(M1530-02-08) A Versatile Room Temperature Stable Lipid Nanoparticle COVID-19 Protein-Based Vaccine

Purpose: Development of a thermostable COVID-19 vaccine is a challenging unmet need which limits the vaccine logistics and accessibility. The goal of present work was to develop liposomal vaccine formulations and manufacturing processes to enable thermo-stable COVID-19 vaccines that can be distributed at room temperature specially during the last mile delivery and to areas where maintaining the cold-chain is problematic.

Methods: Two different COVID-19 vaccines were developed using recombinant spike proteins of SARS-CoV-2 (S1 or S1S2) and Engimata’s patented liposomal nano lipid particle platform technology L-Vac. The vaccines murine immunogenic response was tested by injecting 2 x50 ul of each vaccine intramuscularly (im) in the leg of five female BALB-C mice/ group on days 0 and 14. Five groups of mice were tested; S1 naïve, S1 alone, S1S2 alone, lyophilized S1-L-vac, lyophilized S1S2-L-vac. Serum was collected from the immunized mice as well as naïve mice (negative control) on Days 14 and 28. Antibody response to each vaccine was determined using an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) that was designed for the detection of mouse antibodies against SARS-CoV-2 spike proteins. Vaccine particle size distribution and surface charge were determined by a Precision Detector Dynamic Light Scattering (DLS) instrument PD2000DLS^plus (Precision Detectors) and by Malvern zeta-sizer (Nano-ZS), respectively. Protein conformation of spike proteins were determined by differential scanning calorimetry (nDSF). Lyophilized vaccines were stored at 5C and 25C for 3 months and at 40C for 2 weeks and their stability was assessed by DLS.

Results: The results are listed in Table 1. nDSF results revealed that S1 protein denatures at 49C. In S1S2, two additional denaturation temperatures were observed at 57C and 69C referring to denaturation of S2 domains. Average particle size of S1 and S1S2 liposomal vaccines were 193 (± 4) and 196 (± 3), before and 198 (± 4) and 218 (±7) after lyophilization, respectively. The denaturation temperature of S1 and S1S2 in the lyophilized liposomal vaccines were very similar to those obtained for S1 and S1S2 proteins, except an increase in Tm3 in lyophilized S1S2 liposomes, compared to S1S2 alone suggesting that in these liposomes S2 is interacting with the lipid bilayer of the liposome . All vaccines had a positive surface charge by design and as shown by positive Zeta potential values. ELISA results (Figures 1 and 2) showed that lyophilized liposomal vaccines induced significantly higher IgG response to spike proteins of SARS-CoV-2. The obtained IgG response induced by S1S2 liposomes on Day 28 was 7 times greater than that induced by S1S2 proteins alone and 14 times higher than that obtained for naïve mice at the same dilution factor of sera. Stability studies showed that average particle sizes of lyophilized COVID-19 vaccines D-S1 and D-S1S2 at 25C were at 203 nm ±16 nm and 209 nm ±5 nm, respectively after 5 weeks storage at 25C and increased slightly to 243 nm ±9 nm and 244 nm ±5 nm, respectively over a 3-month period at 25C. The corresponding values for these vaccines after 2 weeks at 40C were 326 ±74 (for D-S1) and 269 ±22 (for D-S1S2) and after 5weeks at 40C, were 262 ±9 (for D-S1) and 355 ±52(for D-S1S2). In the pre-lyo formulations (C-S1 and C-S1S2) after 2 weeks at 40C, the average diameter of vaccine particles had increased to 1,257 nm ± 266 nm and 326 ± 30 nm, respectively showing that freeze-drying enhanced thermal stability of these vaccines at 40C significantly. These pre-lyo solutions were stable for 2 weeks at 25C with average particle sizes of 225 ± 5 nm and 221 ± 3 nm, respectively and without freeze-drying.

Conclusion: In conclusion the results of present study confirm that it is possible to develop a thermostable COVID-19 vaccine using L-Vac platform adjuvant and spike proteins of SARS-CoV-2. Both vaccines showed significantly greater immune response than when S1 or S1S2 spike proteins were administered alone. Vaccines were stable at 25C for at least 1 month and could be distributed at room temperature.

References: 1. Mosharraf, M., Sorayya, A., and Nayar, R., Immunogenic Composition Forming a SARS-COV-2 Vaccine, and a method for its manufacture, US Patent 11278617 Issued March 22, 2022.
2. Mosharraf, M., Sorayya, A., and Nayar, R., Immunogenic Composition Forming a Vaccine, and a method for its manufacture, US Patent 11559577B2 · Granted Jan, 2023.

Acknowledgements: The authors wish to thank HTD Biosystems and MIT Sandbox for funding.

Fig. 1. Serological IgG response to S1 spike proteins of SARS-CoV-2 after 6,400 times dilution.

Fig. 2. Serological IgG response to S1S2 spike proteins of SARS-CoV-2 after 60,000 times dilution.

Table 1. Biophysical characterization of formulations