John T. Bates

Flagellin as an Adjuvant: Cellular Mechanisms and Potential

Flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. An increasing number of studies have demonstrated the effectiveness of flagellin as an adjuvant, as well as its ability to promote cytokine production by a range of innate cell types, trigger a generalized recruitment of T and B lymphocytes to secondary lymphoid sites, and activate TLR5+CD11c+ cells and T lymphocytes in a manner that is distinct from cognate Ag recognition. The plasticity of flagellin has allowed for the generation of a range of flagellin–Ag fusion proteins that have proven to be effective vaccines in animal models. This review summarizes the state of our current understanding of the adjuvant effect of flagellin and addresses important areas of current and future research interest.

Proof of principle for epitope-focused vaccine design

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Direct stimulation of tlr5+/+ CD11c+ cells is necessary for the adjuvant activity of flagellin.

Flagellin is a highly effective adjuvant, but the cellular mechanism underlying this activity remains uncertain. More specifically, no consensus exists as to whether flagellin activates dendritic cells (DC) directly or indirectly. Intramuscular immunization with flagellin-OVA fusion protein resulted in enhanced in vivo T cell clustering in draining lymph nodes and IL-2 production by OVA-specific CD4+ T cells. Immunization with flagellin-OVA also triggered greater levels of Ag-specific CD4+ T cell proliferation than immunization with flagellin and OVA as separate proteins. To determine whether flagellin, in the context of a fusion protein with OVA, was acting directly on DC, we used a combination of CD4+ T cell adoptive transfers and bone marrow chimera mice in which the presence or absence of potential tlr5+/+ CD11c+ cells was controlled by injection of diphtheria toxin. The Ag-specific CD4+ T cell response in mice with CD11c+ cells from a tlr5−/− background and mixed populations of all other hematopoietic cells was dramatically reduced in comparison to mice that had DC from tlr5−/− and wild-type backgrounds. Immunization of MyD88−/−tlr5+/+ mice revealed that the enhanced response following immunization with flagellin-OVA is dependent on signaling via the TLR5-MyD88 pathway as well as enhanced Ag uptake and processing resulting from Ag targeting via TLR5. In summary, our data are consistent with the conclusion that direct stimulation of tlr5+/+ CD11c+ cells is necessary for the adjuvant activity of a flagellin fusion protein and that this adjuvant effect requires signaling through TLR5.

Mucosal adjuvant activity of flagellin in aged mice.

We evaluated the ability of flagellin, a highly effective mucosal adjuvant in mice and non-human primates, to promote mucosal innate and adaptive immunity in aged mice. We found that intratracheal instillation of flagellin induced a stronger respiratory innate response in aged mice than in young mice, and that intranasal instillation of flagellin was equally effective at triggering recruitment of T and B lymphocytes to the draining lymph nodes of young and aged mice. Intranasal immunization of aged mice with flagellin and the Yersinia pestis protein F1 promoted specific IgG and IgA production, but at lower levels and lower avidities than in young mice. Although intranasal instillation of flagellin and F1 antigen increased germinal center formation and size in young mice, it did not do so in aged mice. Our findings are consistent with the conclusion that flagellin can promote adaptive immune responses in aged mice, but at a less robust level than in young mice.

Enhanced Antigen Processing of Flagellin Fusion Proteins Promotes the Antigen-Specific CD8+ T Cell Response Independently of TLR5 and MyD88

Flagellin is a highly effective adjuvant for CD4+ T cell and humoral immune responses. However, there is conflicting data in the literature regarding the ability of flagellin to promote a CD8+ T cell response. In this article, we report that immunization of wild-type, TLR5−/−, and MyD88−/− adoptive transfer recipient mice revealed the ability of flagellin fusion proteins to promote OVA-specific CD8+ T cell proliferation independent of TLR5 or MyD88 expression by the recipient animal. Wild-type and TLR5−/− APCs were able to stimulate high levels of OVA-specific CD8+ T cell proliferation in vitro in response to a flagellin fusion protein containing full-length OVA or the SIINFEKL epitope and 10 flanking amino acids (OVAe), but not to OVA and flagellin added as separate proteins. This effect was independent of the conserved regions of flagellin and occurred in response to OVAe alone. Comparison of IFN-γ production by CD8+ effector cells revealed higher levels of SIINFEKL peptide–MHC I complexes on the surface of APCs that had been pulsed with OVAe–flagellin fusion proteins than on cells pulsed with OVA. Inhibition of the proteasome significantly reduced Ag-specific proliferation in response to OVAe fusion proteins. In summary, our data are consistent with the conclusion that flagellin–OVA fusion proteins induce an epitope-specific CD8+ T cell response by facilitating Ag processing and not through stimulatory signaling via TLR5 and MyD88. Our findings raise the possibility that flagellin might be an efficient Ag carrier for Ags that are poorly processed in their native state.

Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis®), a humanized IgG1 monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2–5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.

Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (Kon) for binding to RSV F protein, while alteration of dissociation rate (Koff) did not significantly affect neutralizing activity. Interestingly, linkage of reduced Kon with reduced potency mirrored the effect of increased Kon found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants.

Reversion of Somatic Mutations of the Respiratory Syncytial Virus–Specific Human Monoclonal Antibody Fab19 Reveal a Direct Relationship between Association Rate and Neutralizing Potency

The role of affinity in determining neutralizing potency of mAbs directed against viruses is not well understood. We investigated the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in the Ab H chain V region of Fab19, a well-described neutralizing human mAb directed to respiratory syncytial virus. Comparison of the affinity-matured Ab Fab19 with recombinant Fab19 Abs that were variants containing reverted amino acids from the inferred unmutated ancestor sequence revealed the molecular basis for affinity maturation of this Ab. Enhanced binding was achieved through mutations in the third H chain CDR (HCDR3) that conferred a markedly faster on-rate and a desirable increase in antiviral neutralizing activity. In contrast, most somatic mutations in the HCDR1 and HCDR2 regions did not significantly enhance Ag binding or antiviral activity. We observed a direct relationship between the measured association rate (Kon) for F protein and antiviral activity. Modeling studies of the structure of the Ag–Ab complex suggested the HCDR3 loop interacts with the antigenic site A surface loop of the respiratory syncytial virus F protein, previously shown to contain the epitope for this Ab by experimentation. These studies define a direct relationship of affinity and neutralizing activity for a viral glycoprotein–specific human mAb.

Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Significance Respiratory syncytial virus is a highly contagious human pathogen, infecting the majority of infants before age 2 y, and is the leading cause of viral bronchiolitis and viral pneumonia in infants and children. An approved prophylactic humanized mouse monoclonal antibody, palivizumab, is currently the standard-of-care treatment for immunocompromised individuals, and competition with palivizumab has been proposed as the basis for measuring effective immune responses for vaccine trials. Using a combination of X-ray crystallography, hydrogen-deuterium exchange, and saturation alanine mutagenesis scanning, we show the structural basis for neutralization by a human antibody at the palivizumab antigenic site. Furthermore, we describe nonneutralizing antibodies that directly compete with palivizumab and another human antibody 14N4. Taken together, the data presented provide unique concepts in structure-based vaccine design. Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix–loop–helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.