

“…The latter result was unexpected because the P40 has been reported to stimulate TNF-a secretion by human moDCs or monocytes. 18,23,25 Secretion of IL-12 was not induced by transduction with Ad-PAMPs, whereas Ad-CD40L potently upregulated this important Th1-inducing cytokine. It has already been shown that flagellin treatment of human DCs does not stimulate IL-12 secretion.…”

“…23 Salmonella flagellin may act as a carrier of epitopes for vaccination against bacteria or viruses 24 and has been shown to enhance immunogenicity of plasmid vaccination. 25 Immunostimulatory properties of the hemagglutininneuraminidase (HN) of the Newcastle disease virus (NDV) have been demonstrated in vitro. 26,27 Mouse fibroblasts transfected with HN or infected with NDV have been shown to stimulate specific T-cell proliferation with greater efficacy than untreated fibroblasts.…”

Molecular vaccines for malaria

“…Conjugation of malaria antigens, Pfs25 and AMA1, to nontoxic Pseudomonas aeruginosa ExoProtein S (rEPA) was shown to increase antibody responses by up to 1000-fold. 102 Codelivery of the toll-like receptor (TLR) ligands 103,104 and fusion of antigens to the C3d component of complement 105,106 have shown promise as molecular adjuvants for infectious disease and cancer and may have applications for malaria vaccines.…”