Introduction: Intentional aerosolization of Yersinia pestis may result in pneumonic plague which is highly fatal if not treated early. Methods: We conducted a phase 1 randomized, double blind (within each group), placebo controlled, dose escalation trial to evaluate a plague vaccine, Flagellin/F1/V, in healthy adults aged 8 through 45 years. Vaccine was administered intramuscularly on Days 0 and 28 at a dose of 1, 3, 6 or 10 mcg. Subjects were observed for 4 h after vaccination for cytokine release syndrome. Reactogenicity and adverse events (AE) were collected for 14 and 28 days, respectively, after each vaccination. Serious AE were collected for the entire study. ELISA antibody and cytokines were measured at multiple time points. Subject's participation lasted 13 months. Results: Sixty healthy subjects were enrolled; 52% males, 100% non-Hispanic, 91.7% white and mean age 30.8 years. No severe reactogenicity events occurred; most AE were mild. No serious AE related to vaccine occurred. A dose response effect was observed to Fl, V and flagellin. The peak ELISA IgG antibody titers (95% CI) after two 10 mcg doses of vaccine were 260.0 (102.6-659.0) and 983.6 (317.3-3048.8), respectively, against Fl and V antigens. The 6 mcg dose group provided similar titers. Titers were low for the placebo, 1 mcg and 3 mcg recipients. A positive antibody dose response was observed to Fl, V and flagellin. Vaccine antigen specific serum IgE was not detected. There were no significant rises in serum or cellular cytokine responses and no significant IgG increase to flagellin after the second dose. Conclusion: The Flagellin/F1JV vaccine exhibited a dose dependent increase in immunogenicity and was well tolerated at all doses. Antibody specific responses to Fl, V and flagellin increased as dose increased. Given the results from this trial, testing higher doses of the vaccine may be merited. (C) 2017 Published by Elsevier Ltd.

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.

Flagellin, the ligand for TLR5, is a potent adjuvant for CD4+ T cell-dependent humoral immune responses and has been reported by several groups to promote CD8+ T cell responses in vivo. However, other groups have failed to detect an effect of flagellin on the CD8+ T cell response. In an attempt to resolve this controversy, we created a protein composed of the SIINFEKL epitope and flanking amino acids fused to the N-terminus of full length Salmonella flagellin for use with TCR transgenic OT-I mice. We found that the CD8+ T cell stimulatory activity of this protein was, as expected, dependent on proteasome processing and was markedly more potent than ovalbumin by itself. However, the adjuvant effect of the fusion protein was TLR5 and IPAF independent. A fusion peptide containing the hypervariable region Salmonella or Pseudomonas flagellin and the SIINFEKL epitope also stimulated antigen-specific CD8+ T cell activation. These results are consistent with the conclusion that flagellin does not promote antigen-specific CD8+ T cell activation. Since the flagellin fusion protein containing the SIINFEKL epitope was far superior to ovalbumin as an inducer of CD8+ T cell activation, it is likely that uptake and processing of the SIINFEKL epitope by antigen-presenting cells is enhanced outside the context of ovalbumin.

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.

A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4 degrees C and at least 168 days at 25 degrees C. At between 29 and 84 days at 37 degrees C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell ( DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 ( SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8(+) and CD4(+) allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.

Department of Microbiology and Immunology, Department of Pediatrics, Section on Comparative Medicine, and Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, and Walter Reed Army Institute of Research Bioproduction Facility, Silver Spring, MD 20910