Elicit: Lipid Nanoparticles in mRNA Vaccine Delivery

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Lipid Nanoparticles in mRNA Vaccine Delivery

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May 5, 2026

How do lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens?

Lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines by encapsulating the mRNA through electrostatic and hydrogen bonding interactions, facilitating cellular uptake via endocytosis, and releasing the mRNA into the cytoplasm when ionizable lipids protonate in acidic endosomes and disrupt the endosomal membrane to enable translation.

Abstract

Lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines through a coordinated physicochemical and biological process. LNPs composed of ionizable lipids, helper lipids, cholesterol, and PEG-lipids form 50-85 nm particles that encapsulate mRNA via electrostatic interactions and hydrogen bonding. Following intramuscular administration, LNPs are taken up by muscle-resident immune cells through endocytosis, with >10% of myeloid and lymphoid cells successfully transduced. The critical delivery step occurs when ionizable lipids protonate in acidic endosomes (pH ~5-6), triggering ion pairing with endosomal phospholipids that disrupts membranes and releases mRNA into the cytoplasm. Nucleoside modifications such as m1ψ reduce innate immune recognition of the mRNA cargo, while optimized formulations achieve 93-95% encapsulation efficiency and mRNA detectability for 14-28 days post-vaccination in humans.

Beyond mRNA delivery, LNPs function as intrinsic adjuvants by triggering rapid neutrophil and dendritic cell recruitment within 24 hours and inducing IL-6 production critical for germinal center responses. This dual functionality produces potent immunogenicity, with optimized formulations achieving 10-fold higher neutralizing titers than standard LNPs and protective immunity at doses as low as 0.25 µg. Rational lipid design enables independent control of delivery efficiency, tissue targeting, immune activation, and stability: the C24 formulation reduces off-target liver expression 6-fold while maintaining potency, adjuvant lipidoids provide TLR7/8 activation, and specific formulations remain stable at 4°C for at least 19 days. These mechanisms collectively explain the exceptional clinical efficacy of LNP-mRNA SARS-CoV-2 vaccines.

Methods

We analyzed 10 sources from an initial pool of 200, using 6 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Lipid Nanoparticle Delivery System, Nucleoside-Modified mRNA, SARS-CoV-2 Antigen Target, Delivery Mechanism Data, Study Type, Delivery System Scope

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How do lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract complete lipid nanoparticle composition and design details for SARS-CoV-2 mRNA delivery, including:

Extract detailed characteristics of the nucleoside-modified mRNA for SARS-CoV-2 vaccine antigens, including:

Extract mechanistic details of how LNPs deliver mRNA into target cells, including:

Extract quantitative measures of mRNA delivery effectiveness specifically for SARS-CoV-2 vaccines, including:

Extract physical and structural characteristics that enable mRNA delivery, including:

Extract biodistribution data showing where LNPs deliver mRNA after administration, including:

Extract data on how LNPs contribute to immune activation beyond mRNA delivery, including:

Extract factors affecting LNP-mRNA stability and delivery performance over time, including:

Results

Characteristics of Included Studies

Study

Full text retrieved?

Study focus

LNP formulation

Study design

Suman Alishetty et al., 2021

Yes

Novel C24 ionizable lipid development

C24 lipid (48:13:37:2 ratio)

Mouse immunogenicity and challenge studies

Afshana Quadiri et al., 2024

Yes

Effect of helper lipid composition

LNP4 with DOPE phospholipid

Hamster protection studies comparing 4 LNP formulations

Afshana Quadiri et al., 2025

Yes

DOPE-containing LNP characterization

LNP4 with DOPE

Hamster immunogenicity and protection

Xuexiang Han et al., 2023

Yes

Adjuvant lipidoid substitution

C12-TLRa adjuvant lipidoid (35:16:46.5:2.5)

Mouse immunogenicity with TLR7/8 agonist lipid

J. Szebeni et al., 2023

Yes

Structural analysis of Comirnaty vaccine

BNT162b2 with ALC-0315

Physicochemical characterization using AFM, cryo-TEM

S. Kent et al., 2024

No

Human pharmacokinetics

Moderna SPIKEVAX

Clinical study in 19 vaccinated subjects

M. Alameh et al., 2021

Yes

Adjuvant activity mechanisms

Proprietary Acuitas formulation

Mouse studies with influenza and SARS-CoV-2 mRNA

Natalia Martinez et al., 2025

No

Monoolein as helper lipid

MO-based modified Moderna LNPs

In vivo delivery efficiency studies

Dorottya Laczkó et al., 2020

Yes

Nucleoside-modified mRNA immunogenicity

Standard LNP formulation

Mouse single-dose immunogenicity

Devdoot Majumdar et al., 2023

No

Mechanisms of muscle-based adjuvancy

Moderna sm-102 formulation

Mouse mechanistic studies of immune response

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All 10 studies investigated lipid nanoparticle delivery systems for nucleoside-modified mRNA encoding SARS-CoV-2 spike protein antigens. Seven studies had full text available, while three were abstract-only. The studies employed diverse approaches including novel lipid development, structural characterization, immunogenicity testing in animal models, and clinical pharmacokinetic analysis in humans.

LNP Composition and Formulation Design

LNP formulations for SARS-CoV-2 mRNA delivery consist of four core lipid components: ionizable lipids, helper lipids, cholesterol, and PEG-lipids. The ionizable lipid component is critical for mRNA complexing and pH-dependent release.

Component

Example formulations

Molar ratios

Design rationale

Ionizable lipid

C24, ALC-0315, C12-TLRa

35-48%

Enhanced endosomal protonation; TLR7/8 activation

Helper lipid

DSPC, DOPC, DOPE, Monoolein

13-16%

Structural stability; pH-dependent mesophase transitions

Cholesterol

Standard component

37-46.5%

Structural support

PEG-lipid

DMG-PEG2000

2-2.5%

Immune evasion and circulation

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The C24 LNP formulation demonstrated a specific composition of 48:13:37:2 for ionizable lipid/DSPC/cholesterol/DMG-PEG2000, formulated in PBS at pH 7.3-7.4. The adjuvant lipidoid formulation used a 35:16:46.5:2.5 ratio and achieved a particle size of approximately 52 nm with low polydispersity (PDI 0.127). Monoolein incorporation as a structural helper lipid induced pH-dependent mesophase transitions promoting inverse hexagonal structures to enhance mRNA release.

LNP morphology varied across formulations. Comirnaty exhibited a granular solid core enclosed by mono- and bilipid layers, with mechanical properties showing soft, compliant structures with stiffness of approximately 9 pN/nm. The particle size ranged from 52 nm to 80-85 nm with polydispersity indices of 0.02-0.06. Surface charge was generally neutral in PBS, though Comirnaty showed a less negative zeta potential of 8.6 ± 5.3. The C24 formulation displayed higher pKa (6.77) compared to MC3 (6.55).

mRNA Cargo Characteristics

The nucleoside-modified mRNA cargo encoded the SARS-CoV-2 spike protein with specific structural modifications. The target antigens included full-length spike protein with deleted furin cleavage site, diproline-stabilized spike protein (S2P), and prefusion-stabilized spike with two proline mutations.

mRNA feature

Specifications

Nucleoside modification

m1ψ (1-methylpseudouridine); Pseudo-U; m1J-5’-triphosphate

Sequence length

3,804 bp; 4,284 nucleotides

Cap structure

Trinucleotide cap1 analog (CleanCap)

Poly-A tail

80-101 adenine residues

Codon optimization

Applied to enhance translation

Encapsulation efficiency

93-95%

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The nucleoside modifications served to reduce immune recognition and enhance stability. The mRNA sequences were codon-optimized for efficient translation in human cells, with coding sequences identical to FDA-approved vaccines mRNA-1273 and BNT162b2 in some formulations.

Cellular Uptake and Delivery Mechanisms

LNPs deliver mRNA through a multi-step process involving cellular uptake, endosomal escape, and cytoplasmic release. The ionizable lipids play a central role in pH-dependent delivery.

Uptake pathways and cell types:

Muscle-resident myeloid and lymphoid cells were directly transduced, with >10% of these cells successfully receiving mRNA. Lung macrophages, epithelial cells, and cancer cells also took up LNPs. Intramuscular administration of empty LNPs elicited strong neutrophil and dendritic responses within 24 hours, indicating rapid immune cell engagement. Skeletal muscle played a role in the early immune response.

Endosomal escape mechanism:

The cellular uptake occurred via endocytosis. Following endocytosis, ionizable lipids became protonated in the acidic endosomal environment. This protonation triggered ion pairing with endosomal phospholipids to open the endosomal membrane. The C24 formulation demonstrated greater endosomal protonation due to its multistage protonation behavior. Neutralization of LNP charge by anionic lipids inside cells disrupted nanoparticle structure, releasing mRNA.

The adjuvant lipidoid formulation enhanced endosomal escape through interaction with TLR7/8 receptors, with structural characterization showing LNPs initially trapped in endo/lysosomes before escaping into the cytoplasm. Monoolein incorporation promoted pH-dependent mesophase transitions that facilitated enhanced mRNA release.

Structural interactions:

The Comirnaty formulation revealed unique mRNA-lipid interaction patterns. Atomic force microscopy demonstrated that molecular strands corresponding to mRNA could be pulled from nanoparticles with stepwise rupture of mRNA-lipid bonds. The neutral intra-LNP core suggested hydrogen bonding rather than ionic interactions as the primary stabilization mechanism. The ionizable lipid ALC-0315 contained free =O and −OH groups capable of hydrogen bonding with nitrogenous bases in mRNA.

Delivery Efficiency and Protein Expression

Quantitative delivery efficiency varied across formulations, with several showing superior performance compared to standard formulations.

Protein expression levels:

The C24 LNP showed 2-fold higher luciferase expression at injection sites compared to MC3 at both high and low doses. Neutralizing antibody titers were 10-fold higher for C24 versus MC3. The adjuvant lipidoid formulation (C12-113/TLRa) outperformed standard C12-113 in mRNA transfection efficiency both in vitro and in vivo, with protein expression lasting over 14 days at injection sites. Monoolein-based LNPs achieved superior mRNA transfection efficiency compared to original Moderna LNPs across diverse cell types.

Dose-response relationships:

The C24 LNP was effective at very low doses, with protection against SARS-CoV-2 occurring at 0.25 µg prime/boost. The C12-113/TLRa formulation consistently outperformed control formulations at any mRNA dose tested.

Tissue specificity:

Enhanced mRNA transfection occurred at both injection sites and inguinal lymph nodes for the adjuvant formulation. The C24 LNP reduced off-target liver expression 6-fold compared to MC3. Monoolein-based LNPs achieved targeted pulmonary delivery by intranasal administration and spleen delivery by intravenous administration.

Biodistribution Patterns

Following intramuscular administration, LNPs demonstrated specific biodistribution kinetics with varying degrees of systemic exposure.

Distribution timeline in humans:

In a clinical study of 19 subjects receiving Moderna SPIKEVAX, both mRNA and ionizable lipid peaked in blood 1-2 days post-vaccination, with median peak levels of 0.19 ng/mL for mRNA and 3.22 ng/mL for ionizable lipid. The mRNA remained detectable for 14-28 days post-vaccination in most subjects. The decay kinetics of intact mRNA and ionizable lipid were identical, suggesting intact lipid nanoparticles recirculated in blood.

Tissue targeting:

The primary target tissues included skeletal muscle at the injection site and draining lymph nodes. Animal studies showed mRNA expression observed within 6-24 hours at injection sites and lymph nodes, with sustained expression over 14 days. Minimal accumulation occurred in major organs including liver, heart, spleen, lung, and kidney.

Factors controlling distribution:

Rapid increase in surface charge near neutral pH limited systemic biodistribution for C24 formulations. The magnitude of mRNA and ionizable lipid detected in blood correlated with boost in PEG antibodies. The ability of monocytes to phagocytose lipid nanoparticles showed an inverse relationship with PEG antibody rise.

Immune Activation and Adjuvant Properties

Beyond mRNA delivery, LNPs demonstrated intrinsic adjuvant activity contributing to robust immune responses.

Innate immune activation:

LNP formulations triggered strong innate immune responses through multiple mechanisms. The ionizable lipid component induced IL-6 cytokine production, critical for T follicular helper cell differentiation. Empty LNPs alone elicited strong neutrophil and dendritic responses within 24 hours of intramuscular administration. Enhanced dendritic cell maturation occurred with specific formulations, particularly LNP4.

Cytokine profiles:

LNPs induced proinflammatory cytokines including IL-6, GM-CSF, IL-1β, IL-5, IP-10, KC, LIF, Lix, and MIP-2. IL-6 production peaked at 4 hours post-injection and remained elevated for at least 24 hours. Increased secretion of IL-6, IL-21, and IFN-γ indicated robust inflammatory responses. The adjuvant lipidoid formulation increased production of TNF-α, IL-12p70, and IL-1β.

TLR activation:

The C12-TLRa adjuvant lipidoid conferred Toll-like receptor 7/8-agonistic activity to LNPs. This TLR7/8 activation enhanced dendritic cell maturation. MyD88-based signaling enhanced mRNA-LNP vaccine potency but was not required for LNP adjuvanticity. The adjuvant activity relied on the ionizable lipid component and IL-6 induction but not on MyD88- or MAVS-dependent sensing.

Reactogenicity:

Injection site inflammation was notably reduced for C24 compared to MC3. The inflammatory responses were primarily localized to injection sites and draining lymph nodes with minimal systemic inflammation for the adjuvant formulation.

Stability and Storage Considerations

LNP-mRNA stability depended critically on storage temperature and formulation design.

Temperature-dependent stability:

The C24 LNP was entirely stable in bioactivity and mRNA integrity when stored at 4°C for at least 19 days. Bioactivity remained stable for two weeks at 4°C but declined by 20-40% at room temperature. Storage at higher temperatures reduced both bioactivity and mRNA integrity, though less so for C24 than MC3. Standard formulations required frozen storage, typically at -80°C, though some could be stored at refrigerated temperatures for up to 30 days.

Mechanisms of degradation:

mRNA degradation occurred through phosphodiester transesterification reactions, which were temperature-dependent. The mechanism involved base-mediated phosphodiester transesterification. mRNA half-lives were significantly longer at 4°C compared to higher temperatures. The LNP environment affected mRNA degradation due to proton partitioning phenomena.

Formulation stability:

The adjuvant lipidoid formulation showed minimal changes in physicochemical parameters after one month of storage at 4°C. High encapsulation efficiency (>93%) contributed to stability. Nucleoside modifications and LNP formulation enhanced translational capacity and biological stability. The hydrogen bonding between mRNA and lipids in some formulations was weaker than ionic interactions, potentially affecting long-term stability.

Synthesis

The mechanisms by which lipid nanoparticles deliver nucleoside-modified mRNA for SARS-CoV-2 vaccines involve an integrated series of physicochemical and biological processes. The delivery cascade initiates with formulation design, where ionizable lipids complex with negatively charged mRNA through electrostatic and hydrogen bonding interactions. Following intramuscular administration, LNPs interact with muscle cells and immune cells through endocytic uptake, with >10% of muscle-resident myeloid and lymphoid cells successfully transduced.

The critical endosomal escape step relies on pH-dependent protonation of ionizable lipids in acidic endosomes, triggering membrane disruption through ion pairing with endosomal phospholipids. Different ionizable lipids achieve this through distinct mechanisms: the C24 formulation employs multistage protonation, while adjuvant lipidoids enhance escape through TLR7/8 receptor engagement. Upon cytoplasmic release, the nucleoside-modified mRNA is translated by cellular ribosomes to produce spike protein antigen.

Beyond simple mRNA delivery, LNPs function as adjuvants by triggering innate immune responses. Empty LNPs alone elicit neutrophil and dendritic cell recruitment within 24 hours, while the ionizable lipid component induces IL-6 production critical for germinal center formation. This dual functionality—mRNA delivery plus immune activation—underlies the exceptional efficacy of LNP-mRNA SARS-CoV-2 vaccines, with some formulations achieving protection at doses as low as 0.25 µg and neutralizing titers 10-fold higher than conventional formulations.

Human pharmacokinetic data reveals that intact LNPs recirculate in blood for weeks post-vaccination, with mRNA detectable for 14-28 days, supporting sustained antigen expression. The formation of PEG antibodies correlates with the magnitude of circulating mRNA/lipid and is inversely related to monocyte phagocytic clearance, suggesting immune-mediated clearance pathways influence delivery kinetics.

Structural innovations continue to optimize this delivery platform. Replacing phospholipids with monoolein induces pH-dependent mesophase transitions promoting inverse hexagonal structures that enhance mRNA release. The C24 formulation reduces off-target liver expression 6-fold while maintaining potency, and adjuvant lipidoids provide TLR7/8 activation to boost cellular immunity. These advances demonstrate how rational lipid design can independently modulate delivery efficiency, tissue targeting, immune activation, and stability—the four key parameters determining LNP-mRNA vaccine performance.

References

Xuexiang Han, M. Alameh, Kamila Butowska, James J Knox, Kendall A. Lundgreen, and 11 more\ (2023).Adjuvant lipidoid-substituted lipid nanoparticles augment the immunogenicity of SARS-CoV-2 mRNA vaccines. Nature Nanotechnology

J. Szebeni, B. Kiss, Tamás Bozó, K. Turjeman, Y. Levi-Kalisman, and 2 more\ (2023).Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA–Lipid Complexes. ACS Nano

Suman Alishetty, Manuel J. Carrasco, M. Alameh, M. Paige, Hooda Said, and 14 more\ (2021).Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution

S. Kent, Shiyao Li, T. Amarasena, A. Reynaldi, W. Lee, and 9 more\ (2024).Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans. medRxiv

Afshana Quadiri, S. Prakash, Latifa Zayou, N. Dhanushkodi, Amruth Chilukuri, and 4 more\ (2024).Spike mRNA Vaccine Encapsulated in a Lipid Nanoparticle Composed of Phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine Induced Potent B- and T-cell Responses Associated with Protection against COVID-19 in Hamsters. bioRxiv

Afshana Quadiri, S. Prakash, Latifa Zayou, N. Dhanushkodi, Amruth Chilukuri, and 5 more\ (2025).A Spike-Based mRNA Vaccine Encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine Containing Lipid Nanoparticles Induced Potent B- and T-Cell Responses Associated with Protection Against SARS-CoV-2 Infection and COVID-19-like Symptoms in Hamsters. Vaccines

M. Alameh, I. Tombácz, Emily Bettini, Katlyn Lederer, Sonia Ndeupen, and 29 more\ (2021).Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses. Immunity

Dorottya Laczkó, Michael J. Hogan, Sushila A. Toulmin, Philip D. Hicks, Katlyn Lederer, and 37 more\ (2020).A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity

Devdoot Majumdar, W. Dowell, Sylvester Languon, Zachary D. Miller, Jake Dearborn\ (2023).Understanding Mechanisms of Adjuvancy in Muscle by mRNA/Lipid Nanoparticles. Journal of Immunology

Natalia Martinez, Haitao Yu, Joshua Iscaro, Hao Wang, Brendan P. Dyett, and 5 more\ (2025).Structure–Function Correlation of Clinically Inspired Lipid Nanoparticles for Lung and Spleen Targeted mRNA Delivery. Advanced Healthcare Materials

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A Spike-Based mRNA Vaccine Encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine Containing Lipid Nanoparticles Induced Potent B- and T-Cell Responses Associated with Protection Against SARS-CoV-2 Infection and COVID-19-like Symptoms in Hamsters

Afshana Quadiri, S. Prakash, Latifa Zayou, N. Dhanushkodi, Amruth Chilukuri, Gemma Ryan, Kelly Wang, Hawa Vahed, A. Chentoufi, L. BenMohamed

Vaccines·

2025·

6 citations

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LNP Composition

- Ionizable lipid type: Proprietary to Cytiva, structure not disclosed - Helper lipids: DSPC, cholesterol; specific ratios not provided - PEG-lipid type: Not specifically mentioned for LNP-4 - Novel or modified lipid components: DOPE (1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine) in LNP-4 - Design rationale: Enhance delivery efficiency and stability - Particle size and polydispersity: Analyzed using DLS, specific values not provided - Formulation buffer and pH conditions: Sterile Ca 2+ and Mg 2+ free PBS; pH 6.4 for mRNA packaging

mRNA Specifications

- Target antigen: SARS-CoV-2 full-length-of-prefusion-stabilized Spike protein with two proline mutations - Specific nucleoside modifications: Full substitution of Pseudo-U - mRNA sequence length and structure: 3804 bp; nucleotide range: 21,504 bp-25,308 bp - Cap structure and poly-A tail details: Polyadenylated (80A) - Codon optimization strategies: Not mentioned - mRNA-to-lipid ratios in formulation: Not mentioned

Cellular Uptake Mechanism

- Cell types: Not specifically mentioned, but implies uptake by cells in general. - Uptake pathways: Endocytosis. - Endosomal escape mechanisms: Neutralization of LNPs' charge by anionic lipids. - Role of ionizable lipids: pH-dependent release; neutral at physiological pH, positively charged in acidic environments. - Timeline: Endocytosis, endosomal escape, translation by ribosomes (no specific timeframe). - Subcellular trafficking: Entry through endosomes, release into cytosol. - Membrane interaction: Electrostatic facilitation due to charge differences.

Delivery Efficiency

- Protein expression levels: Enhanced phenotypic and functional maturation of dendritic cells; induced strong T-cell responses; increased secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; induced higher neutralization IgG titers. - Transfection efficiency percentages: Not explicitly mentioned. - Duration of protein expression: Not explicitly mentioned. - Dose-response relationships: LNP-4 showed higher potency in inducing immune responses and protection. - Comparison with control formulations: LNP-4 outperformed other LNPs (LNP1, LNP2, LNP3). - In vitro vs in vivo delivery efficiency: Both in vitro and in vivo delivery efficiency were assessed in hamsters. - Target tissue specificity: Focus on dendritic cells and T-cell responses.

Structural Properties

- LNP morphology: Spherical delivery vehicles - mRNA-lipid interaction patterns: Ionizable lipids form complexes with negatively charged mRNA - Hydrogen bonding or ionic interactions: Ionizable lipids are positively charged under low pH conditions - Membrane organization: Lipid core matrix composed of phospholipids, cholesterol, and other lipids - Mechanical properties: Not mentioned - Surface charge and zeta potential: Ionizable lipids are positively charged under low pH conditions - Cryo-TEM or AFM structural observations: Not mentioned

Biodistribution Pattern

Not mentioned (the paper does not provide detailed biodistribution data)

Inflammatory Effects

- Innate immune responses triggered by LNPs: Enhanced dendritic cell maturation and strong T-cell responses. - Inflammatory cytokine induction: Increased secretion of IL-6, IL-21, and IFN-γ. - Neutrophil and dendritic cell recruitment: Not mentioned. - Local vs systemic inflammation: Not mentioned. - Adjuvant effects of lipid components: Enhanced immune responses with LNP4. - TLR activation pathways: Not mentioned. - Injection site reactogenicity: Not mentioned. - Timeline of inflammatory responses: Not detailed.

Stability Factors

- Storage temperature requirements: Extremely cold temperatures. - mRNA integrity maintenance: Protected by LNPs from extracellular ribonucleases. - LNP structure preservation: Composed of phospholipids, cholesterol, and other lipids. - Bioactivity retention timeline: Not explicitly mentioned. - Mechanisms of degradation: Degradation by nucleases and self-hydrolysis. - Formulation stability enhancers: Ionizable lipids and phospholipid composition. - Real-time vs accelerated stability data: Not mentioned. - Impact on delivery efficiency: Affected by biophysical parameters of LNPs.

Background: Nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) have emerged as a promising vaccine strategy, especially for COVID-19. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells, the effect of lipid composition on the immunogenicity and protective efficacy of mRNA/LNP vaccines is not well characterized. Studies on using the mRNA/LNP platform for vaccines have largely focused on the nucleic acid cargo with less attention paid to the LNP vehicle. Whether the composition and biophysical properties of LNPs impact vaccine performance remains to be fully elucidated. Methods: In the present study, we used SARS-CoV-2 Spike-mRNA as a prototype vaccine to study the effect of four different LNPs with various lipid compositions. Results: We demonstrate that when the same Spike-mRNA was delivered in the LNP4 formulation based on phospholipid 1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine, it outperformed other LNPs (LNP1, LNP2, and LNP3) that are based on different lipids. Compared to the other three LNPs, LNP4 (i) enhanced the phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses; (iii) increased the secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) provided better protection against SARS-CoV-2 infection and COVID-19-like symptoms in the hamster model. Furthermore, we compared LNP-4 with the commercially available LNPs and found it to provide better T-cell immunity against COVID-19 in hamsters. Conclusion: This study suggests mRNA vaccines encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine containing LNPs induced Potent B- and T cell immunity. The mechanisms by which Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine-based LNPs may activate protective B and T cells are discussed.

1.Introduction

The development of safe and efficacious vaccination against diseases that cause substantial morbidity and mortality is the greatest contribution of any health intervention [1][2] [3] [4]. The beginning of the 21st century marked a revolution in molecular biology and provided insights into microbiology and immunology allowing a greater understanding of vaccine delivery systems [5] [6] [7]. Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced vehicle platforms for the efficient in vivo delivery of nucleoside-modified mRNA vaccines, particularly for COVID-19 [8].

The use of LNPs in COVID-19 mRNA vaccines gained significant attention for delivering the mRNA to cells demonstrating several advantages over viral vectors, including low cytotoxicity and good immunogenicity, simple production, and great scalability [9,10]. mRNAs are inherently unstable and prone to rapid degradation by nucleases and selfhydrolysis. LNPs are efficient for mRNAs' encapsulation, protect them from extracellular ribonucleases, and assist with in vivo and intracellular mRNA delivery [11,12]. Studies on using mRNA/LNP vaccines have largely focused on characterizing the nucleic acid cargos, with less attention being paid to LNP vehicles. While LNPs protect mRNA from degradation and efficiently deliver mRNAs in vivo to antigen-presenting cells, the effect of lipid composition and the biophysical properties of LNPs on the immunogenic and protective efficacy of mRNA/LNPA vaccines remain to be fully elucidated.

LNPs are spherical delivery vehicles that comprise four different lipid components: (i) ionizable lipids, which are primarily required for mRNA complexing; (ii) helper lipids, which enhance the properties of LNPs (stability and delivery efficiency); (iii) cholesterol, which provides structural stability to the LNPs (vehicle); and (iv) surface PEGylation with poly(ethylene glycol) (PEG) or PEG derivatives, which reduces host-immune recognition and improves systemic circulation [13]. These lipids provide several benefits, including formulation simplicity, self-assembly, biocompatibility, high bioavailability, and the potential to transport large payloads [13]. Ionizable lipids are the most essential components of LNPs, as they are neutral in the physiological pH environment but positively charged under low pH conditions. Ionizable lipids form complexes with the negatively charged mRNA and subsequently releases it in the cytosol [14]. The nucleic acid encapsulated in LNPs is delivered through absorption by the cell plasma membrane and the eventual uptake into the cell by endocytosis. After endocytosis, the nucleic acid is released inside the cell. The adsorption of LNPs is facilitated electrostatically because of the difference between the negatively charged cell membrane and the positively charged LNPs [15]. When the LNPs enter the cell, nucleic acid is released from the cationic carrier, and the LNPs' charge is neutralized by anionic lipids found in the cells. This neutralization of the LNPs stops electrostatic attraction between the lipids and nucleic acids, disrupting the structure of the nanoparticles and causing a non-lamellar structure, thus delivering mRNAs with high mRNA encapsulation, greater stability, and pH-sensitive in vivo delivery [16].

Currently, there are several mRNA-based vaccines against SARS-CoV-2 that utilize different LNP compositions, of which two are Pfizer-BioNtech's BNT162b2 and Moderna's mRNA-1273 [17]. Pfizer-BioNtech uses an ionizable cationic lipid called ALC-0315, which they licensed from Acuitas. The nanoparticles used in Pfizer-BioNtech BNT162b2 are ALC-0315, ACL-0159, 1,2-stearoyl-sn-glycerol-3-phosphocholine (DSPC), and cholesterol. ALC-0159 is a polyethylene glycol conjugate, meaning it is a PEGylated lipid [18]. Mod-erna uses its own unique and proprietary ionizable cationic lipid called SM-102. The nanoparticles used in Moderna mRNA-1273 are 1,2-stearoyl-sn-glycerol-3-phosphocholine (DSPC), polyethylene glycol 200-dimyristoyl glycerol (PEG2000-DMG), cholesterol, and SM-102 [12,19]. In both mRNA-based vaccines, the lipids used are 1,2-stearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, a type of PEGylated lipid, and an ionizable cationic lipid. Modifications made to the cholesterol used in these mRNA-based vaccines increases the cellular uptake of LNPs [20]. The PEG-lipid, with its chemical structure of a hydrophilic and hydrophobic region, allows penetration across lipid membranes and influences the size of the particle [21]. The ionizable lipid design has undergone numerous improvements leading to much higher transfection potency. The first ionizable lipid reported AL1 synthesized for nucleic acid delivery, exhibited low therapeutic levels. Improvements to the lipid design resulted in nearly 8000-fold improvements in the therapeutic index [22]. Introducing a ketal group in the ionizable lipid led to 10% increase in gene silencing by LNPs [23].

The challenges remaining for mRNA/LNP vaccines involve the complexity associated with identifying the best formulation. The detailed mechanistic knowledge of how LNPs assist with the endocytosis of mRNA is still lacking. This makes improvements to the design and formulation of LNPs difficult. For most formulations, the bottleneck has not been identified, whether it is endocytosis, endosomal escape, stability of the mRNA, DC activation, or something different [24]. Much research has been performed to improve the endosomal escape and transfection efficiency of LNPs by modifying lipid compositions. However, compared to the extensive research conducted exploring different lipid formulations, the nanostructure of LNPs or the specific packing of lipids and nucleic acids into LNPs has not been fully investigated. Over the past 20 years, ionizable lipid design has undergone numerous improvements. The first ionizable lipid reported AL1, also known as DODAP, did not provide significant therapeutic levels of delivery unless high doses were administered. Improvements to the lipid design resulted in nearly 8000-fold improvements in the therapeutic index [25]. Interestingly, the ester-containing analog, was found to be ineffective while a ketal-containing compound exhibited 10-fold more activity than the effective dose required to achieve 50% gene silencing [20,25]. LNP size plays a critical role in delivery for larger nanoparticles, however research is required to understand whether and how LNP size influences mRNA delivery. A comparative evaluation of different LNPs is, therefore, required to determine the optimal parameters for the desired vaccination. In the present study, we investigated the pre-clinical efficacy of four different LNPs. The four LNPs (described in this study as LNP-1, LNP-2, LNP-3, and LNP-4) differed in their phospholipid compositions or ionizable lipid relative amount, which we found to significantly affect the degree of protection from COVID-19 disease.

In this study, we observed that LNP-4 that contains a phospholipid constituent named as 1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine (DOPE) has higher potency at inducing better immune responses and better protection in hamsters upon SARS-CoV-2 infection. Compared to the other three LNPs, LNP-4 (i) enhanced the phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses; (iii) increased the secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) provided better protection against SARS-CoV-2 infection and COVID-19 in the hamster model. In addition, we compared LNP-4 with a well-established pre-optimized ionizable lipid mix for mRNA-LNP delivery known as GenVoy-ILM TM . The formulation developed by PNI has displayed vaccine stability and efficacy and has been used for both in vitro and in vivo deliveries of mRNA and selfamplifying RNA (saRNA) for infectious disease vaccine research [26][27][28][29]. In this study, we refer to GenVoy-ILM TM as LNP-O (LNP "Off the shelf"). We observed that Spike mRNA encapsulated in LNP-4 elicited higher antibody titers compared to LNP-O upon immunization. In addition, LNP-4-immunized hamsters exhibited significantly higher T-cell responses as well as a display of activation markers on CD4+ and CD8+ T cells in comparison to LNP-O. The findings point to the need for understanding and adjusting lipid formulations to achieve a higher efficacy of LNP-mRNA-based vaccines.

2.Materials and Methods

LNP formulation: Four customized GenVoy-ILM-based lipid nanoparticles containing a common ionizable cationic lipid and with differing compositions of phospholipids, cholesterol, and stabilizer were provided by Precision NanoSystems (PNI) (currently Cytiva) for this project. The 4 LNPs differed in their phospholipid compositions or ionizable lipid relative amounts. LNPs with these compositions are available for purchase from PNI upon request using the identifiers IL00V02, IL00V03, IL00V29, and IL00V41. For the convenience of this study, we used LNP1, LNP2, LNP3, and LNP4 to identify IL00V02, IL00V03, IL00V29, and IL00V41, respectively. Each of these 4 LNPs comprised a unique phospholipid that is the second listed lipid component of the LNP. Different phospholipids are associated with the 4 LNPs as follows: (i) LNP1 (IL00V02) composed of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), (ii) LNP2 (IL00V03) composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), (iii) LNP3 (IL00V29) composed of DPPC (dipalmitoylphosphatidylcholine), and (iv) LNP4 (IL00V41) composed of DOPE (1,2-dioleoylsn-glycero-3-phosphoethanolamine). While the ionizable phospholipid that was used in all the formulations was the same, the amount used between formulations varied. LNP3 (IL00V29) and LNP4 (IL00V41) both have a "higher" amount of ionizable lipid, but the phospholipid composition is different. A single ionizable lipid was used across all formulations. The ionizable lipid and its structure are proprietary to Cytiva. The physical properties associated with the four LNPs are further described in Table 1 . Along with the four customized LNP formulations, we also used for comparison the "Off-the Shelf" available LNP formulation named GenVoy-ILM TM , which is a commercially available proprietary lipid-mix composition comprising DSPC:cholesterol:ionizable-lipid:stabilizer at 10:37.5:50:2.5 mol% for encapsulating nucleic acids, manufactured by Precision NanoSystems Inc (Vancouver, BC, Canada). In this article, this "Off-the Shelf" LNP formulation is called LNP-O. To prepare the lipid encapsulation, the mRNA stock was first diluted in the PNI's formulation buffer. Subsequently, the 4 GenVoy-ILM-based lipid mixes were prepared in ethanol at a 12.5 mM concentration. The LNP formulations were prepared on NanoAssemblr Ignite using a specific formulation scheme, which included a flow rate ratio (FRR) for the mRNA solution to lipid solution of 3:1; total flow rate (TFR) of 12 mL/min; start waste of 1 mL; and end waste of 0.05 mL. After the formulation, the post-chip formulations were diluted to a total 4X volume in sterile Ca 2+ and Mg 2+ free PBS. Amicon ® Centrifugal Filters (Merck KGaA, Darmstadt, Germany) with an MWCO of 30 KDa (LNP1, LNP2, and LNP3) or an MWCO of 100 KDa (LNP4) were used to concentrate formulations and perform ethanol removal and buffer exchange to 1X PBS (pH 7-7.3, Mg 2+ /Ca 2+ -free). mRNA LNPs were filtered manually through a 0.22 µm syringe filter using aseptic techniques and were stored at 4 • C. Finally, the particle size and polydispersity index (PDI) were analyzed using dynamic light scattering (DLS). RNA encapsulation efficiency and concentrations were determined using a RiboGreen plate-based assay.

mRNA synthesis: Sequences of the Spike antigen were derived from the SARS-CoV-2 Omicron sub-variant BA.2 (NCBI GenBank accession number OM617939). Nucleosidemodified mRNAs expressing the SARS-CoV-2 full-length-of-prefusion-stabilized Spike protein with two proline mutations (mRNA-S-2P (size: 3804 bp; nucleotide range: 21,504 bp-25,308 bp)) were synthesized by in vitro transcription using T7 RNA polymerase (MegaScript, Thermo Fisher Scientific, Waltham, MA, USA) on linearized plasmid templates, as previously reported [30]. A modified mRNA transcript with a full substitution of Pseudo-U was synthesized by TriLink Biotechnologies using proprietary CleanCap ® technology. The synthesized polyadenylated (80A) mRNAs were subjected to DNase and phosphatase treatment, followed by silica membrane purification. Finally, the synthesized mRNA was packaged as a 1.00 ± 6% mg/mL solution in 1 mM of sodium citrate, pH 6.4. Purified mRNA was analyzed by agarose gel electrophoresis and kept frozen at -20 • C.

Human Samples: Blood samples were obtained from healthy donors. Consenting adults were screened using a questionnaire determining their demographic information, medication usage, and comorbidities. Participants were excluded from any acquired immunodeficiency or immunomodulating medications (such as steroids or chemotherapy), pregnancy, history of cancer, history of cirrhosis or renal failure, or antibiotic use within 2 weeks of recruitment. Blood samples were taken from individuals aged 18-75 years. The experiments were approved by the Institutional Biosafety Committee at the University of California, Irvine (Protocol number BUA-R112). Written informed consent was obtained from all patients before inclusion.

PBMC isolation: The blood of anonymous healthy donors was diluted with RPMI1640. Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using percoll. After centrifugation, the interphase containing PBMCs was collected and washed twice with RPMI1640. The cells were cultured in tissue culture dishes and incubated at 37 • C, 5% CO 2 .

Generation of human monocyte-derived dendritic cells (MoDCs):

The DCs in PBMC were allowed to adhere to the culture dish. On the next day, the medium was replaced with DC medium containing 800 U/mL of GM-CSF and 50 U/mL of IL4. On day 3 and day 5, a total of 4 mL of fresh DC medium supplemented with 800 U/mL of GM-CSF and 50 U/mL of IL4 was added. On day 7, immature as well as mature DCs were harvested. As moDCs adhere loosely to the culture dish, the use of EDTA or cell scrapers is not needed. Thus, the supernatant was flushed a couple of times to obtain moDCs. Afterward, moDCs were treated with different LNPs for 24-36 h. The cells were harvested and stained for FACS analysis. The supernatant was collected and analyzed for different cytokines.

Flow cytometry: The following anti-human antibodies were used for the flow cytometry assays: CD11c (clone BLY6-BD BioSciences), HLA-DR (clone G46-6-BD BioSciences), CD40 (clone 5C3-Invitrogen), CD86 (clone 2331 (FUN-1)-BD BioSciences), and CD83 (clone HB15e-BD BioSciences). For the surface stain, mAbs against cell markers were added to a total of 1 × 10 6 cells in 1X PBS containing 1% FBS and 0.1% sodium azide (FACS buffer) for 30 min at 4 • C. The cells were washed with FACS buffer and finally suspended in 200 uL of FACS buffer. For each sample, 50,000 total events were acquired on the BD LSRII. Ab capture beads (BD Biosciences) were used as individual compensation tubes for each fluorophore in the experiment. We used fluorescence minus controls for each fluorophore to define positive and negative populations when initially developing staining protocols. Data analysis was performed using FlowJo version 10.10. Statistical analyses were performed using GraphPad Prism version 7.

Hamster mRNA/LNP immunization and SARS-CoV-2 virus challenge: The mRNA/ LNP vaccines were evaluated in the outbred golden Syrian hamster model for protection against the SARS-CoV-2 USA/WA1/2020 strain. The Institutional Animal Care and Use Committee approved animal model usage experiments at the University of California, Irvine (Protocol number AUP-22-086). The recommendations are present in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health performed animal experiments. The total sample size of 6-8-week-old male Syrian golden hamsters used in this was 45. Hamsters divided into nine experimental groups were vaccinated with 1 µg (n = 20) and 3 µg (n = 20) of LNP1, LNP2, LNP3, and LNP4 encapsulating Spike protein intramuscularly in 100 µL doses into the posterior thigh muscle, twice (on day 0 and day 21). The mock group of hamsters was not immunized with any of the mRNA-LNP formulations. Four weeks after the first immunization, blood samples were collected from hamsters under isoflurane anesthesia and spun at 2000× g for 10 min to obtain serum. The hamsters were challenged intranasally with 100 µL of 1 × 10 5 PFU of the SARS-CoV-2 USA/WA1/2020 strain diluted in sterile Dulbecco's modified Eagle's medium (DMEM). Weights were recorded daily until 14 days p.i. Oropharyngeal swabs were collected on days 2, 6, 10, 1and 4 in 500 µL of RNA later for virus titrations. The animals in each group were monitored daily for signs of disease and weighed until 14 days p.i.

In a separate experiment, we compared the efficacy of the LNP4 formulation with the commercially available GenVoy-ILM TM (LNP "Off the shelf"). In this context, we used three groups of hamsters comprising immunization with (i) Spike-mRNA/LNP-4 (n = 6), (ii) Spike-mRNA/LNP-O (n = 6), and (iii) Mock (n = 6).

QuantiFERON assay: The whole heparinized blood sample was collected from each hamster for performing the QuantiFERON assay to determine the secretion of IFN-γ (QuantiFERON SARS-CoV-2, Qiagen). After overnight incubation with SARS-CoV2-specific peptides, supernatant was collected from LNP-stimulated samples in vitro at 12 and 24 h time points for IFN-γ analysis. The cytokine analysis was performed on 96-well flat bottom microplates. ELISA plates were coated with specific capture antibodies. The supernatant obtained from LNP-stimulated moDCs was tested without any dilutions. For hamster ELISA experiments, the sera were collected from blood isolated from immunized hamsters. ELISA was performed on sterile 96-well flat-bottom microplates coated with the Spike antigen in coating buffer overnight at 4 • C. The reaction was terminated by adding 1 M of H 2 SO 4 . The absorbance was measured at 450 nm.

Enzyme-linked immunosorbent assay (ELISA): Serum antibodies for SARS-CoV-2 proteins were detected by ELISA. The 96-well plates (Dynex Technologies, Chantilly, VA, USA) were coated with 100 ng of S protein per well at 4 • C overnight, and then washed three times with PBS and blocked with 3% BSA (in 0.1% PBST) for 2 h at 37 • C [31,32]. After blocking, the plates were incubated with serial dilutions of the sera (100 µL/well, in a two-fold dilution) for 2 h at 37 • C. The bound serum antibodies were detected with HRP-conjugated goat anti-mouse IgG and chromogenic substrate TMB (ThermoFisher, Waltham, MA, USA). The cut-off for seropositivity was set as the mean value plus three standard deviations (3SDs) in HBc-S control sera.

Neutralization Assay: Serum-neutralizing activity was examined, as previously reported in [33][34][35]. The assays were performed with Vero E6 cells (ATCC, CRL-1586) using the SARS-CoV-2 wild-type or Delta strains. Briefly, serum samples were heat-inactivated and three-fold serially diluted (initial dilution: 1:10), followed by incubation with 100 pfu of the wild-type SARS-CoV-2 (USA-WA1/2020) strain for 1 h at 37 • C. The serum-virus mixtures were placed onto a Vero E6 cell monolayer in 96-well plates for incubation for 1 h at 37 • C. The plates were washed with DMEM, and the monolayer cells were overlaid with 200 µL of minimum essential medium (MEM) containing 1% (w/v) of methylcellulose, 2% FBS, and 1% penicillin-streptomycin. Cells were then incubated for 24 h at 37 • C. Vero E6 monolayers were washed with PBS and fixed with 250 µL of pre-chilled 4% formaldehyde for 30 min at room temperature, followed by aspiration removal of the formaldehyde solution and washed twice with PBS. The cells were permeabilized using 0.3% (wt/vol) hydrogen peroxide in water. The cells were blocked using 5% non-fat dried milk followed by the addition of 100 µL/well of diluted anti-SARS-CoV-2 antibody (1:1000) to all wells on the microplates for 1-2 h at RT. This was followed by the addition of diluted anti-rabbit IgG conjugate (1/2000) for 1 h at RT. The plate was washed and developed by the addition of TrueBlue substrate, and the foci were counted using an ImmunoSpot analyzer (CTL, Cleveland, OH, USA). Each serum sample was tested in duplicates.

Histology of animal lungs: Hamster lungs were preserved in 10% neutral buffered formalin for 48 h before transferring to 70% ethanol. The tissue sections were then embedded in paraffin blocks and sectioned at 8 µm thicknesses. Slides were deparaffinized and rehydrated before staining for Hematoxylin and Eosin for routine immunopathology, as previously described [35][36][37].

Virus titration in oropharyngeal swabs: Throat swabs were analyzed for SARS-CoV-2specific RNA by qRT-PCR. As recommended by the CDC, we used ORF1ab-specific primers (Forward-5 ′ -CCCTGTGGGTTTTACACTTAA-3 ′ and Reverse-5 ′ -ACGATTGTGCATCAGCT GA-3 ′ ) and a probe (6FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ) to detect the viral RNA level in oropharyngeal swabs. Briefly, 100 ng of the total nucleic acid eluate was added to a 20 µL total-volume reaction mixture (1x TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), with 0.9 mM of each primer and 0.2 mM of each probe. qRT-PCR was carried out using the ABI StepOnePlus thermocycler (Life Technologies, Grand Island, NY, USA).

Statistical analysis: The experimental groups were compared using the analysis of variance (ANOVA) applied using the multiple comparison-based Tukey test and Student's t-test with GraphPad Prism version 10.1.0 (La Jolla, CA, USA). As we previously described, differences between the groups were identified by ANOVA and multiple comparison procedures [38,39]. The differences between the experimental groups were then identified and expressed as the mean ± SD. Results were considered statistically significant at p < 0.05.

1.Lipid nanoparticles with different compositions vary in their effect on the maturation and activation of dendritic cells:

Much of the immunobiology of dendritic cells (DCs) involves their role as antigen-presenting cells and in the activation of T cells. CD40 is upregulated on activated DCs and CD40L is expressed on activated T cells. The engagement of CD40 on DCs with CD40L induces positive signaling that leads to the expression of CD83/86 and the production of IL-12 in DCs.

We observed a similar phenomenon where the treatment of DCs with different LNPs led to the activation and maturation of DCs. Monocytes isolated from healthy participant's PBMCs (n = 4; age range 24-75 yrs) were treated with GM-CSF/IL-4 for 6 days. On day 7, immature as well as mature DCs were harvested, followed by treatment with a dose of 1.2 µg/mL of four different LNPs for 24 h. We assessed the frequency of surface costimulatory and HLA marker-expressing cells in LNP-treated MDDCs compared to unstimulated cells after 24 h. We found that all LNPs significantly upregulated the percentage of IL12 and CD40-positive human MDDCs compared to the unstimulated control (Figure 1A, B ). How-ever, the upregulation was relatively higher in cultures treated with LNP-4 (Figure 1A, B ). We also observed a significant upregulation in the maturation markers of MDDCs -CD83 and CD86 compared to the unstimulated controls (Figure 1C, D ). However, the upregulation was relatively higher in cultures treated with LNP-4 (Figure 1C, D ).

MDDCs -CD83 and CD86 compared to the unstimulated controls (Figure 1C, D ). However, the upregulation was relatively higher in cultures treated with LNP-4 (Figure 1C, D ).

Next, we tested the production of cytokines following in vitro DC maturation after 6 h and 24 h of incubation with different LNPs. IL-6, IL-21, and IFN-γ were found to be markedly increased after 24 h in cultures stimulated with LNPs and this increase was relatively more pronounced in cultures stimulated with LNP-4 (Figure 1E ). LNPs can induce pro-TFH cytokines, and key cytokines that are efficient in activating innate immune responses like IFN γ and LNP-4 displayed a greater ability compared to other LNPs.

2.Vaccination with mRNA-Spike encapsulated in LNP-4 demonstrated better protection against SARS-CoV-2:

We sought to assess the in vivo protection efficacy of mRNA-Spike LNPs in the well-established SARS-CoV-2 hamster model using the Delta strain. Groups of 8-week-old Syrian golden hamsters were i.m. administrated with either of the two doses of different (1 µg and 3 µg) concentrations of mRNA-Spike LNPs, followed by intranasal (i.n.) challenge with a sub-lethal dose (1 × 10 5 pfu) of the SARS-CoV-2 Delta variant at 14 days following the second immunization (Figure 2 ). Next, we tested the production of cytokines following in vitro DC maturation after 6 h and 24 h of incubation with different LNPs. IL-6, IL-21, and IFN-γ were found to be markedly increased after 24 h in cultures stimulated with LNPs and this increase was relatively more pronounced in cultures stimulated with LNP-4 (Figure 1E ). LNPs can induce pro-TFH cytokines, and key cytokines that are efficient in activating innate immune responses like IFN γ and LNP-4 displayed a greater ability compared to other LNPs.

We sought to assess the in vivo protection efficacy of mRNA-Spike LNPs in the well-established SARS-CoV-2 hamster model using the Delta strain. Groups of 8-week-old Syrian golden hamsters were i.m. administrated with either of the two doses of different (1 µg and 3 µg) concentrations of mRNA-Spike LNPs, followed by intranasal (i.n.) challenge with a sub-lethal dose (1 × 10 5 pfu) of the SARS-CoV-2 Delta variant at 14 days following the second immunization (Figure 2 ). Weight loss analysis showed that all hamsters in the mock group lost body weight and displayed a mean reduction of 4-5% of body weight by day 4 (Figure 3A, D ). Immunized hamsters showed a mean increase of 2-3% (1 µg) and 2-4% (3 µg) in body weight (Figure 3A, D ). However, hamsters immunized with LNP-4 demonstrated better weight gain and overall maximum weight (Figure 3B, E ). The viral titration data show relatively decreased viral RNA copy numbers for hamsters immunized with LNP4 at 1 µg (Figure 3C ) and 3 µg (Figure 3F ) in comparison to other LNPs. Weight loss analysis showed that all hamsters in the mock group lost body weight and displayed a mean reduction of 4-5% of body weight by day 4 (Figure 3A, D ). Immunized hamsters showed a mean increase of 2-3% (1 µg) and 2-4% (3 µg) in body weight (Figure 3A, D ). However, hamsters immunized with LNP-4 demonstrated better weight gain and overall maximum weight (Figure 3B, E ). The viral titration data show relatively decreased viral RNA copy numbers for hamsters immunized with LNP4 at 1 µg (Figure 3C ) and 3 µg (Figure 3F ) in comparison to other LNPs. Weight loss analysis showed that all hamsters in the mock group lost body weight and displayed a mean reduction of 4-5% of body weight by day 4 (Figure 3A, D ). Immunized hamsters showed a mean increase of 2-3% (1 µg) and 2-4% (3 µg) in body weight (Figure 3A, D ). However, hamsters immunized with LNP-4 demonstrated better weight gain and overall maximum weight (Figure 3B, E ). The viral titration data show relatively decreased viral RNA copy numbers for hamsters immunized with LNP4 at 1 µg (Figure 3C ) and 3 µg (Figure 3F ) in comparison to other LNPs.

3.Vaccination with mRNA-Spike encapsulated in LNP-4 induces high IgG titer and neutralizing antibodies:

To assess the immunogenic potential of mRNA/LNP vaccines formulated with four different LNP compositions (Table 1 ), Syrian golden hamsters (n = 5 per group) were vaccinated three weeks apart with two doses of either mRNA/LNP vaccines i.m. Immunogenicity was assessed at approximately 1 week after dose 2 (Day 28); Spikespecific serum immunoglobulin (Ig) G or A binding antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and serum neutralizing antibody titers were measured by a plaque reduction neutralization test (PRNT). Spike mRNA encapsulated in LNP-4 immunization elicited higher antibody titers followed by LNP 3, LNP-2, and LNP-1 (Figure 4A, D ). LNP-1 displayed lower Spike-specific binding titers. In addition to S-specific binding titers, neutralizing antibody responses in sera were evaluated. LNP-4 displayed better neutralizing potential, followed by LNP-1 and LNP-2. LNP-3 displayed a lower neutralizing potential (Figure 4B, E ).

To assess the immunogenic potential of mRNA/LNP vaccines formulated with four different LNP compositions (Table 1 ), Syrian golden hamsters (n = 5 per group) were vaccinated three weeks apart with two doses of either mRNA/LNP vaccines i.m. Immunogenicity was assessed at approximately 1 week after dose 2 (Day 28); Spike-specific serum immunoglobulin (Ig) G or A binding antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and serum neutralizing antibody titers were measured by a plaque reduction neutralization test (PRNT). Spike mRNA encapsulated in LNP-4 immunization elicited higher antibody titers followed by LNP 3, LNP-2, and LNP-1 (Figure 4A, D ). LNP-1 displayed lower Spike-specific binding titers. In addition to S-specific binding titers, neutralizing antibody responses in sera were evaluated. LNP-4 displayed better neutralizing potential, followed by LNP-1 and LNP-2. LNP-3 displayed a lower neutralizing potential (Figure 4B, E ).

4. Vaccination with mRNA-Spike encapsulated in LNP-4 induced a higher level of Interferon-γ: The heparinized, fresh, whole blood collected from each hamster immunized with different LNPs was incubated with SARS-CoV2 Spike-specific peptides for 24 h. The presence of IFN-γ in the plasma samples was determined by ELISA. Peripheral blood obtained from hamsters immunized with LNP-4 showed a higher production of IFN-γ in vitro followed by hamsters immunized with LNP-2, LNP-3, and LNP-1 (Figure 4C, F ).

4.Vaccination with mRNA-Spike encapsulated in LNP-4 induced a higher level of Interferon-γ:

The heparinized, fresh, whole blood collected from each hamster immunized with different LNPs was incubated with SARS-CoV2 Spike-specific peptides for 24 h. The presence of IFN-γ in the plasma samples was determined by ELISA. Peripheral blood obtained from hamsters immunized with LNP-4 showed a higher production of IFN-γ in vitro followed by hamsters immunized with LNP-2, LNP-3, and LNP-1 (Figure 4C, F ).

5. Vaccination with mRNA-Spike encapsulated in LNP-4 confers protection against SARS-CoV-2 Delta variants: We next determined the protective efficacy mRNA/LNP vaccines that incorporate the Spike antigen against the highly pathogenic Delta variant (B.1.617.2). Hematoxylin and Eosin staining of lung sections at day 14 p.i. showed a significant reduction in COVID-19-related lung pathology in the hamsters vaccinated with mRNA/LNP vaccines and LNP-4 compared to mock-vaccinated mice (Figure 5 ). This reduction in lung pathology was observed at a higher level for hamsters vaccinated with 3 µg followed by hamsters vaccinated with a 1 µg dose of the LNP4 composition of the COVID-19 vaccine (Figure 5 ). Taken together, these results indicate that vaccination with our novel COVID-19 vaccine formulated with LNP-4 that constitutes phospholipid DOPE reduces COVID-19-related lung pathology following infection with the pathogenic SARS-CoV-2 Delta variant.

ured by enzyme-linked immunosorbent assay (ELISA). (B) Serum-neutralizing antibody titers obtained from hamsters immunized with 1 µg of Spike-mRNA/LNP as measured by a plaque reduction neutralization test. (C) QuantiFERON assay to determine the induction of IFN-γ cytokine in peripheral blood of hamsters immunized with 1 µg of Spike-mRNA/LNP upon stimulation with Spike peptides. (D) Spike-specific serum immunoglobulin (Ig) G or A binding antibody responses obtained from hamsters immunized with 3 µg of Spike-mRNA/LNP as measured by the enzymelinked immunosorbent assay (ELISA). (E) Serum-neutralizing antibody titers obtained from hamsters immunized with 1 µg of Spike-mRNA/LNP as measured by a plaque reduction neutralization test. (F) QuantiFERON assay to determine the induction of IFN-γ cytokine in the peripheral blood of hamsters immunized with 3 µg of Spike-mRNA/LNP upon stimulation with Spike peptides.

5.Vaccination with mRNA-Spike encapsulated in LNP-4 confers protection against SARS-CoV-2 Delta variants:

We next determined the protective efficacy mRNA/LNP vaccines that incorporate the Spike antigen against the highly pathogenic Delta variant (B.1.617.2). Hematoxylin and Eosin staining of lung sections at day 14 p.i. showed a significant reduction in COVID-19-related lung pathology in the hamsters vaccinated with mRNA/LNP vaccines and LNP-4 compared to mock-vaccinated mice (Figure 5 ). This reduction in lung pathology was observed at a higher level for hamsters vaccinated with 3 µg followed by hamsters vaccinated with a 1 µg dose of the LNP4 composition of the COVID-19 vaccine (Figure 5 ). Taken together, these results indicate that vaccination with our novel COVID-19 vaccine formulated with LNP-4 that constitutes phospholipid DOPE reduces COVID-19-related lung pathology following infection with the pathogenic SARS-CoV-2 Delta variant. 6A ). Immunogenicity was assessed at approximately 1 week after dose 2 (Day 28). Spike-specific serum immunoglobulin (Ig) G or A binding antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and serum-neutralizing antibody titers were measured by a plaque reduction neutralization test (PRNT). Spike mRNA encapsulated in LNP-4 immunization elicited higher antibody titers compared to LNP-O (Figure 6B ). LNP-O displayed lower S-specific binding titers. In addition to S-specific binding titers, neutralizing antibody responses in sera were evaluated. LNP-4 displayed a slightly better neutralizing potential compared to LNP-O (Figure 6C ). LNP-O displayed a slightly lower neutralizing potential. The heparinized, fresh, whole blood collected from each hamster immunized with different LNP-4 vs. LNP-O levels was analyzed for T-cell responses. LNP-4-immunized blood showed significantly better overall T-cell responses as well as a display of activation markers on CD4 + and CD8 + T cells (Figure 6D ). lower degree of hemorrhages. Images were captured at 4× and 10× resolutions. Syrian golden hamsters (n = 6 per group) were vaccinated three weeks apart with two doses of either mRNA/LNP vaccines i.m. (Figure 6A ). Immunogenicity was assessed at approximately 1 week after dose 2 (Day 28). Spike-specific serum immunoglobulin (Ig) G or A binding antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and serum-neutralizing antibody titers were measured by a plaque reduction neutralization test (PRNT). Spike mRNA encapsulated in LNP-4 immunization elicited higher antibody titers compared to LNP-O (Figure 6B ). LNP-O displayed lower S-specific binding titers. In addition to S-specific binding titers, neutralizing antibody responses in sera were evaluated. LNP-4 displayed a slightly better neutralizing potential compared to LNP-O (Figure 6C ). LNP-O displayed a slightly lower neutralizing potential. The heparinized, fresh, whole blood collected from each hamster immunized with different LNP-4 vs. LNP-O levels was analyzed for T-cell responses. LNP-4-immunized blood showed significantly better overall T-cell responses as well as a display of activation markers on CD4 + and CD8 + T cells (Figure 6D ).

4.Discussion

The need for mRNA encapsulation arises from the fragility of mRNA as a standalone. mRNA molecules are inherently unstable and can be easily degraded by both extracellular nucleases and cellular enzymes. The immune system perceives mRNA as a foreign sub-stance and tries to eliminate it when it is injected into the body. Even before the mRNA reaches the target cells, where it can be translated into proteins, this occurs very quickly. To limit the degradation and chemical reactions that result in the degradation of modified mRNA, current mRNA-based vaccines targeted against SARS-CoV-2 must be stored at extremely cold temperatures. In contrast, encapsulating mRNA in lipid nanoparticles (LNPs) protects it from degradation and immune recognition, allowing it to reach the target cells and be translated into proteins. LNPs in mRNA-based vaccines are designed to fuse with the cell membrane, allowing the encapsulated mRNA to enter the cell. A viral protein is subsequently synthesized by the ribosomes from the mRNA, which is subsequently presented on the cell surface for immune recognition. The vaccine can achieve a stronger immune response when the mRNA is encapsulated in LNPs rather than injected alone. In addition to prolonging the duration of protein expression, LNPs also provide the immune system more time to recognize and respond to viral proteins [40].

LNPs typically consist of a lipid core matrix that solubilizes the mRNA, as well as stabilizing surfactants or emulsifiers. The lipid core matrix of LNPs is usually composed of a mixture of lipids, including phospholipids, cholesterol, and other lipids, such as triglycerides, diglycerides, monoglycerides, and fatty acids [41]. These lipids include an ionizable cationic lipid whose positive charge can bind to the negatively charged mRNA, a PEGylated lipid meant for stability, and a phospholipid along with cholesterol designed for structural support. The lipid components facilitate the LNPs containing mRNA to enter a cell through an endosome. When the LNP is inside the acidic endosome, the ionizable lipids become positively charged and help to release the LNP and modified mRNA into the cell's cytoplasm. The modified mRNA, now free, is translated by the ribosomes to create proteins. mRNA-encoded antigens more closely resemble the structure and presentation of viral proteins expressed during a natural infection. mRNA vaccines are vector-less and thus can avoid the potential for diminished immunogenicity with repeat dosing sometimes observed with vector-based vaccines. Moreover, mRNA vaccines for respiratory pathogens, such as SARS-CoV-2, have been established, demonstrating robust immune responses and high real-world effectiveness against disease. However, the most effective LNPs for vaccination need to be uncovered and investigated further.

In the present study, we underline the crucial role of delivery systems, i.e., lipid nanoparticles, in the efficacy of vaccines. LNPs improve the pharmacokinetics of the mRNA vaccine, such as its distribution and stability in the body, which can enhance the vaccine's overall efficacy. However, LNPs need to be tailored with a specific lipid composition, which can maximize the effective immune response by promoting certain pathways of the immune system. Therefore, in this preclinical study, using SARS-CoV-2 as a model pathogen, the immunogenicity and protective efficacy of different LNPs encoding the SARS-CoV-2 specific Spike were evaluated. In this study, we expand upon the effect of LNP formulation on the induction of immune responses. Studies have shown that there is an intrinsic ability of LNPs to promote IL-6 secretion in mice and subsequent T FH cell induction [42]. Here, we show that one specific LNP formulation (LNP4) induced higher maturation and the activation of DCs as measured by the frequency of co-stimulatory surface receptors and the production of cytokines and chemokines. By enhancing APC maturation and cytokine production, LNP-4 likely promotes a stronger and more durable immune response to the vaccine antigen compared to other LNPs. Additionally, a twodose primary i.m. vaccination regimen of mRNA (Spike)-LNP elicited systemic immune responses and resulted in lower SARS-CoV-2 infection levels and disease severity versus mock-vaccinated controls after the viral challenge. Vaccination with mRNA/LNP4 showed improved protection against SARS-CoV-2. Based on the findings from this study, we used LNP-4 while formulating all our mRNA/LNP-based COVID-19 vaccines, which we found to be highly efficient in providing antigenicity and immunity against multiple SARS-CoV-2 variants of concern [43].

We observed that using LNP-4 that contains a phospholipid constituent named 1,2dioleoyl-sn-glycero-3-Phosphoethanolamine (DOPE) has higher potency at inducing better immune responses and better protection in hamsters upon SARS-CoV-2 infection. In addition, we also assessed the immunogenic potential of LNP-4 in comparison to a commercially available ("off the shelf") LNP, which we have referred to as LNP-O. We observed that Spike mRNA encapsulated in LNP-4 elicited higher antibody titers compared to LNP-O. LNP-O displayed lower S-specific binding titers. The heparinized, fresh, whole blood collected from each hamster immunized with different LNP-4 vs. LNP-O samples was analyzed for T-cell responses. LNP-4 immunized blood showed significantly better overall T-cell responses as well as a display of activation markers on CD4 + and CD8 + T cells.

The biophysical parameters of LNP largely impact its immunogenicity, and an understanding of the same is an important step for enabling the rapid development of potent mRNA/LNP vaccines. Different mRNA vaccine formulations developed for protection against infectious conditions have entered clinical studies to evaluate their effectiveness. The size of LNPs for example can have an impact on their immunogenicity, as demonstrated by studies such as the one conducted by Hassett et al. and Brewer et al. [44]. In the study by Hassett et al., the researchers investigated the effect of different biophysical factors on the immunogenicity of a cytomegalovirus (CMV) mRNA/LNP vaccine in mice. They found that LNP size had the strongest correlation with immunogenicity, with larger LNPs (up to 100 nm) leading to higher antibody titers. Similarly, Brewer et al. evaluated the antibody response to ovalbumin (OVA)-loaded liposomes of different sizes (100, 155, 225, and 560 nm) in mice [45] and found that smaller vesicles elicited higher levels of IgG1, which is indicative of a Th2-type immune response. In the present study, among the studied four LNPs, we have shown that LNP4 (IL00V41), which comprised DOPE phospholipid and having a relatively smaller size than other LNPs, provides better protection than other LNPs composed of DSPC, DOPC, and DPPC (Table 1 ). It is imperative to mention that this slight difference in size affects the immune response [44]. The finding from this study highlights the importance of focusing on incremental improvements, such as tweaking a single component like the phospholipid type or particle size, rather than attempting to overhaul multiple variables simultaneously. These small, deliberate refinements can collectively lead to significant advancements over time. Most importantly, these experiments formed our basis of LNP selection, and we used LNP-4 for antigen screening against SARS-CoV-2 [43]. Furthermore, we observed that vaccination with mRNA encoding the Spike protein of SARS-CoV-2 encapsulated in LNP-4 induces high levels of IgG antibodies and neutralizing antibodies against the virus. Additionally, peripheral blood obtained from hamsters immunized with LNP-4 showed a higher production of IFN-γ followed by hamsters immunized with other LNPs. LNP4 (IL00V41) comprising DOPE (1,2-dioleoylsn-glycero-3-phosphoethanolamine) and the amount of ionizable lipid used in the formulations were "higher" for LNP4 (IL00V41). Because the same antigen (Spike) and same dosing/immunization regimen were used in this study, we established the protective efficacy of various vaccine formulations. However, the mechanism of action of LNPs was not examined in this study. Nevertheless, our study points out the importance of the development and screening/testing of LNP formulations for favorable immunostimulatory profiles in studies such as these. The size and phospholipid dependence of LNPs for their efficacy are areas of ongoing research, with many aspects not fully understood. The role of specific phospholipid mixtures and their off-target effects are not fully understood. Future studies will address these important aspects.

5.Conclusions

Identifying the optimal formulation of lipid nanoparticles is challenging, as it depends on the specific molecule being encapsulated. The size and lipid composition need to be tailored to enhance delivery efficiency and stability. In the present case of mRNA vaccines for SARS-CoV-2, four types of LNPs were used. We slightly modified their formulations to determine the most effective LNP to be used in our mRNA-LNP vaccine. Based on the outcomes of this study, we used LNP4 to formulate our broad-spectrum multiantigen mRNA/LNP-based pan-coronavirus vaccine. Our findings reveal that LNP4, with a relatively small particle size, contained DOPE and the highest proportion of ionizable lipids, significantly improving immune efficacy.

availability

Data Availability Statement: Data are contained within the article.

annex

Informed Consent Statement: Not applicable.

Conflicts of Interest:

Studies of this report were supported by Public Health Service Research grants AI158060, AI150091, AI143348, AI147499, AI143326, AI138764, AI124911, and AI110902 from the National Institutes of Allergy and Infectious Diseases (NIAID) to L.B. L.B. has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company's Scientific Advisory Board. L.B.'s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies. Precision Nanosystems formulated the 4 LNPs described in this study. The details about the LNPs are confidential and protected under proprietary rights. There is no conflict between Precision Nanosystems in terms of publishing these experimental results without revealing any proprietary information.

Acknowledgements

Acknowledgments: Studies of this report were supported by Public Health Service Research grants AI158060, AI150091, AI143348, AI147499, AI143326, AI138764, AI124911, and AI110902 from the National Institutes of Allergy and Infectious Diseases (NIAID) to L.B. L.B. has an equity interest in TechImmune, LLC., a company that may potentially benefit from the research results and serves on the company's Scientific Advisory Board.L.B.'s relationship with TechImmune, LLC., has been reviewed and approved by the University of California, Irvine by its conflict-of-interest policies.

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