(M1430-11-60) Characterization of Protein Antigens in Liposomal Vaccines Using Differential Scanning Fluorimetry

Purpose: Vaccines contain microgram quantities of antigens. This makes characterization of the antigens and their interaction with the vaccine problematic. The purpose of this study was to evaluate the potential of using differential scanning fluorimetry to evaluate the interaction between protein antigens and liposomal adjuvants in different vaccine designs.

Methods: Different vaccines were developed using recombinant spike proteins of SARS-CoV-2 (S1 or S1S2) and L-Vac liposomal adjuvant. In one vaccine design, the antigens were mixed with the pre-made liposomal adjuvants of opposing charge to create liposomal particles with the protein antigen attached to their exterior surface. These were referred to as A-S1 and A-S1S2. In another design, each spike protein was entrapped in the lipid-blend, extruded, and freeze-dried. These were referred to as C-S1 and C-S1S2 before and D-S1 and D-S1S2 after freeze-drying, respectively. Differential scanning fluorimetry (nano DSF) was used to determine the denaturation temperature (Tm) of the proteins before and after interaction with or entrapment in the liposomes. Each sample was run at least in duplicate without sample manipulation. The samples were heated from 20°Cto 90°C at 1.5°C /min and the denaturation temperatures of proteins were characterized using the instrument software. Liposomes and proteins alone were run as controls.

Results: The results are shown in Table 1. As it is shown in Table 1, one melting temperature was obtained for protein S1 at 49°C (±0°C). For protein S1S2, three melting temperatures were obtained at 50°C (±1°C), 57°C (±2°C), and 69°C (±0°C), n=3 per sample. After the proteins were entrapped in the liposomes and freeze-dried, the melting temperatures of the S1 domain shifted slightly to 51°C (D-S1) and 51°C (±2°C) (D-S1S2). Also, in the D-S1S2 vaccines, Tm2 and Tm 3 shifted to higher temperatures of 59°C (±4°C) and 73°C (±3°C), respectively, suggesting that in these vaccine designs, the S2 domains of the S1S2 protein are interacting with the lipid bilayer. Fluorescence Ratio by nDSF showed that each protein regains its initial conformation after freeze-drying of vaccines.

Conclusion: It was possible to evaluate the interaction between the recombinant spike protein antigens of SARS-COV-2 at 10 ug/mL and the lipid bilayer of liposomal adjuvant L-Vac without any sample manipulation using nDSF. It was shown that in vaccines made of S1-liposomes, the S1 domain was interacting with the protein, where as in vaccines containing S1S2 protein antigen, the S2 domains interacted with the lipid bilayer more than the S1 domain. The extent of this interaction was independent of the location of the spike proteins (on the exterior surface or entrapped in the liposomes) and similar results were obtained in both cases.  These results illustrate the utility of using nanoDSF as a powerful tool to investigate the conformation and stability of protein antigens on liposomal based vaccines.


Table 1. Biophysical Characterization Data