Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from <i>Yersinia pestis</i> as Next Generation Plague Vaccines

<div><p>Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism <i>Yersinia pestis</i> poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH₂-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced T_H1 and T_H2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of <i>Y. pestis</i> CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.</p></div

Designing monomeric F1 mutants.

<p>(<b>A</b>) 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>B</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 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p

New plague immunogen designs.

<p>Schematic of various approaches used to design plague immunogens. See text for details. (<b>A</b>) <i>Y. pestis</i> 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>B</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. (<b>C</b>) Deletion of the putative immunomodulatory sequence (aa residues 271–300) of V antigen. (<b>D</b>) Mutagenesis of Asn35 and Ile67 to produce an oligomerization deficient YscF. (<b>E</b>) 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. (<b>F</b>) 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.</p

An oligomerization deficient YscF mutant.

<p>(<b>A</b>) Schematic of native YscF and YscF35/67 mutants. (<b>B</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. (<b>C</b>) Purity of YscF and YscF35/67 proteins as analyzed by SDS-PAGE and Coomassie blue staining of the peak fractions.</p

Construction of mutated F1-V immunogens.

<p>(<b>A</b>) 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g002" target="_blank">Figure 2</a>. (<b>B</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 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) 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. (<b>D</b>) 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p

Induction of proinflammatory cytokines by F1mut-V and F1mut-V10 immunogens.

<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a>.</p

The mutated and T4 displayed plague antigens provided complete protection against <i>Y. pestis</i> CO92 in a Brown Norway rat model of pneumonic plague.

<p>(<b>A</b>) Vaccine formulations used in various groups, twelve rats per group. The rats were immunized as per the basic scheme shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g006" target="_blank">Figure 6</a>. The soluble antigens (groups 2–4) were adjuvanted with alhydrogel. The T4 displayed groups contained no adjuvant. (<b>B</b>) Survival of vaccinated rats against intranasal challenge with 5,000 LD₅₀ of <i>Y. pestis</i> CO92. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.</p

The soluble monomeric F1 mutant protein elicits robust antibody titers and provides complete protection in a mouse model of pneumonic plague.

<p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were evaluated in a mouse model. (<b>A</b>) Balb/c mice, twelve per group, were vaccinated with various plague antigens adjuvanted with alhydrogel. (<b>B</b>) Immunization scheme. (<b>C</b>) 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). (<b>D</b>) Survival of immunized mice against intranasal challenge with 90 LD₅₀ of <i>Y. pestis</i> CO92. The survived mice were re-challenged with 9,800 LD₅₀ at day-48 post-first challenge. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> 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.</p

The T4 nanoparticle displayed plague immunogens induced robust immunogenicity and protective efficacy against pneumonic plague.

<p>The immunogenicity and protective efficacy of T4 displayed plague immunogens were evaluated in a mouse model using the same immunization scheme shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g006" target="_blank">Figure 6B</a>. (<b>A</b>) 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>B</b>) Antigen-specific antibody (IgG) titers as determined by ELISA. (<b>C</b>) Survival of vaccinated mice against intranasal challenge with 90 LD₅₀ of <i>Y. pestis</i> 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.</p