Figure 1.
Schematic of various approaches used to design plague immunogens. See text for details. (A) Y. pestis surface components targeted for vaccine design. F1 is the structural unit of the capsular layer. V forms a pore at the tip of the injectisome needle and facilitates translocation of Yops into the host cell. YscF is the structural unit of the injectisome needle. (B) Reorientation of the NH2-terminal β-strand of F1 to generate monomeric F1. “n” and “n+1” refer to the F1 subunits the β-strands belong to; the red strands to “n” subunit and the blue strand to the “n+1” subunit. (C) Deletion of the putative immunomodulatory sequence (aa residues 271–300) of V antigen. (D) Mutagenesis of Asn35 and Ile67 to produce an oligomerization deficient YscF. (E) Structural model of bacteriophage T4. The enlarged capsomer shows the major capsid protein gp23* (green; “*” represents the cleaved form) (930 copies), Soc (blue; 870 copies), and Hoc (yellow; 155 copies). Yellow subunits at the five-fold vertices correspond to gp24*. The portal vertex (not visible in the picture) connects the head to the tail. (F) Display of F1mut-V-Soc fusion protein on the Hoc− Soc− phage particle. Models of the enlarged capsomers before and after F1mut-V display are shown.
Figure 2.
Designing monomeric F1 mutants.
(A) Schematic of native F1, F1mut1, and F1mut2 recombinant constructs. The donor β-strand of F1 is shown in pink, the T cell epitope region in blue, and the rest of the F1 coding sequence in green. The numbers correspond to the aa residues of F1. Native F1 has one hexa-histidine tag (orange) at the NH2-terminus, whereas F1mut1 and F1mut2 have two hexa-histidine tags, one at the NH2-terminus and another at the COOH-terminus. (B) Expression and solubility analysis. The recombinant F1 proteins were over-expressed by adding IPTG to 1 mM final concentration. The samples at 0, 1, or 2 h time points were analyzed by SDS-PAGE (15% gel) and Coomassie blue staining. The positions of F1 protein bands are marked with red arrows. The samples at 1 h or 2 h time points were analyzed for solubility using the B-PER reagent. S, soluble fraction (supernatant from 12,000 g centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (C) Purification of F1mut1. The F1mut1 recombinant protein was purified from the cell-free lysates by HisTrap affinity chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The molecular weight of F1mut1 peak fraction was calculated from the calibration curve constructed by gel filtration on the same column of standard proteins of known molecular weight [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1mut1 protein after SDS-PAGE and Coomassie blue staining of the peak fraction. Similar results were obtained with the F1mut2 recombinant protein. See Materials and Methods for additional details.
Figure 3.
Construction of mutated F1-V immunogens.
(A) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents the coding sequence of V antigen, and yellow, the putative immunomodulatory sequence that is part of V sequence. Rest of the colors represents the same as described in legend to Figure 2. (B) Expression and solubility analysis of F1-V constructs were performed using the B-PER reagent. The samples were analyzed by SDS-PAGE and Coomassie blue staining. The positions of the F1-V protein bands are marked with red arrows. S, soluble fraction (supernatant from 12,000 g centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (C) F1-V, F1mut-V and F1mut-V10 were purified by HisTrap column chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The calibration graph was generated by passing various molecular weight standards through the same column [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1-V, F1mut-V, and F1mut-V10 proteins following SDS-PAGE and Coomassie blue staining of the peak fractions. The color of arrows corresponds to the color of the elution profiles of various proteins. (D) Stability of F1-V and F1mut-V proteins was tested by treatment with increasing amounts of trypsin at room temperature overnight. The ratios shown above the gel correspond to the ratios of F1-V or F1mut-V proteins to trypsin (wt∶wt). See Materials and Methods for additional details.
Figure 4.
An oligomerization deficient YscF mutant.
(A) Schematic of native YscF and YscF35/67 mutants. (B) Purification of YscF and YscF35/67 mutant proteins. The gel filtration profiles showed that the native YscF eluted as a broad peak spanning the entire high molecular weight range and the mutated YscF35/67 eluted as two peaks, one as a high molecular weight aggregate near the void volume, and another at 22 kDa corresponding to the size of a dimer. (C) Purity of YscF and YscF35/67 proteins as analyzed by SDS-PAGE and Coomassie blue staining of the peak fractions.
Figure 5.
Engineering of F1, V, and YscF antigens and display on phage T4 nanoparticle.
(A) Schematic of Soc-fusions. Soc is shown in red and YscF in brown. Rest of the colors represents the same as shown in Figures 2 and 3. V-Soc, V was fused to the NH2-terminus of Soc; V-Soc-YscF, V was fused to the NH2-terminus and YscF to the COOH-terminus of Soc; F1mut-V-Soc, F1mut-V was fused to the NH2-terminus of Soc; F1mut-V-Soc-YscF, YscF was fused to the COOH-terminus of F1mut-V-Soc; F1mut-V10-Soc, F1mut-V10 was fused to the NH2-terminus of Soc. (B) The Soc fusion proteins in panel A were over-expressed and purified as described in Materials and Methods. The purity of the proteins was evaluated by SDS-PAGE and Coomassie blue staining. Lanes: M, molecular weight standards; 1, V-Soc; 2, F1mut-V-Soc; 3, F1mut-V10-Soc; 4, V-Soc-YscF; 5, F1mut-V-Soc-YscF. (C) Display of F1mut-V-Soc on phage T4. Approximately 3×1010 Hoc−Soc− phage particles were incubated at the indicated ratios of F1mut-V-Soc molecules to capsid binding sites and display was carried out as described in Materials and Methods. Lanes: Phage control, Hoc− Soc− phage used in the experiment (the position of gp23* band is labeled with a black arrow); U and B represent the unbound and phage-bound fractions. See the appearance of F1mut-V-Soc band in the bound lanes (red arrow), which is not present in the phage control. (D) Saturation binding curve of F1mut-V-Soc. The density volumes of bound and unbound proteins from SDS-PAGE (C) were determined by laser densitometry and normalized to that of gp23* present in the respective lane. The copy numbers were determined in reference to gp23* (930 copies per capsid). The data were plotted as one site saturation ligand binding curve and fitted by non-linear regression using the SigmaPlot10.0 software and the calculated binding parameters are shown. Kd, apparent binding constant; Bmax, maximum copy number per phage particle. (E and F) Cryo-electron micrograph of wild-type control phage T4 (E) and phage T4 decorated with F1mut-V (F). Arrows point to a layer of fuzzy projections around the perimeter of the capsid in the F1mut-V decorated phage. (G) Various Soc fusion proteins displayed on phage T4 for immunizations. Lanes: M, molecular weight standards; Phage control, Hoc− Soc− phage used in the experiment; 1, V-Soc; 2, F1mut-V-Soc; 3, V-Soc-YscF; 4, F1mut-V-Soc-YscF; 5, F1mut-V10-Soc. Red arrows show the positions of various displayed protein bands. Presence of a second fainter and shorter band in lanes 3 and 4 (blue arrows) indicate that some of the C-terminally fused YscF was cleaved off by nonspecific proteolysis.
Figure 6.
The soluble monomeric F1 mutant protein elicits robust antibody titers and provides complete protection in a mouse model of pneumonic plague.
The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were evaluated in a mouse model. (A) Balb/c mice, twelve per group, were vaccinated with various plague antigens adjuvanted with alhydrogel. (B) Immunization scheme. (C) Antigen-specific antibody (IgG) titers were determined by ELISA, using purified V (I), F1mut2 (II), or YscF35/67 (III) as the coating antigen. No significant cross-reactivity was observed between the antibodies produced against one plague antigen versus a different plague antigen that was coated on the ELISA plate. Error bars represent S.D. “***” denotes p<0.001 (ANOVA). (D) Survival of immunized mice against intranasal challenge with 90 LD50 of Y. pestis CO92. The survived mice were re-challenged with 9,800 LD50 at day-48 post-first challenge. See Materials and Methods for additional details. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.
Figure 7.
The T4 nanoparticle displayed plague immunogens induced robust immunogenicity and protective efficacy against pneumonic plague.
The immunogenicity and protective efficacy of T4 displayed plague immunogens were evaluated in a mouse model using the same immunization scheme shown in Figure 6B. (A) The T4 displayed plague immunogen groups, twelve mice per group. The Soc-fused plague immunogens were displayed on T4 phage particles and were directly used for vaccination without any adjuvant. (B) Antigen-specific antibody (IgG) titers as determined by ELISA. (C) Survival of vaccinated mice against intranasal challenge with 90 LD50 of Y. pestis CO92. The survived mice were re-challenged with 9,800 LD50 at day-48 post-first challenge. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.
Figure 8.
The T4 displayed plague immunogens generated balanced TH1
(IgG1) and TH2 (IgG2a) responses. The immunization scheme is shown in Figure 6B. Seven days after the second boost, sera were collected and IgG1 (A) and IgG2a (B) titers were determined by ELISA. F1mut-V was used as the coating antigen, since it covers all the epitopes present in both F1mut-V and F1mut-V10. Note that the sera of the control T4 phage-immunized mice showed higher background. This was because T4 phage, as demonstrated in previous studies, induces a strong antibody response to its components. Consequently, the sera from T4 phage- immunized mice will have increased amounts of IgGs compared to the pre-immune sera, giving more non-specific background at low dilutions of the sera. Data shown are the antibody titers of 12 mice in each group with S.D. (error bars). *, p<0.05; ***, p<0.001 (ANOVA).
Figure 9.
The F1mut-V and F1mut-V10 mutants show comparable immunogenicity and protection against pneumonic plague.
The immunogenicity and protective efficacy of F1mut-V and F1mut-V10 were compared both as adjuvanted soluble antigens or adjuvant-free T4 nanoparticle decorated antigens. (A) The vaccine formulations used in the study, eight mice per group. (B) Total F1-V specific antibody titers as determined by ELISA. Note that the sera of the control T4 phage-immunized mice showed higher background than the pre-immune sera, probably because T4 phage induces a strong antibody response to its components which raises the levels of the IgGs in the sera and gives more non-specific background at low dilutions. (C) Survival of vaccinated mice against intranasal challenge with 5,350 LD50 of Y. pestis CO92. The survived mice were re-challenged with 20,000 LD50 at day-88 post-first challenge. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.
Figure 10.
Induction of proinflammatory cytokines by F1mut-V and F1mut-V10 immunogens.
Seven days after the second boost (day-49), mice (5 per group) were sacrificed and spleens were harvested. The splenocytes were cultured and stimulated by purified F1-V protein. Cytokines levels were determined as described in Materials and Methods.
Figure 11.
The mutated and T4 displayed plague antigens provided complete protection against Y. pestis CO92 in a Brown Norway rat model of pneumonic plague.
(A) Vaccine formulations used in various groups, twelve rats per group. The rats were immunized as per the basic scheme shown in Figure 6. The soluble antigens (groups 2–4) were adjuvanted with alhydrogel. The T4 displayed groups contained no adjuvant. (B) Survival of vaccinated rats against intranasal challenge with 5,000 LD50 of Y. pestis CO92. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.