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.2013 Jan 30;5(2):224-48.
doi: 10.3390/toxins5020224.

Disruption of the putative vascular leak peptide sequence in the stabilized ricin vaccine candidate RTA1-33/44-198

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Disruption of the putative vascular leak peptide sequence in the stabilized ricin vaccine candidate RTA1-33/44-198

Laszlo Janosi et al. Toxins (Basel)..

Abstract

Vitetta and colleagues identified and characterized a putative vascular leak peptide (VLP) consensus sequence in recombinant ricin toxin A-chain (RTA) that contributed to dose-limiting human toxicity when RTA was administered intravenously in large quantities during chemotherapy. We disrupted this potentially toxic site within the more stable RTA1-33/44-198 vaccine immunogen and determined the impact of these mutations on protein stability, structure and protective immunogenicity using an experimental intranasal ricin challenge model in BALB/c mice to determine if the mutations were compatible. Single amino acid substitutions at the positions corresponding with RTA D75 (to A, or N) and V76 (to I, or M) had minor effects on the apparent protein melting temperature of RTA1-33/44-198 but all four variants retained greater apparent stability than the parent RTA. Moreover, each VLP(-) variant tested provided protection comparable with that of RTA1-33/44-198 against supralethal intranasal ricin challenge as judged by animal survival and several biomarkers. To understand better how VLP substitutions and mutations near the VLP site impact epitope structure, we introduced a previously described thermal stabilizing disulfide bond (R48C/T77C) along with the D75N or V76I substitutions in RTA1-33/44-198. The D75N mutation was compatible with the adjacent stabilizing R48C/T77C disulfide bond and the T(m) was unaffected, whereas the V76I mutation was less compatible with the adjacent disulfide bond involving C77. A crystal structure of the RTA1-33/44-198 R48C/T77C/D75N variant showed that the structural integrity of the immunogen was largely conserved and that a stable immunogen could be produced from E. coli. We conclude that it is feasible to disrupt the VLP site in RTA1-33/44-198 with little or no impact on apparent protein stability or protective efficacy in mice and such variants can be stabilized further by introduction of a disulfide bond.

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Figures

Figure 1
Figure 1
Ricin LD50 in the intranasal challenge model of BALB/c mice. (A) Dose- and time-dependence of survival. Ten female BALB/c mice were challenged with the indicated amounts of ricin and observed twice per day for a period of two weeks; (B) Time- dependence of calculated LD50. The data shown in Panel A were submitted to probit analysis to determine the LD50 of ricin at every 0.5-day intervals following ricin challenge and the calculated LD50 values were plotted versus time. The expression “LD50” with an arrow indicates that we regarded a challenge with 2.5 μg/kg ricin with a 13 day post-challenge observation window as a representative LD50 in this model.
Figure 2
Figure 2
Mouse lung histopathology after intranasal ricin challenge. Ten female BALB/c mice were challenged intranasally with 10 μg/kg ricin, and on day 2, post-challenge mice were anesthetized and their lungs processed for histopathology. The panels above are representative photographs of the pathological changes in mice exposed to ricin. (A) Inflammatory cell infiltrates (large arrowheads) surround bronchi (**) and blood vessels (*), and many alveoli are filled with proteinaceous fluid (edema) evident as pink staining material (small arrowheads). Note also the presence of perivascular edema and hemorrhage affecting the vessel adjacent to the small bronchus; (B) Alveoli are largely filled by fibrin (arrowhead), and there are also some degenerate neutrophils and necrotic debris evident; (C) Only a limited region of the epithelium lining a bronchus remains viable (arrowheads); the remainder is necrotic. Note the edema of the peribronchial connective tissue and infiltration by neutrophils, many of which are degenerate. The pathological changes evident can be compared to the normal mouse lung histology shown in (D).
Figure 3
Figure 3
Protection provided by different doses of RTA1-33/44-198 vaccine against intranasal challenge with 10 LD50 ricin. Groups of 20 mice were vaccinated i.m., either with 2.5 μg, 10 μg, or 40 μg/mouse RTA1-33/44-198, in the presence of Rehydrogel adjuvant with two booster immunizations two weeks apart after priming. One group of mice received only the adjuvant (sham vaccine). Four weeks after the last booster immunization, the mice were intranasally challenged either with 10 LD50 ricin in PBS or PBS lacking ricin (sham challenge). Mice were observed twice daily for survival (A), once daily for body weights (B) and once daily for disease signs (C). Qualitative grading of the disease between 0 and 4 was as described in Materials and Methods. In panels (A) and (B), symbols represent group mean values and vertical bars show one standard deviation.
Figure 4
Figure 4
Protection provided by different doses of RTA1-33/44-198 vaccine against intranasal challenge with 10 LD50 ricin. Groups of 20 mice were vaccinated i.m., either 2.5 μg, 10 μg, or 40 μg/mouse RTA1-33/44-198, in a prime-booster-booster strategy two weeks apart. One group of mice received only the adjuvant, but not the immunogen (sham vaccine). Four weeks after the last booster immunization, the mice were challenged intranasally either with 10 LD50 ricin in PBS or PBS lacking ricin (sham challenge). On day 2 post-challenge, 10 mice in each group were sacrificed, and protein concentration in the BALF and total blood glucose concentrations were determined. The protein and glucose concentrations were repeated on day 13. (A) Protein concentrations in the BALF on day 2 post-challenge; (B) Protein concentrations in the BALF on day 13 post-challenge; (C) Blood glucose concentrations on day 2 post-challenge; (D) Blood glucose concentrations on day 13 post-challenge. In all panels, the columns represent group mean values, and the vertical bars show one standard deviation.p values above the lines with a knob on the left were calculated using an unpairedt-test to compare group mean values between the sham vaccine group and each group of vaccinated animals following ricin exposure;p values above the lines with a knob on the right were calculated to compare group mean values between the sham challenge group with each group of vaccinated animals.
Figure 5
Figure 5
Apparent thermal stability of VLP mutants of the RTA1-33/44-198 immunogen. (A) Temperature melting curves. Changes in molecular structure of the proteins induced by elevated temperatures were monitored by circular dichroism (C,D) spectroscopy at 222 nm. Data are expressed in percent of the initial ellipticity observed at 10 °C; (B) Melting temperatures associated with 50% loss of ellipticity of the molecules. Error bars indicate one standard deviation of the data of three independent experiments.
Figure 6
Figure 6
Comparison of the VLP sites among ricin and three different RTA-based immunogens (labeled in figure). The B-cell epitope recognized by the Univax R70 antibody is shown in magenta. The L161–I175 epitope bound by human neutralizing antibodies characterized by Castelletti [21] is shown in green. The T-cell epitope is shown in blue; this epitope is found to be fully helical in the truncated 1-33/44-198 disulfide-bonded immunogens.
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References

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