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

Abstract

Flagellin is a key component of the flagella of many pathogens, including Pseudomonas aeruginosa. Flagellin is an attractive vaccine candidate because it is readily produced and manipulated as a recombinant protein and has intrinsic adjuvant activity mediated through TLR5. Although DNA vaccines encoding native Pseudomonas B-type (FliC) or A-type (FlaA) flagellin are strongly immunogenic, the resultant Ab response interferes with the interaction of homologous flagellin with TLR5. This reduces the ability of the host to clear homologous, but not heterologous, flagellin-expressing P. aeruginosa. To circumvent this problem, a DNA vaccine encoding a mutant FliC R90A flagellin was developed. The mutant Ag encoded by this vaccine was highly immunogenic, but its ability to interact with TLR5 was reduced by >100-fold. Vaccination with this flagellin mutant DNA vaccine induced cross-reactive Abs against both FliC and FlaA, but few Abs capable of interfering with TLR5 activation. The flagellin mutant DNA vaccine provided excellent protection against both FliC- and FlaA-expressing P. aeruginosa. These findings suggest that vaccines against flagellated pathogens should avoid inducing Abs against TLR5 and raise the possibility that flagellated bacteria evade host elimination by facilitating the production of Abs that reduce the host’s ability to mount an innate immune response.

Deletion of Flagellin’s Hypervariable Region Abrogates Antibody-Mediated Neutralization and Systemic Activation of TLR5-Dependent Immunity

“…Therefore, in vivo blockade of TLR signaling by MAMP-specific neutralization Abs is a novel mechanism for down-regulating innate immunity. Our results are consistent with the report by Saha et al (31), which suggested that ex vivo Ab blockade of the TLR5 activation motif of Pseudomonas aeruginosa flagellin reduces its efficacy to induce lung innate responses. Interestingly, we found that neutralizing epitopes in S. typhimurium FliC are embedded within the conserved region (regions 1-173 and 401-494) that also carries the activation motif 89 -96 (8, 9).…”

A Fusion Protein Vaccine Containing OprF Epitope 8, OprI, and Type A and B Flagellins Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa

“…The titers of flagellin-, OprI-, and OprF-specific IgG antibodies following immunization with OprF 311-341 -OprI-flagellins are in most cases two logs higher than those reported in other studies (12,14,22,50,51,67,68,70). For example, von Specht et al (62) required three immunizations with 70-fold more antigen to achieve equivalent antibody responses.…”

“…aeruginosa possesses two types of flagellins, type A and type B, that differ in amino acid composition and length of the hypervariable region. P. aeruginosa flagellins have the unique property of being potent adjuvants as well as protective antigens (8,32,42,50). Previous work has established flagellin as a potent adjuvant in mice (1, 3, 9, 10, 23, 33-35, 45, 53, 56) as well as cynomolgus and African green monkeys (24,36).…”

Evaluation of Flagella and Flagellin of Pseudomonas aeruginosa as Vaccines

“…In a rat model of P. aeruginosa-induced pneumonia, administration of human monoclonal antibodies (MAbs) to flagella provided protection against infection and decreased lung injury (24), and another set of human MAbs provided protection in a murine model of pneumonia during neutropenia (28). A DNA vaccine encoding recombinant type a or type b P. aeruginosa flagellin also induced protective immunity against lethal P. aeruginosa lung infection (33), although this study curiously found better heterologous protection than homologous protection when DNA encoding wild-type flagellin was incorporated into the vaccine. A fusion protein of outer membrane protein F (OprF) residues 311 to 341, mature OprI residues 21 to 83, and flagellins a and b (termed OprF311-341-OprI-flagellins) generated significant immune responses in mice and promoted enhanced clearance of strain PA01 in a pulmonary challenge model (42).…”