Purification and characterization of flagellin-F1-V.
The production and purification of flagellin-F1-V were done according to currently accepted GMP. An initial analysis revealed that flagellin-F1-V accumulates in inclusion bodies and can be solubilized in the presence of 1.75 M guanidine HCl (Fig. ). By washing the inclusion bodies with 50 mM Tris HCl, pH 8.0-5 mM EDTA and then washing them with the same buffer containing TX-100 (twice) and the starting buffer-1.75 M guanidine HCl, it is possible to obtain a highly enriched preparation of flagellin-F1-V. The flagellin-F1-V solubilized from the inclusion bodies was subjected to two rounds of size-exclusion chromatography on a Sephacryl S-400 HR column with a buffer containing 50 mM Tris HCl, pH 7.5, 150 mM NaCl, and 8 M urea. Fractions were collected beginning after the exclusion volume. The final product was a clear, colorless solution with a protein concentration of 1.25 mg/ml and an endotoxin content of 24 EU/ml (19.2 EU/mg protein or 1.92 ng endotoxin/mg protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the final product revealed a major band at approximately 90 kDa and two minor bands at 70 to 80 kDa (Fig. ). Western blot analysis with rabbit antiserum against flagellin revealed that the major band as well as the minor bands contained flagellin (data not shown). Furthermore, a single N-terminal amino acid sequence that was identical to that of S. enterica serovar Enteritidis FliC was obtained (data not shown). The final product contained proteins of 87,836, 87,891, and 87,942 Da in a ratio of 68%:25%:7%. Two minor peptides of 5,574 and 5,331 Da were present at levels of 1% or less. Consistent with the findings of those analyses, two-dimensional gel electrophoresis revealed the presence of three proteins of similar molecular masses and pI values in the range of 5.5 to 6 (data not shown). A hydrolyzed sample of flagellin-F1-V containing 1.54 nmol of PTC-lysine did not contain detectable PTC-homocitrulline, a finding that is consistent with the absence of carbamylation. Additional analyses demonstrated that the final product contained less than 10 μM guanidine and IPTG.
In vitro TLR5-specific signaling activity of flagellin-F1-V.
To evaluate the TLR5-specific signaling activity of the purified flagellin-F1-V, we incubated TLR5-negative and TLR5-positive RAW 264.7 cells with increasing concentrations of flagellin-F1-V or flagellin (as a positive control) and determined the amount of TNF-α produced over 4 h. As shown in Fig. , flagellin-F1-V, like flagellin, induced TNF-α production in a TLR5-specific manner. The potencies of the two proteins were quite similar, indicating that the addition of the F1 and V sequences to the flagellin did not have a significant effect on the TLR5 binding domain of flagellin.
FIG. 2. Concentration-dependent stimulation of TNF-α production by flagellin-F1-V in cultures of TLR5-positive RAW 264.7 cells. TLR5-positive RAW 264.7 cells (closed circles and closed triangles) and TLR5-negative RAW 264.7 cells (open circles and open (more ...) Induction of an antigen-specific humoral immune response using flagellin-F1-V.
To evaluate the ability of flagellin-F1-V to induce a humoral immune response against the F1 and V antigens, groups of seven female BALB/c mice were immunized i.m. with 0.3, 1, 3, or 10 μg of flagellin-F1-V. Twenty-eight days later, the mice were boosted by use of the same route and doses and were then bled 14 days later. Plasma was prepared and assayed for anti-F1 and -V IgG antibodies by ELISA. As shown in Fig. , flagellin-F1-V induced a very robust humoral immune response against the F1 and V antigens, with titers approaching 3 × 106. Similar results were obtained when the flagellin-F1-V was administered i.n. (data not shown).
FIG. 3. Flagellin-F1-V immunization of BALB/c mice results in a robust antigen-specific IgG response against F1 and V. Groups of seven BALB/c mice were immunized twice i.m. with increasing doses of flagellin-F1-V. Ten days later, the animals were bled and the (more ...) Y. pestis challenge of mice.
To assess the protective effect of the flagellin-F1-V vaccine against respiratory challenge with Y. pestis CO92, groups of BALB/c mice were given flagellin-F1-V or PBS i.m., as described above, and were then challenged i.n. with approximately 150 MTD50s of Y. pestis CO92. The PBS-treated mice succumbed within 3 days after challenge, whereas all of the mice immunized with flagellin-F1-V survived with few, if any, signs of morbidity (Fig. ). Complete protection was also obtained with mice immunized i.n. (data not shown).
FIG. 4. Immunization of BALB/c mice with flagellin-F1-V protects against a lethal respiratory challenge with Y. pestis. Groups of 10 BALB/c mice were immunized twice i.m. with 10 μg flagellin-F1-V (open circles) or PBS only (closed circles). Two weeks (more ...)
In a subsequent challenge study, immune and control mice were challenged with approximately 150 MTD50s of Y. pestis CO92 and were then killed after 3 days to assess the levels of Y. pestis in their blood, liver, lungs, and spleen. As shown in Table , control mice had very large numbers of CFU in all of the sites. However, there was no detectable CFU at any site in mice immunized with flagellin-F1-V. Taken together, the challenge studies clearly demonstrate that flagellin-F1-V is a highly protective vaccine and promotes sterile immunity against Y. pestis given i.n.
Bacterial burden in BALB/c mice challenged with Y. pestisa
Stability of flagellin-F1-V.
To assess the stability of flagellin-F1-V, samples of the protein were incubated at 4°C, 25°C, or 37°C; and at various times aliquots were assayed for in vitro TLR5-stimulating activity, in vivo adjuvant activity, and protective effect against i.n. challenge with Y. pestis. As shown in Table , the TLR5-stimulating activity of flagellin-F1-V (as measured by the RAW 264.7 cell assay) was stable for at least 297 days when it was stored at 4°C and at least 168 days when it was stored at 25°C. In contrast, the TLR5-stimulating activity decreased significantly when flagellin-F1-V was stored at 37°C for greater than 29 days and was most evident after 168 days. Although the activity was reduced by approximately 80% at 168 days, the half-maximal response was still obtained at 1.5 × 10−10 M. The stability observed at 4°C and 25°C was also observed when the samples were assayed for the ability to promote an antigen-specific IgG response against the F1 and V antigens in BALB/c mice (Table ). As with the TLR5-stimulating activity of material stored at 37°C, flagellin-F1-V lost significant activity at the higher temperature. By day 168, the efficacy of the vaccine was approximately 15% of that for the day 0 sample. It is of interest that the drop in the anti-V IgG titer was markedly greater than that of the anti-F1 titer. This finding may indicate that the V antigen in the fusion protein is more sensitive to temperature than the F1 antigen.
Temperature stability of flagellin-F1-Va
We also examined the effect of temperature on the ability of flagellin-F1-V to promote a protective response against respiratory challenge with Y. pestis. When BALB/c mice were immunized twice with 10 μg flagellin-F1-V stored at 4°C, 25°C, or 37°C for 56 or 112 days, all of the mice were fully protected (data not shown). This finding is consistent with the results of the dose-response experiment presented in Fig. , in which we observed that 1 μg of flagellin-F1-V was as effective as 10 μg in eliciting a maximal humoral response.
As a first step in developing an understanding of the physical changes in flagellin-F1-V that were responsible for the loss of bioactivity at 37°C, we used dynamic light scattering to assess any potential changes in the overall structure of the fusion protein that might be associated with a decrease in adjuvant activity or immunogenicity. As shown in Fig. , flagellin-F1-V appeared to assume a coil-like monomeric structure of approximately 25 nm that was maintained at 4°C and 25°C for at least 168 days. However, at 37°C, the protein diameter increased to approximately 40 nm by day 29 and remained at this diameter over the next 139 days. This change may reflect oligomerization of the protein. Since the full biologic function at 37°C was retained for 29 days, it is likely that this change may be a precursor to other changes (not detected by dynamic light scattering) that ultimately reduce bioactivity.
FIG. 5. Dynamic light-scattering analysis of flagellin-F1-V incubated at 4°C, 25°C, and 37°C. Samples of flagellin-F1-V incubated at 4°C (—), 25°C (- - -), or 37°C (-·- ·) were subjected (more ...) Induction of an antigen-specific humoral immune response in African green monkeys immunized with flagellin-F1-V.
In a prior study (17
), we demonstrated that flagellin is a highly effective adjuvant in cynomolgus monkeys, promoting a robust humoral immune response against the F1 and V antigens of Y. pestis
. In the current study, we examined the effectiveness of the flagellin-F1-V vaccine in adult male African green monkeys (average weight, approximately 6 kg). Prior to immunization, these animals had relatively low titers against the F1 and V antigens (Table ). However, two immunizations with flagellin-F1-V generated a dramatic increase in the titers of anti-F1 and -V IgG antibodies in these animals. The mean anti-F1 IgG antibody titers increased from 3.7 × 103
± 3.6 × 103
to 4.39 × 105
± 1.68 × 105
, and the anti-V IgG titers from 8.51 × 103
± 14.6 × 103
to 2.73 × 106
± 2.14 × 106
Flagellin-F1-V induces an antigen-specific adaptive immune response in nonhuman primatesa
In an initial experiment, we immunized 4- to 6-month-old African green monkeys (weight, approximately 1 kg) with the dose of flagellin-F1-V given to the adults (200 μg) and found that they, like the adults, also exhibited a robust antigen-specific response to F1 and V (data not shown). In a subsequent experiment, we evaluated lower doses in the range of 25 to 100 μg and found that the monkeys responded as well to 25 μg as they did to 100 μg (data for 25 μg are shown in Table ).
Finally, we examined the effect of flagellin-F1-V in cynomolgus monkeys (age, approximately 1 year). As shown in Table , all of the cynomolgus monkeys exhibited robust F1- and V-specific IgG responses to flagellin-F1-V.