key: cord-0773622-s3edk157 authors: Ai, Liangxia; Li, Yafei; Zhou, Li; Zhang, Hao; Yao, Wenrong; Han, Jinyu; Wu, Junmiao; Wang, Ruiyue; Wang, Weijie; Xu, Pan; Li, Zhouwang; Wei, Chengliang; Chen, Haobo; Liang, Jianqun; Guo, Ming; Huang, Zhixiang; Wang, Xin; Zhang, Zhen; Xiang, Wenjie; Lv, Bin; Peng, Peiqi; Zhang, Shangfeng; Ji, Xuhao; Li, Zhangyi; Luo, Huiyi; Chen, Jianping; Lan, Ke; Hu, Yong title: Lyophilized mRNA-lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2 date: 2022-02-11 journal: bioRxiv DOI: 10.1101/2022.02.10.479867 sha: a245d0303693b7ca942edb17dcb3e880fde46918 doc_id: 773622 cord_uid: s3edk157 Advanced mRNA vaccines play vital roles against SARS-CoV-2. However, due to the poor stability, most current mRNA delivery platforms need to be stored at −20 °C or −70 °C. Here we present lyophilized thermostable mRNA loaded lipid nanoparticles, which could be stored at room temperature with long-term stability. We demonstrate the applicability of lyophilization techniques to different mRNA sequences and lipid components. Three lyophilized vaccines targeting wild-type, Delta and Omicron SARS-CoV-2 variant were prepared and demonstrated to be able induce high-level of IgG titer and neutralization response. In the Delta challenge in vivo experiment, the lyophilized mRNA vaccine successfully protected the mice from infection and clear the virus. This lyophilization platform could significantly improve the accessibility of mRNA vaccine or therapeutics, particularly in remote regions. After years of research and development, mRNA delivery systems made their breakthrough and became the frontrunners that prevent coronavirus disease 2019 . Compared with inactivated vaccines and recombinant protein vaccine, mRNA vaccines could be easily and quickly updated to target different variants, and with comparable first-class protective efficacy as well [1] . The sequence-independent manufacturing process saved a lot of time and cost to develop a new vaccine, especially in the pandemic situation, just as the situation fighting against the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [2, 3] . Current mRNA therapeutics heavily depend on lipid or lipid-like delivery systems to improve their in vivo transfection efficacy [4] . Several lipid components form the nanoparticles carrying the mRNA to targeted organelles. Although mRNA therapeutics show such superiority, challenges about stability still impede their accessibility. Cryogenic preservation and transportation are needed for two current licensed mRNA vaccines, BNT162b2 (-80 o C ~-60 o C) and mRNA-1273 (-20 o C) [5] . The demanding requirements come from the complex interactions among multiple lipid components and the instability of mRNA, which is sensitive to oxygen, moisture, enzymes, pH and even more [6] . As the exact mechanisms have not been solved, how to improve the stability of mRNA therapeutics is still challenging [7] . Lyophilization is a process that removes water by sublimation under vacuum at a low temperature [8] . It's a relatively mild drying method and could improve the stability of vulnerable macrobiomolecules or colloidal nanoparticles [9] . The lyophilized mRNA could be stored at 4 o C or at room temperature for long time [10, 11] . However, drying of mRNA-lipid nanoparticles (mRNA-LNP) is more sophisticated, as the freezing and the dehydration process would induce mechanical force and deform the vehicle structure, leading to vehicle aggregation, mRNA breakage or leakage [12] . Moreover, some research showed that even the mRNA-LNP remained their integrity and encapsulation efficiency, the in vivo transfection efficacy was greatly reduced after lyophilization due to some unknown reasons [13] . Here we present an optimized lyophilization technique, which could effectively sustain the physiochemical properties and bioactivity of mRNA-LNP and achieve long-term storage at 2 o C ~8 o C. The improved thermostability was verified with LNPs containing different mRNA molecules, demonstrating its wide applicability. Furthermore, we utilized this technique to prepare the first lyophilized, thermostable mRNA-LNP vaccines with encoding the antigen of wild-type (WA1, Lyo-mRNA-WT), Delta (Lyo-mRNA-Delta) or Omicron COVID-19 variant (Lyo-mRNA-Omicron) and confirmed their high-leveled antibody responses and prevention ability. Noticeably, we optimized the mRNA sequences in theses lyophilized mRNA vaccines. The lyophilized vaccine could both induce strong immune response and produce high levels of neutralizing antibody titer. The mRNA-Delta and mRNA-Omicron vaccine also showed crossprotection ability against other variants. In the challenge study of mRNA-Dela vaccine against Delta strain, the vaccine also demonstrated to fully protect the mice from infection and clear the virus. Lipids were purchased from Xiamen Sinopeg Biotech Co., Ltd. Cholesterol was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Ethanol was obtained from Aladdin. Citric acid and trisodium citrate were obtained from Sigma-aldrich. SARS-COV-2 WA1, Delta and Omicron pseudovirus using recombinant replication-deficient vesicular stomatitis virus (VSV) vector that encodes Luciferase instead of VSV-G (VSVΔG-Luc) were obtained from Vazyme. SARS-COV-2 NTD-RBD region were used as antigens in this work. The respective mRNAs were produced in vitro by standard T7 RNA polymerase-mediate transcription reaction and added with Cap1, then purified though Fast Protein Liquid Chromatography. Luciferase mRNA were synthesized as the same way and purified by MEGAclear™ Transcription Clean-Up Kit (Invitrogen). The lipid nanoparticles (LNPs) were prepared by mixing an aqueous phase containing the mRNA with an ethanol phase containing the lipid mixtures using a T-junction mixing device as reported previously [14] . In brief, the mRNA was dissolved in a 100 mM citric buffer (pH 4.0). The lipid mixture was dissolved in anhydrous ethanol with the molar ratio of ionizable lipid:1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC): cholesterol: PEG-lipid=46.3:9.4:42.7:1.6. The N:P ratio was kept as 6:1. And the ethanol and aqueous phases were mixed at a volume ratio of 3:1 in the T-junction device. After mixing, LNPs were dialyzed against a buffer at pH 7.4 for 6 hours. After that, LNPs were sterilized via a 0.22 μm filter and stored at 4 o C for further use. The average diameter, polydispersity (PDI) and zeta potential were measured with a NS-90Z device (Malvern Panalytical). The cryo-TEM images were obtained from Southern University of Science and Technology. The concentration of leaked mRNA (Cleak) was determined with a fluorescence detection kit, following the manufacture's protocols. LNP was also lysed with 0.4% Triton X-100 to determine the concentration of total mRNA (Ctotal). The encapsulation efficiency was calculated as the following equation: LNP solution was added with cryoprotectant and filled in a penicillin bottle, then the mixture was lyophilized with a freezer dryer (Pilot-2H, Boyikang) using a designed procedure. The The mRNA integrity was measured with a gel retardation assay [15] and microfluidic capillary electrophoresis (Agilent Fragment Analyser) [16] , as previously reported. HEK 293T/17 and ACE2-expressing 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM L-glutamine, 10% FBS (sigma-aldrich) and 1% penicillin/streptomycin (BI) at 37 o C and 5% CO2. Female BALB/c mice age of 6-8 weeks and heterozygous B6/JGpt-H11 em1Cin(K18-ACE2) /Gpt mice All samples' inactivation was performed according to IBC approved standard procedures for removal of specimens from high containment. Luciferase mRNA loaded LNPs (mRNA-Luc LNPs) and its lyophilized product, Lyo-mRNA-Luc LNPs were examined with their in vivo transfection efficiency. The encapsulated mRNA concentration was adjusted to 100 μg/mL and freeze-dried. Lyo-mRNA-Luc LNPs were reconstituted using the same volume of nuclease-free water with before drying. Mice were imaging system (FluoView400, Boluteng). Three kinds of LNPs with mRNA encoding the antigen of WA1 (Lyo-mRNA-WT), Delta (Lyo-mRNA-Delta) or Omicron COVID-19 variant (Lyo-mRNA-Omicron) were prepared and used for mice immunization experiment. 0.5 mL LNP solutions containing 0.1 mg/mL encapsulated mRNA were freeze-dried with a penicillin bottle. Lyophilized LNPs were reconstituted using 0.5 mL nuclease-free water. Mice were received two doses of LNPs through intramuscular injections of 20 μL, 50 μL, or 100 μL reconstituted LNPs. The dilution water was also nucleasefree. The blood samples were collected and centrifuged at 1200 g and 4 o C for 10 min. The supernatant serum was separated, aliquoted, and frozen at −80°C. Neutralizing antibody titers were tested according to the reported methods [17] . Briefly, serum samples were diluted and mixed with a certain amount (1. under anesthesia from each vaccination, and 7 dpi from blank LNP control group. The lung and brain tissues were collected to determine viral titer by qPCR. Data were collected at least six independent experiments for in vivo experiment. All other experiments were performed at least three times independently unless specified. Statistical analysis was mainly processed using Prism software version 8.3 (GraphPad Software Inc., San Diego, CA) and analyzed by one-way or two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Statistically significant differences were considered when p < 0.05. Four kinds of LNPs used in this paper were prepared through a classic T-junction mixing process [14] . LNPs containing Luc mRNA (mRNA-Luc LNPs), WT mRNA (mRNA-WT LNPs), Delta mRNA (mRNA-Delta LNPs) or Omicron (mRNA-Omicron LNPs) all showed narrow size distribution and high encapsulation efficiency (EE), as listed in Table 1 . From the cryo-TEM images (Figure 1A) , we could observe the uniform, lamellar and vesicular structure of the LNPs. After lyophilization, the dried LNPs looked like a white fluffy cake (Figure S1 , supporting information) and could be dissolved in water easily and quickly (<10 seconds). The reconstituted solution was uniform and translucent, just as the freshly-prepared LNP solutions. The size, polydispersity index (PDI) and encapsulation efficiency of LNPs (Table 1) only changed slightly, indicating that the optimized lyophilization process didn't changed their basic physical properties. The mRNA integrity was also well-maintained (>90%, Figure S2 , supporting information), demonstrating the lyophilization process didn't cause damage to the mRNA structure. We then evaluated the thermostability of Luc LNPs by incubating then at 4 o C, 25 o C, or at 40 o C for different time ( Figure 1B, 1C) . The products didn't exhibit any change and sustained the size and encapsulation efficiency at 4 o C and 25 o C after 18 days, indicating the high stability of the product. At 40 o C, the size increased, but the encapsulation efficiency and mRNA integrity ( Figure 1D ) were maintained. It could be estimated that the lyophilized LNPs could be stored at a 2~8 o C refrigerator for a long time. The in vivo transfection efficiency of lyophilized LNPs was first evaluated with Luciferase mRNA. As seen in Figure 1E Next, a mRNA-Delta vaccine was prepared to further evaluate its efficacy against SARS-COV-2 Delta and other variants based on the lyophilized mRNA-LNP formulation (Lyo-mRNA-Delta). As show in Figure 3A , after immunizing intramuscularly twice with 5 µg dosage at a 2-week interval, the sera obtained 14 days after the second vaccination showed a very strong Delta-RBD specific IgG antibody response in the six-week-old female BALB/cJ mice, with a geometric mean titer of 2.37×10 6 . Potent neutralizing activity was also induced at this time point, titers against Delta were up to 9340 reciprocal ID50, while cross against Omicron, WA1 were 3278 and 30874, respectively, as showed in a vesicular stomatitis virus-based SARS-CoV-2 pseudovirus neutralization assay ( Figure 3B ). In contrast to the phenomenon of 20-to 130fold neutralization reduction for ancestral spike vaccines elicited sera [18] [19] [20] [21] [22] , the titers of lyophilized mRNA-Delta vaccine against Omicron were still high, consistent with data from other studies [23, 24] . Interestingly, the mRNA-Delta vaccine stimulated much higher neutralizing antibody titers to wild-type SARS-COV-2 pseudovirus than Delta itself. We also developed a lyophilized mRNA vaccine against Omicron (Lyo-mRNA-Omicron) since it has a significant transmission advantage over Delta and become the dominant variant of concern in the current wave of COVID-19 [25] . To investigate its antigenicity, mice were twodose injected intramuscularly by an accelerated immunization process at day 0 and day 7. Sera were collected one week after the second dose and pseudovirus titers were analyzed. Neutralizing antibody titers against Omicron increased to a high value as 4758. Still, it could induce certain neutralization response against Delta though lower than Omicron. In summary, our lyophilized mRNA vaccines were able to trigger robust antibody response against SARS-COV-2 virus. The viral load in right lung tissue of Lyo-mRNA-Delta vaccinated animals remained at the level of low limit of quantification 7 dpi. In contrast, a high level of viral load existed in the right lung tissue of animals in blank LNP control group, which up to approximately 1011 copies/g ( Figure 4D ). Importantly, the viral load in right lung tissue had almost undetectable in all Lyo-mRNA-Delta vaccinated mice 14 dpi (Below detection limit). The viral load in brain tissues in 5 μg animal groups displayed similar patterns as those in lung tissues. And 10 μg Lyo-mRNA-Delta showed remarkable protective effect on viral dissemination and amplification ( Figure 4E ). Together, these data suggest that lyophilized mRNA-Delta vaccine is highly immunogenic and can fully protect the challenged mice from SARS-COV-2 infection. The mRNA vaccine has several advantages, such as rapid design of antigen, potential for quick, inexpensive and scalable manufacturing, and inducing strong humoral and cellular immunity, which make it a powerful weapon against infectious diseases, especially against those caused by virus that are easy to mutation. The advantage and efficacy of mRNA vaccines has also been proved by two approved and marketed mRNA vaccines against COVID-19, Comirnaty and Spikevax. While the storage and transport of the current approved mRNA vaccines require ultracold temperature, which limit its accessibility. In this study, we successfully achieved the lyophilization of mRNA-LNP vaccine. The particle size, encapsulation efficiency, poly dispersity index and zeta potential after reconstitution only changed slightly compared with freshly-prepared LNPs. And the bioactivity and immunogenicity were well-maintained, meeting the mRNA vaccine specification. The In conclusion, the lyophilized mRNA-LNP vaccine has the advantage of both high immunogenicity and accessibility, and very suitable for prevention of epidemic such as SARS-COV-2. We are actively promoting its clinical trial application. Supporting Information is available from the author. reconstituted Lyo-mRNA-Omicron. 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