Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Figures

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  Figure 1. Primary structure analysis of the 3 NRRV antigens. Representative deconvoluted mass spectra for analysis of intact proteins under reducing condition for (a) P[4], (b) P[6], and (c) P[8]. Representative total ion chromatogram from peptide map analysis of reduced, alkylated, and chymotrypsin digested proteins for (d) P[4], (e) P[6], and (f) P[8]. See Supplemental Figure S1 for intact mass analysis data under nonreducing condition and sequence coverage (100% for both reducing and nonreducing conditions) from peptide map analysis.

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  Figure 2. Higher-order structure (HOS) analysis of each of the 3 NRRV protein antigens at 10°C in 10 mM PBS, pH 7.2. Representative FTIR absorbance spectrum of the amide I region and Fourier self-deconvoluted peaks for (a) P[4], (b) P[6], (c) P[8] antigens, and (d) secondary structure composition of each antigen obtained from Fourier self-deconvolution of FTIR spectrum. (e) Far-UV CD spectra and (f) intrinsic tryptophan fluorescence emission spectra overlay for the 3 antigens. Error bars denote 1SD from triplicate measurements.

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  Figure 3. Size, aggregation, and heterogeneity analysis of each of the 3 NRRV antigens in 10 mM PBS, pH 7.2. (a) Representative SV-AUC sedimentation coefficient distribution profiles from 0 to 14 Svedbergs, inset shows c(s) distribution from 4 to 14 Svedbergs for better visualization of aggregate species peak(s). (b) Representative SE-HPLC chromatograms for 3 antigens with inset showing the peaks corresponding to fragments and/or aggregate species. (c) SDS-PAGE analysis under nonreducing and reducing (+DTT) conditions. (d) Representative HIC chromatograms and (e) representative RP-HPLC chromatograms of the 3 antigens overlay with inset showing the minor peaks for better visualization. DTT, dithiothreitol; SV-AUC, sedimentation velocity analytical ultracentrifugation.

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  Figure 4. Higher-order structure (HOS) stability and aggregation propensity of the NRRV antigens in 10 mM PBS, pH 7.2, as a function of thermal stress (10°C-90°C). (a) Far-UV CD normalized mean residue ellipticity at 216 nm, (b) intrinsic tryptophan fluorescence normalized MSM peak intensity, (c) representative DSC thermograms of each antigen, (d) extrinsic ANS fluorescence MSM peak intensity, (e) static light scattering intensity at 295 nm, and (f) OD350 values for P[4] (black), P[6] (red), and P[8] (blue) antigens. Bar graphs on the right side of each panel show thermal onset (Tonset) and thermal melting temperature (Tm) values for each antigen. Error bars represent 1SD from triplicate measurements. DSC, differential scanning calorimetry; MSM, mean spectral center of mass.

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  Figure 5. Biophysical stability profile from radar chart analysis of each NRRV antigen versus temperature across the pH range of 3.0 to 8.0. Sample buffer is 20 mM citrate phosphate buffer containing 150 mM NaCl at indicated pH value. (a) P[4], (b) P[6], and (c) P[8] show radar charts generated from multivariate stability data sets shown in Supplemental Figure S2. Radar chart key is provided in the bottom panel (see text for further explanation).

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  Figure 6. Forced degradation (Asn deamidation) studies of each NRRV antigen after incubation at pH 9.0 25°C for 6 d. (a) Monomer species mass from intact mass analysis of reduced (red) and nonreduced (black) samples for each antigen. (b) Representative total ion chromatogram from peptide map analysis of reduced, alkylated, and chymotrypsin digested P[4] samples, pH 7.2 4°C (top) and pH 9.0 25°C (bottom), native Asn peaks are pointed by red arrows and deamidated Asn peaks are pointed out by blue arrows (see Supplemental Fig. S3 for peptide map data with P[6] and P[8] antigens). (c) Percentage deamidation of different Asn residues in each antigen obtained from the peptide mapping analysis under reducing conditions. Error bars represent 1SD from triplicate vials.

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  Figure 7. Forced degradation (non-native disulfide formation) studies with P[6] antigen after incubation at pH 9.0 25°C for 6 d (a) SDS-PAGE analysis, (b) intact mass analysis, and (c) RP-UHPLC analysis of the stressed and control samples, further, MS1 spectra confirming the molecular masses of the eluting peaks to the P[6] monomer and dimer species are shown. Refer to Supplemental Figures S4 and S5 for similar data with P[4] and P[8] antigens, respectively. RP-UHPLC, RP ultra-high pressure chromatography.

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  Figure 8. Forced oxidation studies with NRRV antigens as a function of hydrogen peroxide concentration. Representative (a) deconvoluted mass spectra for intact mass analysis, and (b) peptide map analysis of P[6] antigen under reducing (+DTT) condition. See Supplemental Figures S6-S8 for the complete data sets of the 3 antigens. (c) Hydrogen peroxide effect on the relative percent oxidation of 2 of 2 methionine residues for P[6], P[8], and 2 of 3 methionine residues for P[4]. (d) % total area and retention time of main peak in RP-HPLC chromatograms of each antigen, see Supplemental Figure S9 for individual chromatograms. Error bars represent 1SD from triplicate measurements.

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  Figure 9. Structural modeling of the 3 NRRV protein antigens. (a) Schematic description of the protein composition and nomenclature of each NRRV antigen, and (b) 3D structure prediction of each NRRV antigen using I-TASSER modeling; DVP8* protein is shown in gray, GSGSG linker in cyan, and P2 epitope in black. Residues susceptible to chemical degradation under stressed conditions are highlighted (Cys—red, Asn—green, Met—magenta).