Flagellin-F1-V Fusion Protein Is an Effective Plague Vaccine in Mice and Two Species of Nonhuman Primates

Abstract

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°C and at least 168 days at 25°C. At between 29 and 84 days at 37°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.Yersinia pestis, a gram-negative coccobacillus, causes an acute and often fatal disease that may appear in one of three major manifestations: bubonic, septicemic, or pneumonic disease. Transmission to humans most commonly occurs via a bite by infectious fleas and is associated with regional lymphadenopathy or bubo. In the United States, bubonic plague accounts for 80 to 90% of the cases of Y. pestis infection. Pneumonic plague is the most dangerous form of the disease and is generally fatal if an individual does not receive appropriate treatment within 18 h after the onset of respiratory symptoms. Pneumonic plague would be the most likely outcome in the case of a bioterrorism attack.In view of the seriousness of infection with Y. pestis, a substantial effort has been focused on the development of protective vaccines. Killed bacteria have been used in plague vaccines for over 100 years, and a formalin-inactivated bacterial vaccine has been licensed in the United States. However, its efficacy has not been properly evaluated in well-controlled studies. Nonetheless, a retrospective study of armed services personnel who served in Vietnam provided indirect evidence for efficacy in the prevention of bubonic plague (8). However, no evidence in support of a protective effect against the pneumonic form of the disease was obtained. Although a live attenuated strain of Y. pestis (strain EV76) has been evaluated (31), more recent studies have focused on the development of acellular vaccines, in particular, vaccines containing the fraction 1 capsular antigen ...

Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin

“…37,38,[40][41][42] The current data support the conclusion that the adjuvancy of flagellin is more dependent on TLR5 than the NLRC4 inflammasome. 32 However, they also suggested that deficiency in the NLRC4-mediated activity of flagellin might make it a better adjuvant for enhancing the antibody response.…”

“…Flagellin functions as an immunogen to induce strong immune response against itself and co-administered antigens. [16][17][18][37][38][39][40][41][42] Based on its biological properties, flagellin or flagellin-based fusion proteins are being developed as therapeutic agents or vaccines to treat infectious diseases, toxic exposures, and even cancer. [43][44][45][46][47] In recent years, studies have shown that the immune activities of flagellin, such as adjuvancy, are dependent on TLR5 recognition and the NLRC4 inflammasome.…”

Potential adjuvantic properties of innate immune stimuli

“…116 As described above, as a consequence of the potent effects of flagellin on both the innate and adaptive limbs of the immune system, the protein has indeed been demonstrated to have potent adjuvantic effects in experimental vaccine constructs. [117][118][119][120][121][122] However, the safety profile of flagellin is yet to be unequivocally established. Of particular note, it has been observed that the administration of flagellin at doses comparable to or lower than that of bacterial LPS induces prominent local and systemic immune/inflammatory responses in vivo.…”

Over-activation of TLR5 signaling by high-dose flagellin induces liver injury in mice

“…Flagellin-triggered responses exert various effects on different cells, tissues and organs; these responses vary greatly according to the dose, physiological condition of the cell, the animal used and the experimental setting. 15,17,26 However, the beneficial flagellinactivated responses for host defenses and the potential adverse effects of flagellin in different organs can be contradictory. Therefore, the flagellin-triggered pathophysiological responses should be explored further to provide a clearer understanding of the correlation between the pattern recognition receptors (PRRs)-flagellin interaction and its pathophysiological effects to enhance the application of recombinant flagellin as a prophylactic and therapeutic agent.…”