The goals of this study were to compare the immunogenicity and functionality of flagellin and flagella as possible candidates for a vaccine against P. aeruginosa
pneumonia and also to determine if such immunogens might give rise to antibodies that could interfere with the host's ability to detect the presence of flagellated pathogens via TLR5 activation (15
). Prior work with animals (3
) and CF patients lacking detectable P. aeruginosa
) has validated the vaccine potential of flagella. Vaccines based on monomeric flagellin have shown some efficacy in animals when a DNA vaccine encoding P. aeruginosa
flagellin lacking TLR5 agonist activity is used (33
), as has a multimeric construct of OprF(311-341)-OprI-flagellin fusion proteins (42
) in models of burn wound and pulmonary infection. However, no direct comparison of the protective efficacy against infection by immunization with flagellin or flagella has been reported for P. aeruginosa
or, as far as we can determine, for other microorganisms. In vitro
studies suggest that the protective activity of antibody to flagella or flagellin correlates with inhibition of motility (24
) and opsonic killing (42
). Here we found that polymeric flagella, which by nature contain, in addition to the polymerized FliC protein, components such as the FliD cap protein and possible basal body components, were clearly superior to monomeric flagellin at inducing antibodies that inhibited motility, mediated opsonic killing, and provided protective immunity against acute lung infection. Also, the specificity of the protection for strains expressing only the homologous flagellar type indicated that potentially contaminating antigens in flagellar preparations isolated from P. aeruginosa
cells were not contributing to the protective effects observed. Additionally, immunization with flagella induced lower titers of antibody that could interfere with the TLR5 agonist activity of flagellin and flagella, but only antibody to type a flagella and flagellin inhibited TLR5 activation by whole bacterial cells. These findings suggest a superiority of intact flagella over monomeric flagellin as a component of a P. aeruginosa
In evaluating effects on P. aeruginosa
motility and opsonic killing in vitro
, we found flagellar-type-specific activity with antibodies to type b antigens, whereas the antibodies raised to type a flagella had some activity against type b strains. Since amino acid segments of the two flagella types are partially similar, it is possible that the type a flagellum vaccine was better able to induce antibodies to these shared components. Another potential basis for these differences may be glycosylation of flagellar proteins (5
), wherein the type a flagellum is glycosylated by larger and more heterogeneous oligosaccharides than type b (40
), possibly enhancing its immunogenicity. In addition, the lack of the glycan groups on the recombinant monomeric flagellins could also partly explain the poor ability of the antibodies raised to these proteins to promote opsonic killing. Nonetheless, for both motility inhibition and opsonic killing, the intact flagella were superior to monomeric flagellin at inducing antibodies mediating these in vitro
correlates of protection.
In the mouse pneumonia model, we showed that passive immunization with antibodies to flagellin did not confer protection against infection with either type of P. aeruginosa
. This outcome is different from that in the work of Saha et al. (33
), who reported that immunization with a flagellin DNA vaccine protected against heterologous but not homologous bacterial challenge. This is curious in that almost all prior studies showed protection following flagellar vaccination was type specific (3
). The DNA vaccine construct may be superior at stimulating cross-protective humoral and/or cellular immune responses compared to intact flagellin protein, or active immunization with a DNA vaccine may provide results superior to those of passive immunization. Weimer et al. (42
) obtained enhanced clearance of P. aeruginosa
strain PA01 following immunization of mice with a construct of OprF311-341-OprI-flagellins, although the challenge dose was insufficient to cause mortality in the controls immunized with OprF311-341-OprI vaccine lacking flagellins. Similarly, immunization of young African green monkeys with this construct elicited serum antibodies that when passively administered to mice could promote clearance of P. aeruginosa
from the lungs (41
). However, since our goal was to compare flagellin-mediated immunity to that induced by polymeric flagella, we were able to validate a superior effect of the latter in passive transfer studies wherein injection of rabbit antibodies raised to flagella demonstrated high levels of protection in mice against lethal lung infection.
Active vaccination with intact flagella also showed high levels of type-specific, LPS-independent protection in a mouse pneumonia model and modest but less protection against motile clinical isolates from CF patients. These isolates were obtained from CF patients enrolled in the flagellar vaccine trial (12
) who, in spite of vaccination, nonetheless became colonized with P. aeruginosa
. We suspected these strains might be less susceptible to protection mediated by flagella derived from strains PAK and PA01, and the findings in our mouse studies bear this out. This could have important consequences for future vaccine trials incorporating flagella as vaccines and suggests that there may be additional flagellar components needed in a comprehensive vaccine, such as those expressing different subtype antigens on type a flagella (3
). Since virtually all prior studies in this area have only evaluated protection against strain PAK or PA01, conclusions about the utility of flagellin or flagella as a vaccine have to be tempered with the lack of a comprehensive demonstration of efficacy against multiple strains, including clinical isolates, and, now that they are available, clinical isolates from flagellum-vaccinated CF patients unable to resist colonization by P. aeruginosa
Another concern related to use of flagellin or flagella as vaccines is whether they will induce antibodies that interfere with TLR5-mediated innate immunity (15
), which has been suggested by the studies of Saha et al. (33
). However, the experimental design of that study is not informative as to the potential for antibody to flagella or flagellin to interfere with innate immunity during an actual infection with live P. aeruginosa
cells. These investigators used purified, recombinant flagellin to enhance TLR5-mediated innate immunity by first incubating it with either antibody to wild-type flagellin or antibody raised to the R90A flagellin variant lacking the TLR5 binding domain. They subsequently inoculated BALB/c mice with this flagellin-antibody mixture 2 h prior to challenge with live P. aeruginosa
PA01. The flagellin mixed with antibody to wild-type flagellin was less able to confer protection from lung infection than was flagellin mixed with antibody to the R90A variant, presumably due to inhibition of TLR5-mediated innate immune responses. However, this experiment did not indicate if antibody to flagellin inhibited TLR5 activation from whole bacteria during infection, thus increasing the animal's susceptibility to infection.
When testing the activation of TLR5 by P. aeruginosa bacterial cells, strain PAK was a more potent activator of the receptor than was strain PA01, consistent with the findings using purified flagella from these strains. However, for unclear reasons, while antibody to both flagella and flagellin could inhibit in a dose-dependent manner TLR5 activation mediated by strain PAK, neither of them could inhibit activation mediated by PA01 or by two other type b flagellum strains. Since there was no luciferase signal from cells infected with the ΔfliC strains, other P. aeruginosa PAMPs, such as LPS, were not active in this assay. The mechanisms that might explain the inability of antibody to flagella or flagellin to inhibit the activation of TLR5 by type b strains is not clear, but it may not necessarily be related to preventing TLR5 binding but rather perhaps to some other property of antibody to flagella or flagellin, such as inhibition of motility. In this regard, type a strains such as PAK may be less able to interact with the cells if motility is inhibited by the antibody to flagella, whereas the type b strains may either be less inhibited in their overall motility by antibody or use an alternative means of locomotion, such as pilus-mediated twitching motility, to interact with cells, or the antiserum to type b flagella or flagellin may be less potent at inhibiting motility than antibody to type a flagella, although this was not apparent in our in vitro motility inhibition assays.
Taken together, it seems that flagella, composed primarily of the FliC protein but also containing the type-specific FliD cap protein and basal body components, would be a better candidate for a vaccine against P. aeruginosa than flagellin, since immunization with flagella was demonstrated to be more proficient in generating flagellar-type-specific antibodies that inhibit motility, mediate opsonic killing, and protect against acute P. aeruginosa lung infection. In addition, although the antibodies to flagella inhibited activation of TLR5, they were less potent than antibodies to flagellin in this regard, minimizing the potential for interference with the induction of innate immunity mediated by activation of TLR5. Of note, however, use of flagella from strains PAK and PA01 as vaccines provided only modest protection against clinical isolates from flagellar-vaccine-immunized CF patients who nonetheless became colonized with flagellated P. aeruginosa. This suggests additional flagellar antigens may need to be incorporated into a comprehensive vaccine or strains other than the prototype PAK and PA01 may need to be utilized as a source for the flagellar antigens to find some that either are more immunogenic or give rise to more cross-reactive antibodies. Identifying the optimal formulation of the components of flagella needed for a maximally effective P. aeruginosa vaccine should enhance the utility of this approach for future evaluations.