Study examines lyophilized mRNA-lipid nanoparticle vaccines with long-term stability

Vaccines have been proven to be efficacious in controlling the spread of coronavirus disease 2019 (COVID-19). Among the different kinds of vaccines available, mRNA vaccines became the frontrunners in mitigating the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to its high efficacy and sequence-independent manufacturing process—which could be quickly updated against a new variant, together with a low manufacturing cost.

Currently approved mRNA vaccines depend on lipid or lipid-like delivery systems for their transfection efficacy. Due to the demanding requirements of the lipid components and sensitivity of the mRNA to moisture, oxygen, pH and enzymes, cryogenic preservation and transportation (-20°C to -70°C) of the mRNA vaccines is necessary to maintain its stability. This impedes its accessibility in remote areas.

Lyophilization is a mild drying method that removes water by sublimation at low temperatures under a vacuum. This process could improve the stability of lipid nanoparticles as well as increase the storage temperature of mRNA to 4°C or room temperature for long durations. However, lyophilization of the mRNA- lipid complex is a sophisticated process as it would induce mechanical forces potent enough to deform the vehicle structure.  This can cause vehicle aggregation, mRNA breakage or mRNA leakage. Studies have revealed that this process can even reduce the transfection efficacy of the mRNA vaccine.

The study

A study was posted to the bioRxiv* preprint server where an optimized lyophilization technique was proposed, which was hypothesized to be able to effectively sustain the bioactivity and physicochemical properties of the mRNA-lipid nanoparticles (mRNA-LNP) as a result of which it could be stored at 2°C~8°C for long durations.

The wide applicability of this process was demonstrated by verifying the thermostability conferred by it with LNPs containing different mRNA molecules. The proposed lyophilization technique was further utilized to prepare a thermostable mRNA-LNP vaccine – encoding the antigen for Wild type, Delta and Omicron variants of SARS-CoV-2. The prevention ability and the high leveled antibody response were confirmed.

For this research, the SARS-CoV-2 N-terminal domain (NTD)-receptor-binding domain (RBD) region was used as an antigen. LNPs were prepared and characterized. Lyophilization was carried out by adding LNP solution with the cryoprotectant, filling the mixture in a penicillin bottle and carrying out the process with a freezer dryer – using a designed procedure. This resulted in a powder that was characterized and stored at 4°C for further use.

The stability of the lyophilized LNPs was measured by incubating them at either 4°C, 25°C or 40°C for a different time. The integrity of the mRNA was measured using microfluidic capillary electrophoresis and a gel retardation assay. ACE2- expressing 293T and HEK 293T/17 cells were cultured.

The in vivo transfection efficacy of Luciferase mRNA-loaded LNPs (mRNA- Luc LNPs) and its lyophilized product Lyo-mRNA- Luc-LNPs was examined. For the immunization experiment in mice, three kinds of LNPs were prepared with mRNA encoding the antigen of WA1, Delta or Omicron variants of SARS-CoV-2. An ELISA, a Pseudotyped virus neutralization assay, a SARS-CoV-2 Delta neutralization assay and a SARS-CoV-2 Delta challenge in K18-hACE2 KI transgenic mice were then carried out.

Results

All the four LNPs used in the study showed high encapsulation efficacy (EE) and narrow size distribution. After lyophilization, the EE, size and polydispersity index (PDI) didn’t change indicating that the process didn’t alter the basic physical properties. The integrity of the mRNA was also maintained which suggested that the process didn’t damage the mRNA structure.

On incubating the Luc LNPs for different times to evaluate the thermostability, no change was found. The EE was sustained at 4°C and 25°C after 18 days. However, this parameter increased at 40°C, although the EE and mRNA integrity was maintained. From these findings, it could be estimated that after lyophilization by the given process, the LNPs could be stored in a refrigerator for long durations at 2~8°C.

On assessing the in vivo transfection efficacy of the lyophilized LNPs, well-maintained bioactivity of the mRNA-Luc LNPs was seen. The immunogenicity of the lyophilized mRNA-WT LNPs was assessed following a prime-boost regimen where mice were injected intramuscularly with mRNA-WT LNPs or Lyo-mRNA-wild-type (WT) LNPs. The immunization was done at 0 and on day-7. It was found that all the vaccine-induced high IgG titers. The pseudotyped virus assay demonstrated that the lyophilization process didn’t affect the bioactivity of the antibodies.

On evaluating a prepared mRNA-Delta vaccine for its efficacy against Delta and other variants of SARS-CoV-2 in mice, it was found that the vaccine induced much higher neutralizing antibody titers against an antigen of the wild-type (WA1) than the Delta variant.

On testing the antigenicity of the lyophilized mRNA vaccine against omicron (Lyo-mRNA-Omicron), robust neutralization antibody titers against Omicron, along with a lower degree of neutralization response against the Delta variant were detected.

On evaluating the efficacy and immunogenicity of Lyo-mRNA- Delta in K18-hACE2 KI mice it was found that the vaccine fully protected the mice from the SARS-CoV-2 Delta infection, proving its high immunogenicity.

The study proved the high immunogenicity and stability of lyophilized mRNA-LNP vaccines. Therefore, it was inferred that this vaccine was a suitable choice for use as a measure of control for the COVID-19 pandemic, and hence, its clinical trials may be promptly initiated.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Nidhi Saha

I am a medical content writer and editor. My interests lie in public health awareness and medical communication. I have worked as a clinical dentist and as a consultant research writer in an Indian medical publishing house. It is my constant endeavor is to update knowledge on newer treatment modalities relating to various medical fields. I have also aided in proofreading and publication of manuscripts in accredited medical journals. I like to sketch, read and listen to music in my leisure time.