Journal: Scientific Reports

Article Title: Ensilication Improves the Thermal Stability of the Tuberculosis Antigen Ag85b and an Sbi-Ag85b Vaccine Conjugate

doi: 10.1038/s41598-019-47657-9

Figure Lengend Snippet: Ensilication protects Sbi III-IV-Ag85b against thermal denaturation. ( a ) CD spectra showing partially reversible thermal unfolding of Sbi III-IV-Ag85b. CD spectra at 5 °C, 20 °C, 85 °C and upon return to 20 °C are displayed in the inset figure. Deconvolution was performed using DichroWeb. ( b ) Boltzmann sigmoidal fit of CD thermal denaturation curve at 222 nm indicating a melting temperature of 46.3 °C. ( c ) Temperature-induced loss of Sbi III-IV-Ag85b structural integrity visualised by SDS-PAGE. ( d ) Time-course dynamic light scattering size distribution by intensity plot of Sbi III-IV-Ag85b ensilication in 50 mM Tris pH 7 showing a maximal particle size of 55 nm. ( e ) Sbi III-IV-Ag85b ensilication trials for visualisation of sol-gel precipitates showing that MgCl 2 , CaCl 2 , glycine and Sbi III-IV improve precipitate formation. Water and Sbi III-IV alone were included as negative and positive controls respectively. ( f ) SDS-PAGE analysis showing protein levels in the released solution and unensilicated supernatant following ensilication of Sbi III-IV-Ag85b under the four conditions depicted. See Supplementary Table for protein concentrations. ( g ) Anti-Sbi western blot analysis of C3 activation in normal human serum following incubation with native or released Sbi III-IV-Ag85b. Higher molecular weight bands depicting opsonisation of native and released Sbi III-IV-Ag85b with C3 activation products are evident. All samples are at a concentration of 27 μM except for the released sample containing Sbi III-IV which is at 18 μM. ( h ) Alternative pathway activity analysis showing comparable complement depletion activity for native Sbi III-IV-Ag85b (4 µM) and Sbi III-IV-Ag85b (4 µM) released following ensilication under the conditions shown. Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. A one-way ANOVA with Dunnett’s multiple comparisons analysis confirmed no significant differences in activity between native Sbi III-IV-Ag85b and the released Sbi III-IV-Ag85b samples ( P > 0.05). ( i ) CD spectroscopy showing the near-identical secondary structures of native and released Sbi III-IV-Ag85b. Spectra are representative of 3 replicates. Deconvolution was performed using DichroWeb. ( j ) Alternative pathway assay depicting comparable functional activities for native Sbi III-IV-Ag85b (4 μM) and Sbi III-IV-Ag85b (4 µM) released subsequent to ensilication in 50 mM glycine pH 7.4. Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. An unpaired t-test confirmed no significant difference in activity between native and released Sbi III-IV-Ag85b ( P > 0.05). ( k ) SDS-PAGE profile illustrating thermal denaturation and aggregation in native but not released Sbi III-IV-Ag85b samples. ( l ) Alternative pathway activity analysis displaying retention of complement depletion activity in released but not in native thermally-treated Sbi III-IV-Ag85b (4 μM). Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. A one-way ANOVA with Dunnett’s multiple comparisons analysis was used to analyse differences in activity compared to native untreated Sbi III-IV-Ag85b. * P < 0.01. DLS, CD and opsonisation analysis of thermally-treated native and released Sbi III-IV-Ag85b can be found in Supplementary Figs – respectively. Full-length gel images of ( c , f , k ) are included in Supplementary Figs – and full-length western blot image of ( g ) is shown in Supplementary Fig. .

Article Snippet: 10 μl of pooled complement human serum (Patricell) was mixed with an equal volume of untreated or thermally-treated native or released Sbi III-IV-Ag85b or Sbi III-IV.

Techniques: Circular Dichroism, SDS Page, Western Blot, Activation Assay, Incubation, Molecular Weight, Concentration Assay, Activity Assay, Standard Deviation, Functional Assay

Journal: Scientific Reports

Article Title: Ensilication Improves the Thermal Stability of the Tuberculosis Antigen Ag85b and an Sbi-Ag85b Vaccine Conjugate

doi: 10.1038/s41598-019-47657-9

Figure Lengend Snippet: Thermostabilisation of Sbi III-IV using ensilication. ( a ) Thermal melt circular dichroism spectroscopic study showing reversible thermal unfolding of Sbi III-IV. CD spectra at 5 °C, 20 °C, 85 °C and upon return to 20 °C are displayed in the inset figure. Deconvolution of CD data was performed using DichroWeb. ( b ) Boltzmann sigmoidal fit of CD thermal denaturation curve at 222 nm depicting a melting temperature of 35.6 °C. ( c ) Temperature-induced loss of Sbi III-IV structural integrity visualised by SDS-PAGE. ( d ) Thermal denaturation-mediated loss of Sbi III-IV (0.5 μM) complement depletion activity demonstrated by alternative pathway activity analysis. Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. A one-way ANOVA with Dunnett’s multiple comparisons analysis was used to analyse differences in activity compared to native untreated Sbi III-IV., * P < 0.01. ( e ) FESEM image of ensilicated Sbi III-IV powder at 20,000x magnification. Scale bar is shown at the bottom of the image. See Supplementary Fig. for FESEM images at 650x and 5,000x magnification. ( f ) CD spectra showing similar secondary structures for native and released Sbi III-IV. Spectra are representative of 10 replicates. Deconvolution of CD data was performed using DichroWeb. ( g ) Anti-Sbi western blot analysis illustrating comparable C3 activation by native and released Sbi III-IV (both at 27 μM). ( h ) Alternative pathway activity analysis demonstrating comparable C3 depletion activities for native and released Sbi III-IV (both at 0.5 μM). Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. An unpaired t-test confirmed no significant difference in activity between native and released Sbi III-IV ( P > 0.05). ( i ) SDS-PAGE illustrating thermal degradation of native but not released Sbi III-IV. A small amount of dimeric Sbi III-IV is present in all released samples. ( j ) Alternative pathway activity analysis showing temperature-induced loss of function in native but not in released Sbi III-IV (0.5 μM) samples. Data are displayed as mean values (n = ≥ 2) ± standard deviation from the mean. A one-way ANOVA with Dunnett’s multiple comparisons analysis was used to analyse differences in activity compared to native untreated Sbi III-IV. * P < 0.01. DLS, CD and opsonisation analysis of thermally-treated native and released Sbi III-IV can be found in Supplementary Figs – respectively. Full-length gel images of ( c , i ) are included in Supplementary Figs and . Full-length western blot image of ( g ) is shown in Supplementary Fig.

Article Snippet: 10 μl of pooled complement human serum (Patricell) was mixed with an equal volume of untreated or thermally-treated native or released Sbi III-IV-Ag85b or Sbi III-IV.

Techniques: Circular Dichroism, SDS Page, Activity Assay, Standard Deviation, Western Blot, Activation Assay