
==== Front
Mol Pharm
Mol Pharm
mp
mpohbp
Molecular Pharmaceutics
1543-8384
1543-8392
American Chemical Society

37491979
10.1021/acs.molpharmaceut.2c01024
Article
Delivery and Expression of mRNA in the Secondary Lymphoid Organs Drive Immune Responses to Lipid Nanoparticle-mRNA Vaccines after Intramuscular Injection
Takanashi Asuka
https://orcid.org/0000-0003-0224-3308
Pouton Colin W. *
https://orcid.org/0000-0002-8840-8654
Al-Wassiti Hareth *
Drug Delivery, Disposition and Dynamics (D4), Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), Parkville, VIC 3052, Australia
* Email: colin.pouton@monash.edu.
* Email: harry.al-wassiti@monash.edu.
26 07 2023
07 08 2023
26 07 2024
20 8 38763885
29 11 2022
27 04 2023
26 04 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Lipid nanoparticles (LNPs) are the prime delivery vehicle for mRNA vaccines. Previous hypotheses suggested that LNPs contribute to innate reactogenicity and lead to the establishment of a vaccine adaptive response. It has not been clear whether LNP adjuvancy in the muscle is the prime driver of adaptive immune responses or whether delivery to secondary lymphatic organs is necessary to induce strong adaptive responses. To address this, we formulated reporter gene (NLuc) or OVA mRNA into LNP or coadministered the mRNA with empty LNP. After IM injection, we correlated the delivery with adaptive immune responses. Additionally, we investigated humoral responses to modified mRNA encoding the SARS-CoV-2 spike protein. Compared to unformulated mRNA encoding nanoluciferase, with or without co-administered empty LNPs, LNP-formulated mRNA resulted in high levels of nanoluciferase in the secondary lymphoid organs. Similarly, LNP-mRNA encoding ovalbumin led to a cellular immune response against OVA while free mRNA, with or without empty adjuvanted LNPs, caused little or no immune response. Finally, only mice injected with LNP-formulated mRNA encoding SARS-CoV-2 spike protein elicited robust cellular and humoral immune responses. Our results suggest that the mRNA delivery and transfection of secondary lymphatic organs, not LNP adjuvancy or RNA expression in muscle, are the main drivers for adaptive immune response in mice. This work informs the design of next-generation mRNA delivery systems where better delivery to secondary lymphatic organs should lead to a better vaccine response.

mRNA
nonviral delivery
lipid nanoparticles
delivery strategy
vaccine
COVID19
Monash University 10.13039/501100001779 NA document-id-old-9mp2c01024
document-id-new-14mp2c01024
ccc-price
Special Issue

Published as part of the Molecular Pharmaceuticsvirtual special issue “Emerging Trends in Molecular Pharmaceutics across Australasia”.
==== Body
pmcIntroduction

mRNA vaccines have emerged as a robust technology to induce protective immunity against infectious diseases1 and have stimulated interest in therapeutic applications of mRNA technology.2 Intramuscular injection of mRNA for subsequent translation into protein was demonstrated more than 30 years ago,3 though delivery of mRNA to other organs remained a limiting factor that prevented the widespread use of mRNA for vaccination. Early attempts to inject free mRNA into muscle resulted in modest or low immunological responses.4 However, new delivery systems such as lipid nanoparticles (LNPs) improved vaccine responses after intramuscular injection and have unlocked the potential of mRNA vaccines.5

LNP formulations were successfully used to deliver siRNA,6 prior to their deployment to deliver mRNA,7,8 particularly mRNA vaccines during the SARS-CoV-2 pandemic.1,9,10 LNPs are designed to encapsulate and protect nucleic acids in particles formed using ionizable cationic lipids, helper lipids, and PEGylated lipids. In addition, contemporary LNPs appear to promote endosomal escape after internalization by cells and release mRNA into the cytoplasm, although there is likely to be an opportunity for improved effciency.6,11,12 Importantly, LNPs are effective in protecting mRNA from nuclease degradation.13

Several research groups have studied protein expression after injection of either LNP-mRNA or LNP-DNA formulations using various routes of administration,5,14−17 but there have been few attempts to probe the association between lymphatic delivery and immune response. Intramuscular administration is the predominant route of administration of commercial vaccines approved for human use, including the recently approved COVID-19 mRNA vaccines. There is a need for more research on the fate of such vaccines and their mechanisms of action after intramuscular injection. Gene expression studies using reporters have been used as a proxy to evaluate the delivery and, ultimately, vaccine effectiveness in preclinical models.5,18,19 Often, these studies assess muscle expression or whole-animal bioluminescence but rarely study the expression in lymphoid tissues such as lymph nodes or the spleen.20 LNPs produce robust vaccination responses in individuals, but it is unclear what mechanism LNPs unlock that explains the potency of LNP-mRNA products. In a study using unmodified mRNA, LNP encapsulation enhanced gene expression at the intramuscular injection site. Lutz et al. considered that muscle expression might recruit immune cells to the muscle resulting in antigen sampling,21 and it has been hypothesized that infiltrating cells then take up the antigens expressed in the muscle subsequent to the delivery of mRNA or DNA.22 This view follows analogous hypotheses that seek to explain the mechanisms of action of non-mRNA vaccine modalities when local inflammation can attract infiltrating immune cells.23,24 While LNPs can be inflammatory, it is not clear if their reactogenic nature is directly responsible for driving adaptive immune responses.25

To address the need for mechanistic knowledge, we conducted a series of experiments to probe the correlation between the translation of mRNA in various organs and the resulting immune response. We injected free or LNP-formulated mRNA or DNA encoding the reporter protein Nanoluciferase (NLuc) for gene expression studies. We also co-injected empty LNPs with free mRNA to determine the role of the formulation. We used a cellular response model to correlate the gene expression studies with analogous studies of the immune response to ovalbumin. We also investigated humoral responses to modified mRNA encoding the SARS-CoV-2 spike protein. Based on our data, we propose that mRNA delivery to and expression in lymphoid tissues is the critical determinant of strong vaccine responses.

Experimental Section

Materials

For in vivo gene expression studies, the plasmid DNA used in this study was pNL 1.1 CMV (PlasmidFactoryGmbH & Co. KG, Germany). The mRNAs used in this study were produced using HiScribe T7 ARCA mRNA Kit (NEB, Australia) on linearized plasmids containing ORF encoding NLuc, NLucP, FLuc (Promega Inc., Australia), or full OVA protein. The LNP was prepared using 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) (Avanti Polar Lipids Inc., USA), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) (MedChemExpress, USA), Cholesterol (Sigma-Aldrich, Germany), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids Inc., USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000) (Avanti Polar Lipids Inc., USA), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) (Avanti Polar Lipids Inc., USA). Nano-Glo Luciferase Assay System and Glo Lysis Buffer, 1× (Promega, USA) were used to quantify the luminescence in the gene expression study.

The OVA protein and OVA 257–264 synthetic peptide (SIINFEKL) (Sigma-Aldrich, Germany) were used in the in vivo cytotoxic t-cell killing assay. eBioscience CFSE, for flow cytometry, was used for fluorescent cell staining dye (Thermofisher Scientific, USA). EIA/RIA Assay Microplate (Corning) was used as the plates and 1-Step Ultra TMB-ELISA Substrate Solution and Stop Solution (Thermofisher Scientific, USA) for ELISA assay.

mRNA Production

Unmodified mRNA was produced using a HiScribe T7 ARCA mRNA Kit (New England BioLabs, Australia) as recommended by the manufacturer. Nucleoside-modified mRNA was produced using a cap-1 structure (Trilink, USA). The length of the mRNA was analyzed using denaturing formaldehyde gel electrophoresis using 500–1000 ng of mRNA and was stored at −80 °C until use. The concentration of mRNA was measured using Nanodrop (Thermofisher Scientific, USA).

Preparation of LNP

Ionizable cationic lipid, phosphatidylcholine (DSPC), cholesterol, and PEG lipids were dissolved in ethanol with a molar ratio of 50:10:38.5:1.5 for mRNA and DNA. The nucleic acids were dissolved in acetate buffer solution at a pH of 3.8–4. LNPs were assembled by mixing the nucleic acid solution with the lipids at an N/P ratio of 6, where N/P represents the ratio of ionizable nitrogen atoms to phosphate groups in the mixture. A Nano Assembler Benchtop (Precision Nanosystems, Vancouver, Canada) and a NanoAssembler cartridge (Precision Nanosystems, Vancouver, Canada) were used to produce the LNP particles, which were processed by overnight dialysis with either PBS or TRIS buffer. The dialysed particles were concentrated to the desired concentration using Amicon filters with MWCO of 50 kDa (Merck Millipore, Massachusetts, United States).

Animal Work

All animal experiment procedures described were conducted under the approval of the Monash Institution of Pharmaceutical Science Animal Ethics Committee. C57BL/6J male and Balb/c female mice at the age of 7–20 weeks were used for all in vivo experiments.

Determination of NLuc in Mouse Tissue

The Nano-Glo Luciferase Assay System (Promega, USA) was used to determine the NLuc, NLuc Pest (NLucP) expression from tissues. Luciferase Assay System (Promega, USA) was used to determine the firefly luciferase (FLuc) expression from tissues. The tissues were isolated 1, 7, and 28 days post-injection and were stored at −80 °C immediately after isolation. The isolated tissues were homogenized using a gentleMACS Dissociator and suspended in Glo Lysis Buffer, 1×, to prepare the tissue lysates. The luminescence of the NLuc reporter protein in various tissue lysates was determined according to the manufacturer’s protocol. Luminescence was measured using an Envision plate reader (PerkinElmer, USA).

In Vivo Cytotoxic T-Cell Killing (IVCTK) Assay

An in vivo cytotoxic T-cell killing assay was conducted to investigate the cellular immune responses of C57BL/6J mice after administration of different mRNA formulations. Splenocytes from donor unvaccinated C57BL/6J mice were harvested. Splenocytes were then split into two equal populations to produce target and nontarget cells.

Target cells were prepared by exposing cells to OVA257–264 peptide (1 μg/mL) and also by exposure to 5 μM carboxyfluorescein succimidyl ester (CFSE) which labels cells by covalent coupling. Cells unpulsed with OVA peptide were incubated with 0.5 μM CFSE to produce a population of nontarget cells labeled with lower intensity fluorescence. The two populations of cells were mixed in equal ratios. From the mixture, 2 × 107 cells were injected intravenously into C57BL/6J mice that had either been injected with saline or immunized with vaccine, 7 days prior to transfer of target and non-target splenocytes. Twenty hours later, mice were euthanized, and their spleens were isolated to examine the presence of target and non-target cells by flow cytometry. Induction of cell-mediated target cell killing was determined by comparing the survival of target cells versus nontarget cells.

Flow Cytometry

A BD FACSCanto II (BD Biosciences, CA) was used for flow cytometry to determine the ratio of OVA-pulsed to DMSO-pulsed splenocyte populations remaining in the recipient mouse spleen. The following formula was used to determine the OVA-specific T cell killing in each mouse:

where RU:P,negative control and RU:P,experimental are the ratios of unpulsed to pulsed splenocytes in the negative control mouse and experimental mouse, respectively.

Injection Schedule for IVCTK Assay

The mice were injected with different mRNA formulations encoding full length ovalbumin or the injection vehicle control (PBS). Single-cell splenocyte suspensions were prepared using isolated spleens and a 70 μm strainer. Splenocytes were treated with red blood cell lysis buffer incubated at 37 °C for 2 min. The cells were resuspended in PBS + 2% FBS. The single-cell suspension was divided into two tubes with equal volumes. One tube was treated with OVA257–264 peptide (1 μg/mL), and the other was treated with just the appropriate DMSO-containing solvent. Both groups of cells were incubated for 1 h at 37 °C. The tubes were labeled with high and low concentrations of CFSE (5 and 0.4 μM), respectively. The two tubes were mixed in a 1:1 ratio, resuspended in PBS, and injected intravenously (2 × 107 mixed cells in 200 μL of PBS) into recipient mice, 6 days after the mice had been immunized. On the following day, the recipient mice were euthanized, and splenocytes were isolated for analysis by flow cytometry.

Antibody Titer ELISA Assay

An enzyme-linked immunosorbent assay (ELISA) was conducted to determine the humoral responses after administration of different mRNA formulations to Balb/c mice. The mice received different mRNA formulations on days 0 and 21 to simulate a typical prime and boost protocol. Blood was collected at different time points during the course of the experiment, by cheek bleed prior to the terminal bleed after euthanasia. Blood samples were processed to isolate serum for assay of antibody titer. The ELISA was performed using the plates coated with the spike protein of the Wuhan SARS-CoV-2.

Corning 96 Well EIA/RIA Assay Microplates were used to perform the serum analysis. 50 μL of 0.5 μg/mL of Spike-His recombinant protein (synthesized at the Peter Doherty Institute) in PBS was used for antigen coating. The coated plates were stored overnight at 4 °C, followed by the addition of 200 μL of blocking solution (PBS + 2% BSA) at room temperature for 1 h. TBST was used to wash the plates three times, and the plates were placed in an airtight bag at 4 °C.

Serum was diluted using PBS + 0.2% BSA. 50 μL of diluted serum was incubated at room temperature for 1.5 h. 300 μL of TBST was used to wash the plates. To develop and stop the signal, TMB 1-Step Ultra and Stop solutions were used, respectively, for titer measurement. The measurement absorbance was set to 450 nm for evaluation.

Injection Schedule of Antibody Titer Assay

The mRNA formulations used in this experiment were produced using mRNA encoding the whole spike protein of the ancestral (Wuhan) SARS-CoV-2. Mice were injected with various mRNA formulations or a negative control at day 0. The serum samples from the mice were obtained through cheek bleeding and the collection of blood in heparin tubes for days 0, 21, and 56.

The blood was at rest for 30–60 min at room temperature before centrifuging the sample at 1000g at 4 °C to isolate the serum. The collected serum was aliquoted and stored at −80 °C until use.

Results

LNP Formulation Protects mRNA from RNases

LNPs were formulated using an ionizable lipid (DODAP), DSPC, cholesterol, and DSPE-PEG. The microfluidic mixing method produced small particles with narrow size distributions and high encapsulation efficiency. Dynamic light scattering (DLS) indicated that LNPs had an average particle diameter of 60.9 nm and a low polydispersity index (PDI) of 0.17. The ribogreen assay indicated that the encapsulation efficiency of mRNA by the LNP was 90.7% (Table 1), consistent with the qualitative indication of encapsulation shown using agarose gel electrophoresis (Figure 1A). Compared to the sample of free mRNA (Figure 1A, lane 2) in the gel, LNP-mRNA displayed a faint band indicating high encapsulation of the mRNA (Figure 1A, lane 4). The mRNA encapsulated in the LNPs maintained its integrity after extraction (Figure 1A, lane 5). Encapsulation by LNPs also ensured the protection of mRNA against RNases (Figure 1A, lane 6) while RNases degraded free mRNA (Figure 1A, lane 3).

Figure 1 Characterization of LNPs. (A) mRNA agarose gel electrophoresis: RNA ladder (lane 1), free mRNA (lane 2), free mRNA after exposure to RNase A (lane 3), LNP-mRNA (lane 4), phenol-chloroform extraction of mRNA from an LNP (lane 5), phenol-chloroform extraction of mRNA from an LNP after exposure to RNase A (lane 6). (B) Relative light units (RLU) determined in calf muscle homogenate were measured using a plate reader at 1, 7, or 28 days after injection of 10 μg of unmodified mRNA encoding either Nanoluciferase (NLuc), Nanoluciferase Pest (NLucP), or Firefly luciferase (FLuc). The dotted line in the graph indicates the average limit of detection of all the luciferase assays. The data are in mean ± SEM of n = 3 mice for (B). (C) Schematic of the gene expression study of LNP-mRNA using Biorender software.

Table 1 Particle Size Measurements and Encapsulation Efficiencya

formulation	Zavg ± SD (nm)	PDI ± SD	EE ± SD (%)	zeta ± SD (mV)	
LNP-mRNA	60.9 ± 7.8	0.17 ± 0.07	90.7 ± 1.6	–0.64 ± 0.17	
a The particle size measurement of a representative sample of LNP-mRNA using dynamic light scattering and encapsulation efficiency of LNP. PDI = Polydispersity Index and EE = Encapsulation Efficiency. The representative data are shown in mean ± SD with n = 3.

Delivery of mRNA Encoding NLuc Results in Sustainable Translation of Protein

NLuc has the desirable property of being a very sensitive reporter protein, but we were concerned that the protein stability could outlive the stability of the mRNA, giving a false impression of the half-life of the transcript. To address this, we first evaluated the kinetics of NLuc used in this study with other luciferase reporters (Figure 1B). The dotted line in the graph indicates the typical limit of detection obtained from the luciferase assays.

10 μg of unmodified mRNA encoding either NLuc, NLuc Pest (NLucP), or firefly luciferase (FLuc) were administered into the gastrocnemius (calf) muscle in a volume of 50 μL. NLucP incorporates a protein destabilizing domain engineered to reduce the stability of the encoded proteins. We determined the relative amount of reporter proteins in the calf muscles 1, 7, and 28 days after injection. NLuc levels were similar 1 and 7 days after injection and were 10-fold lower on day 28 (Figure 1B). FLuc levels were higher at 7 days than 1 day after the injection and also were observed to be approximately one order of magnitude lower at 28 days. Relative luminescence levels for FLuc were 3–4 orders of magnitude lower than for NLuc, reflecting the high sensitivity of the NLuc reporter. The relative luminescence determined after injection of NLucP mRNA was significantly different from NLuc. Luminescence after 1 day was over 100 times lower for NLucP than NLuc at the same time point. After 7 days, the NLucP signal was reduced by more than one order of magnitude, and there was a further 10-fold reduction by 28 days. Given that both NLuc and NLucP levels were reduced by a similar relative amount from day 7 and day 28, it is difficult to draw conclusions as to whether the protein stability of NLuc affects our assessment of the expected kinetic profile of protein translation in muscle.

We concluded that NLuc is valuable for studies of mRNA translation in different organs, in that its high sensitivity provides a broad dynamic range and allows the detection of mRNA translation in tissues that receive low levels of mRNA. For further studies, we used NLuc without the PEST fusion domain, as our study was primarily concerned with the relative levels of translation in various tissues.

LNP Delivers mRNA to the Lymphatic Tissues, while Free mRNA Results in More Localized Expression within Muscle Tissue

Once we validated the stability of NLuc protein, we evaluated the tissue distribution of NLuc resulting from intramuscular injection of free or formulated mRNA or DNA. In this study, we used unmodified mRNA. Mice received 10 μg formulated or free NLuc mRNA into the calf muscle (CM). We isolated tissues after 1 or 7 days after injection and determined the reporter protein levels, as relative luminescence per unit mass of tissue, using a standard NLuc assay (Promega) (Figure 2A). Free mRNA induced similar gene expression profiles at 1 and 7 days. The highest concentration of NLuc was found in the calf muscle (CM) at both time points. Free mRNA and LNP-mRNA produced similar levels of NLuc in the CM (Figure 2A). We also observed much higher levels of the reporter protein in both draining and nondraining lymph nodes (DLN and nDLN), spleen (SP), and liver (LV) after injection of LNP-mRNA when compared to injection of free mRNA one day after the injection. With the exception of the CM, the NLuc level was lower in all tissues on day 7. Nevertheless, NLuc levels produced by LNP-mRNA in DLN, nDLN, SP and LV were higher than those produced by free mRNA at both time points.

Figure 2 Relative NLuc levels (expressed as relative light units per unit mass of tissue) found in various tissues 1 or 7 days after intramuscular injection of 10 μg of mRNA or DNA. Draining lymph node (DLN), non-draining lymph node (nDLN), the calf muscle (CM), spleen (SP), kidney (KD), liver (LV), quadriceps muscle (QM), lung (LG), and heart (HT) at 1 and 7 days after injection of 10 μg of free mRNA or LNP-mRNA (Figure 2A) and free DNA or LNP-DNA (Figure 2B). Data are presented as mean ± SEM (n = 6 mice). The dotted line represents the typical limit of detection from all luciferase assay plates analyzed. **p < 0.01 and *p < 0.1 as determined by two-way analysis of variance (ANOVA) with Tukey’s post hoc test.

We conducted parallel gene expression studies after the delivery of plasmid DNA (Figure 2B). Similar to the results obtained with free mRNA in Figure 2A, free DNA produced very little NLuc expression other than in CM at both time points. DNA formulated in LNPs resulted in different gene expression profiles to the corresponding LNP-mRNA particles.

High or moderate NLuc was produced in CM or DLN, respectively, at both time points, similar to our observations with LNP-mRNA. However, the other tissues we sampled produced negligible NLuc after administering LNP-DNA particles.

Only LNP-Formulated mRNA Resulted in High Cellular and Humoral Immune Responses

In this study, we administered intramuscular injections to mice using either free mRNA, LNP-mRNA, or mRNA combined with empty LNP (eLNP + Free mRNA). In this study, we compared LNPs produced using one of two alternative ionizable lipids (DODAP or DLin-MC3-DMA). DLin-MC3-DMA (MC3) is a commonly used ionizable lipid used in the FDA-approved Onpattro siRNA product and is well-studied in the mRNA vaccine context. This lipid is generally assumed to be more active than DODAP, though the published data available is limited. For all latter experiments, we used empty LNPs with an average particle size of 54 nm (PDI = 0.17) as determined using a Malvern Zetasizer. Each formulation contained 10 μg mRNA encoding NLuc. Tissues were isolated after a day to check the NLuc levels in tissues (Figure 3), which were consistent with the data shown in Figure 2.

Figure 3 Relative NLuc levels in various tissues and its correlation with targeted cytotoxic T cell-based killing caused by vaccination with ovalbumin mRNA. (A) Tissue NLuc levels expressed as relative light units (RLU) per unit mass of tissue in draining lymph node (DLN), calf muscle (CM), spleen (SP), and liver (LV), 14 h after injection of 10 μg of either free mRNA, eLNP (DODAP) + Free mRNA, LNP (DODAP)-mRNA, eLNP (MC3) + Free mRNA, or LNP (MC3)-mRNA. Data are presented as mean ± SEM (n = 5 mice). The dotted line represents the average limit of detection from all luciferase assay plates analyzed. (B) Pie charts show the relative contribution of each organ to the total luminescence (RLU) without normalization. (C) Fraction of OVA-specific CD8+ T cells eliminated in mice immunized with PBS (negative control), free mRNA, eLNP (DODAP) + Free mRNA, LNP (DODAP)-mRNA, and eLNP (MC3) + Free mRNA or LNP (MC3)-mRNA). eLNP = empty LNP. A total of 30 μg of mRNA encoding OVA was administered to mice in each case. The data are expressed as mean ± SEM (n = 6 mice). The graphs display **p < 0.01, ***p < 0.001, and ****p < 0.0001 as determined by one-way ANOVA with Tukey’s post hoc test.

NLuc levels found in CM were similar for free mRNA, eLNP + Free mRNA, and LNP-mRNA (i.e., coadministration of free mRNA and empty LNPs) for both lipids. However, NLuc levels in draining lymph node (DLN), spleen or liver were distinctly different. LNP-mRNA formulated using DODAP produced NLuc levels, expressed as RLU per unit mass, of 1.06 × 105, 4.9 × 101, and 2.39 × 102 in draining lymph node, spleen, or liver, respectively, whereas eLNP + Free mRNA produced much lower levels (3.5 × 102, 1.6, and 1.5 RLU in the draining lymph node, spleen, or liver, respectively (Figure 3A)). The same trend of NLuc levels was produced for LNP-mRNA formulated using MC3 but with higher RLU per unit of tissue mass. LNP-mRNA using MC3 showed 7.87 × 105, 2.48 × 104, and 3.06 × 104 RLU in the draining lymph node, spleen or liver, respectively (7.4, 496, and 128 times greater than LNP (DODAP)-mRNA in the corresponding tissues). However, eLNP (MC3) + Free mRNA showed NLuc levels that are 9.47 × 102, 1.14 × 103, and 7.4 × 103 times lower than LNP (MC3)-mRNA in draining lymph node, spleen, and liver, respectively. The results suggest that mRNA must be encapsulated within LNPs to distribute to distant organs of interest.

Next, we used a standard in vivo cytotoxic T-cell killing assay to study the cellular immune response following injection of Free mRNA, LNP-mRNA, or eLNP + Free mRNA. We injected 30 μg doses of mRNA encoding ovalbumin (OVA) into the calf muscle (Figure 3B). In this experiment, free mRNA injection produced no detectable killing of the target splenocytes. Similarly, approximately 3% killing was observed after injection of eLNP (DODAP) + Free mRNA. eLNP + Free mRNAs formulated using DLin-MC3-DMA resulted in the killing of 1–22% of target cells at an average of 10.5%. When mRNA was encapsulated into LNP, the extent of cell killing was significantly greater. LNP (DODAP)-mRNAs resulted in an average killing of 31.9% of target cells, while LNP (MC3)-mRNAs resulted in complete T-cell killing.

To investigate humoral immunity, we used N1-methyl pseudouridine-modified mRNA encoding the ancestral (Wuhan) whole spike protein of SARS-CoV-2 as a model to investigate the response to mRNA formulations. Mice received prime (day 1) and boost (day 21) intramuscular injections of an LNP formulated with 3 μg of Spike mRNA using DLin-MC3-DMA. In addition, we have used DMG-PEG instead of DSPE-PEG in this formulation. Responses were determined by ELISA using plates coated with Wuhan spike protein. The data indicate that both on day 21 (Figure 4A left) and day 56 (Figure 4A right), only LNP-formulated mRNA produced responses above the limit of detection (Figure 4A). The boost dose elevated the log antibody titer for LNP-mRNA from an average of 2.9 ± 0.3 on day 21 to 4.9 ± 0.3 on day 56. The titer produced by free mRNA was below the limit of detection over a dose range of 3 or 30 μg (up to 10-fold greater than the dose administered in the LNP formulation). This suggests that only LNP-formulated mRNA can produce a humoral immune response. eLNP (MC3) + Free mRNA resulted in no detectable titers. A similar trend was shown using an ELISPOT assay where LNP-formulated mRNA produced 1.3 × 104 ± 3.3 × 103 of IFN-γ activity while the level detected after injection of free mRNA was close to or at the limit of detection at both doses (Figure 4B,C).

Figure 4 Humoral responses in mice after administration of free mRNA, eLNP (MC3) mRNA, or LNP (MC3)-formulated mRNA. (A) Antibody binding to ELISA plates, expressed as optical density values, using different dilution factors of serum collected from mice after administration of either Free mRNA (3 μg), free mRNA (30 μg), eLNP + Free mRNA (3 μg), or LNP-mRNA (3 μg) at Day 21 (left) or Day 56 (right). (B) Corresponding end-point titers at Days 21 and 56 from (A). (C) Number of SARS-CoV-2 whole spike protein-specific interferon-y producing splenocytes detected using IFN-γ ELISPOT assay at Day 56. The data are expressed as mean ± SEM (n = 4 mice). eLNP = empty LNP. The dotted line in the graph indicates the average limit of detection from the blank values of all the assays. ***p = 0.0001 and ****p < 0.0001 as determined by ordinary one-way ANOVA with Tukey’s post hoc test.

Discussion

LNPs are an essential component of successful mRNA vaccines, but the precise mechanisms by which they contribute to the induction of robust immune responses still need to be clarified. We found that LNP-formulated mRNA injected by the intramuscular route produced comparable levels of the reporter protein in the calf muscle to levels produced by free mRNA. This was consistent with the findings reported by Pardi et al.5 It is not clear how muscles take up large amounts of mRNA compared to other organs, but as we have shown, the protein translation level and duration depend on the protein itself. We decided to conduct an experiment to understand the stability of NLuc in vivo by comparing the translation of NLuc, FLuc, and NLucP. We found that NLuc was present at high levels for at least 28 days, but this may have been due to an unusual degree of protein stability of NLuc.26 Fusing NLuc with PEST signal (NLucP) was expected to accelerate degradation.27 The overall level of NLucP found in muscle was lower at all time points, but the kinetics of loss of protein between day 7 and day 28 did not appear to be markedly different, suggesting that the use of the NLuc reporter was justified and did not lead to a false impression of the time course of translation. The presence of NLucP proteins even after 7 days suggests other factors may be involved. While further studies to understand the pharmacokinetics of expression using various protein reporters would be valuable and could benefit from comparisons between NLuc, NLucP, or FLuc, we decided that the use of NLuc was a good choice for studies focused on biodistribution and delivery due to NLuc remarkable sensitivity.

Our gene expression and cellular immune response studies used unmodified mRNA. Unmodified mRNA elicited strong expression and cytotoxic activities according to previous reports.21,28 In contrast, modified mRNA was used for our humoral immune response studies to evaluate antibody titers similar to the approved vaccines.29 All gene expression experiments used unmodified mRNA, whereas the vaccine experiments used modified mRNA. During the gene expression studies, it was uncertain whether unmodified or modified mRNA was suitable for prophylactic vaccine purposes in the mRNA field at that time. It became more certain that modified mRNA was the better candidate for prophylactic vaccines while we continued our investigation. However, to keep the study consistent, we decided to have unmodified mRNA for gene expression experiments. Furthermore, it is important to note that in vivo cytotoxic T-cell responses using unmodified mRNA and antibody titer responses using modified mRNA demonstrated the same conclusion. Free mRNA and empty LNP + mRNA do not mount a significant immune response.

We show that the organ expression of mRNA differs greatly between LNP-formulated nucleic acid and free nucleic acid. Only LNP-formulated mRNA was significantly expressed in organs other than muscle following intramuscular injection (Figure 2), particularly in draining lymph nodes and spleen. Until recently, due to a lack of sensitivity, there were no detailed protein translation data resulting from IM-injected mRNA in organs other than in muscle. A previous report revealed limited detection of protein translation in the lymph nodes and spleen,30 but it has been unclear what role mRNA translation serves in these tissues to induce a vaccine response.

We also observed mRNA expression in non-draining lymph nodes using LNP-mRNA but not mRNA alone. The non-draining inguinal lymph node was isolated from the opposite side of the injection site. This observation opens the possibility that the particles may be entering the lymph nodes from the bloodstream. Finally, we did not observe DNA expression in organs other than the muscle. Free DNA has been shown to drive expression in the muscle, although a clear understanding of the mechanism of nuclear entry has not been established.31−33 Nuclear access could be explained by elevated pressure in the muscle after IM injection, which stretches membranes and enables the DNA to enter muscle nuclei. However, LNP-encapsulated DNA has little or no expression in organs outside the muscle, at least at the doses used in this study. We hypothesize that expression in muscle is not the critical factor for vaccine activity and it is the ability of mRNA to lead to antigen production in immune cells that distinguishes mRNA from DNA.

In contrast, we observed encouraging expressions of LNP-mRNA in secondary lymphatic organs, which translated to the induction of robust cellular and humoral immune responses. Free mRNA lacked the ability to mount a sufficient cellular response, as shown from the OVA model of in vivo cytotoxic T-cell killing assay, consistent with previous reports using unformulated mRNA.13,34 Notably, the combination of free mRNA and empty LNP (eLNP + Free mRNA) using MC3 lipid also gave a minimal immune response, while the delivery of LNP-formulated mRNA resulted in a complete response using the same lipid formula. DODAP-formulated mRNA had high killing activity, albeit lower than that produced by DLin-MC3-DMA. This is not surprising given that DODAP delivery of NLuc mRNA leads to lower expression activity, as reported in a previous study35 and our work here (Figure 3A).

It is critical to note that there were no major differences in muscle expression between DODAP or MC3-formulated mRNA (<1.5×). However, large differences could be seen between DODAP and MC3 in secondary lymphatic organs such as the DLN (>10×) or the spleen (>100×). The increased expression in those lymphatic organs correlates better with vaccine responses seen when MC3 and DODAP were compared. Those results strengthen the suggestion that the expression of secondary lymphatic organs is necessary for inducing adaptive immune response.

The ionizable lipids used in LNPs were found to drive a pro-inflammatory environment, which has been linked with the establishment of adaptive immune responses.21,22,36 While inflammatory molecules may cause such responses, another report has shown that LNPs induce no inflammatory responses but exhibit sufficient adjuvant activity to promote adaptive immunity.25,37 Our data suggest that the key concept in inducing an adaptive immune response may lie in fact, with the effective mRNA delivery to secondary lymphatic organs. In our work, empty LNP co-administered with free mRNA was insufficient to stimulate the adaptive immune system compared with LNP-formulated mRNA (LNP-mRNA, Figure 3). The fundamental difference between the two conditions is that only LNP-formulated mRNA is delivered efficiently to the spleen, liver, and nondraining and draining lymph nodes as shown from Figure 3A. Furthermore, the pie chart gives us the indication that spleen delivery may have more impact on the immune response than the lymph nodes. As the relative contribution of spleen increases from LNP (DODAP) mRNA to LNP (MC3), there is a significant increase in the T-cell killing. It should be pointed out that further study is needed to understand how much the liver contributes to enhancing or reducing the immune response. That being said, this clear expression in lymphatic organs correlates directly with an adaptive cellular immune response driven by T-cell killing. Although empty LNPs may be capable of inducing a pro-inflammatory environment at the site of injection and can indeed drain to lymphatic organs, our data suggest that this reactogenicity by supplementing LNP is not entirely responsible for driving a strong adaptive immune response.

Confirming the hypothesis, ELISAs were unable to detect antibody titers after injection of free mRNA or free mRNA + empty LNPs even after two immunizations. A previous study on nonhuman primates revealed similar results.21 Consistent with our in vivo cytotoxic T-cell killing experiment, our data suggest that mRNA must be adequately formulated with the LNP to achieve sufficient humoral immunity. Increasing the dose of free mRNA encoding the SARS-CoV-2 spike protein from 3 μg to a high-dose 30 μg had also no impact on antibody titer, although this is expected to increase expression in muscle.

Using a stable protein such as NLuc can be advantageous for studies involving the quantification of mRNA expression and biodistribution in various organs.26 However, the expression levels can be saturated at very high doses and may distort the quantification of gene expression. We limited the dose to 10 μg of NLuc mRNA for this study. In contrast, NLuc fused to the PEST signal may be useful as an indicator of the half-life of mRNA or to evaluate the rate of protein translation and decay. Expression half-life and pharmacokinetics were outside the scope of this study but have been reported in other studies.5 A detailed understanding of translation pharmacokinetics will be valuable in the future, particularly in lymphatic organs. mRNA translation kinetics would be valuable in particular when a new mRNA entity is studied or new UTRs are investigated.38

Our findings can be viewed as the groundwork for future research in understanding the mechanism of LNP-formulated mRNA delivery or next-generation LNPs that lead to better vaccines. Our data suggest that the focus should be on mRNA translation in lymph nodes and the spleen rather than in the muscle. This will lead to a better interpretation of formulation performance eliciting an immune response, at least in preclinical animal models. We propose a simplified model for the LNP mechanism of action (Figure 5) based on delivery to the relevant immune cells within secondary lymphatic organs. Using this model, future improvements in the delivery of mRNA and enhanced expression in the lymphatic organs will lead to two significant advantages. First, improved delivery of mRNA to secondary lymphatic organs will cause most of the dose to be utilized where it is needed to mount an immune response. As a result, similar vaccine responses can be achieved with lower doses. Second, shifting the mRNA expression and delivery from the muscle to the lymphatic organs will reduce local reactogenicity. Local injection adverse reactions are a characteristic of the available SARS-COV-2 vaccines,39 likely caused by LNP adjuvancy and mRNA expression in the muscle after injection. Additionally, preferential delivery to the lymphatic organs will reduce the dose needed to mount a similar vaccine response, ultimately reducing the overall dose-dependent reactogenicity.40 It should be noted that the biodistribution and cellular immune response studies were carried out using male mice while the humoral immune response study used female mice. It is conceivable that biodistribution could be subtly different in female mice. However, the biodistribution data obtained from other study using male mice show similar results to that of the female mice in other studies.5,30,41 There could be differences in the biodistribution, but we have no evidence suggesting otherwise. Furthermore, preclinical and clinical studies have shown both sexes elicit comparable antibody responses with no or only minor differences.42−44 Nonetheless the small difference, if any, in vaccine response between two sexes do not affect the major findings or conclusions derived from encapsulated and free mRNA biodistribution.

Figure 5 Model for mRNA vaccine delivery. A simplified model of the mechanism of action of the LNPs is supported by evidence from this study. The model shows mRNA delivery by empty LNPs (eLNP) and free mRNA (top) leads to high muscle but a very low level of gene expression in the secondary lymphatic organs. This consequently leads to little to no immune response despite high expression in muscle and the presence of adjuvanted empty LNPs. In contrast, formulated LNP mRNA leads to the induction of effective immune responses and those strongly correlate with expression in secondary lymphatic organs such as the draining lymph nodes. Biorender was used to create the illustration.

One major obstacle is the translatability of biodistribution data to human clinical settings. The lack of organ biodistribution in humans and large animals complicates the understanding of this phenomenon. Important questions remain in studying LNP mRNA distribution and expression in large animals- which portion of the dose can drain into the draining lymph and how much of it escape to the bloodstream after intramuscular injection. However, it appears clear that muscle expression does not lead to sufficient vaccination. Future studies in humans and large animal models such as sheep can help shed light on the contribution of draining lymph nodes versus the spleen in antigen expression and vaccination.

Few questions remain from studying the delivery phenomenon. How does the LNP reach the non-draining lymph nodes? And which of the lymphatic organs, spleen or lymph nodes, drive the majority of cellular and humoral immune responses? And how do these findings in animals correlate with what happens in other larger animals and humans? These questions require future investigation to shed light on new ways to improve and build better delivery systems for mRNA vaccines.

Conclusions

Our results demonstrate that mRNA delivery and translation in lymphoid tissue correlates better with the induction of the adaptive immune responses. Though intramuscular injections of free mRNA, free mRNA plus empty LNP, and LNP-formulated RNA all result in robust translation of protein in muscle, only the LNP-formulated mRNA led to strong adaptive immune response likely due to higher expression in lymphoid organs other than the muscle. Our study suggests that enhanced delivery and expression of mRNA to lymphoid tissues should be the objective for the next-generation mRNA vaccine delivery systems.

Statistical Analysis

The particle characteristics presented in the table were expressed in mean ± standard deviation, with each data point representing n = 3 samples. For all in vivo experiments, the data are expressed in mean ± SEM, with each data point showing n = 3–6 samples unless stated otherwise. A two-way ANOVA with Tukey’s post hoc test was used to evaluate the statistical significance of all gene expression experiments. An ordinary one-way ANOVA plus Tukey’s post hoc test was used to evaluate the statistical significance of in vivo cytotoxic T cell killing activity and antibody titer responses. A p-value of less than 0.05 was considered to be statistically significant in all tests.

Data Availability Statement

The authors verify that the data supporting the results in this study are available within the article. Raw data that make up the results and support the findings of this study are available from the corresponding author, upon reasonable request.

Author Contributions

A.T. and H.A. conducted the experiments. A.T., H.A., and C.W.P. conceived the study and wrote the manuscript.

The authors declare no competing financial interest.

Acknowledgments

A.T. was the recipient of a PhD scholarship from Monash University. We acknowledge the infrastructure provided by the Faculty of Pharmacy and Pharmaceutical Sciences (Monash University). We acknowledge and thank MIPS Commercialisation Incubator Program and mRNA Victoria for financial support to C.W.P. and H.A. in conducting some of the mRNA studies.
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