The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines.

Recent years have witnessed incredible growth in RNA therapeutics, which has benefited significantly from decades of research on lipid nanoparticles, specifically its key component-the ionizable lipid. This comment discusses the major ionizable lipid types, and provides perspectives for future development. Need for ionizable lipidsBroadly speaking, ribonucleic acid (RNA) therapeutics include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and single-guide RNAs (sgRNAs)-mediated CRISPR-Cas9 system, which can manipulate essentially any gene of interest through distinct modes of action 1 . However, RNA therapeutics are susceptible to nucleases and cannot permeate cells due to their large size and negative charge. Delivery of RNAs to target cells by clinically translatable lipid nanoparticles (LNPs) provides vast opportunities to tackle a series of life-threatening diseases including COVID-19 2 . LNPs typically consist of four components-ionizable lipid, phospholipid, cholesterol, and PEGylated lipid, among which, the ionizable lipid plays a major role in protecting RNAs and facilitating their cytosolic transport. Ionizable lipids are positively charged at acidic pH to condense RNAs into LNPs, but are neutral at physiological pH to minimize toxicity. They can be protonated in the acidic endosome after cellular uptake, and interact with anionic endosomal phospholipids to form cone-shaped ion pairs that are not compatible with a bilayer (Fig. 1). These cationicanionic lipid pairs drive the transition from the bilayer structure to the inverted hexagonal H II phase, which facilitates membrane fusion/disruption, endosomal escape and cargo release into the cytosol 3 . Since 2008, ionizable lipids with diverse chemical identities have been created. Systematic categorization of these lipids based on their structures can greatly benefit the field and facilitate the development of next-generation ionizable lipids. Currently, there are five major ionizable lipid types that are widely used for RNA delivery (Fig. 1). Unsaturated ionizable lipidsTail saturation greatly influences the fluidity and delivery efficiency of ionizable lipids. Increasing the tail unsaturation from 0 to 2 cis double bonds correlates with an increased tendency for bilayer lipids to form a nonbilayer phase 4 , leading to enhanced membrane disruption and

The SARS-CoV-2 Spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization-escape that could compromise vaccine efficacy, sera from Spike-immunized mice, nonhuman primates and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD “up” conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88-or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

SARS-CoV-2 mRNA vaccination induces robust humoral and cellular immunity in the circulation; however, it is currently unknown whether it elicits effective immune responses in the respiratory tract, particularly against variants of concern (VOCs), including Omicron. We compared the SARS-CoV-2 S–specific total and neutralizing antibody responses, and B and T cell immunity, in the bronchoalveolar lavage fluid (BAL) and blood of COVID-19–vaccinated individuals and hospitalized patients. Vaccinated individuals had significantly lower levels of neutralizing antibody against D614G, Delta (B.1.617.2), and Omicron BA.1.1 in the BAL compared with COVID-19 convalescents despite robust S-specific antibody responses in the blood. Furthermore, mRNA vaccination induced circulating S-specific B and T cell immunity, but in contrast to COVID-19 convalescents, these responses were absent in the BAL of vaccinated individuals. Using a mouse immunization model, we demonstrated that systemic mRNA vaccination alone induced weak respiratory mucosal neutralizing antibody responses, especially against SARS-CoV-2 Omicron BA.1.1 in mice; however, a combination of systemic mRNA vaccination plus mucosal adenovirus-S immunization induced strong neutralizing antibody responses not only against the ancestral virus but also the Omicron BA.1.1 variant. Together, our study supports the contention that the current COVID-19 vaccines are highly effective against severe disease development, likely through recruiting circulating B and T cell responses during reinfection, but offer limited protection against breakthrough infection, especially by the Omicron sublineage. Hence, mucosal booster vaccination is needed to establish robust sterilizing immunity in the respiratory tract against SARS-CoV-2, including infection by the Omicron sublineage and future VOCs.

The SARS-CoV-2 Spike protein acquired a D614G mutation early in the COVID-19 pandemic that appears to confer on the virus greater infectivity and now globally is the dominant form of the virus. Certain of the current vaccines entering phase 3 trials are based on the early D614 form of Spike with the goal of eliciting protective neutralizing antibodies. To determine whether D614G mediates neutralization-escape that could compromise vaccine efficacy, sera from Spike-immunized mice, nonhuman primates and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 Spike on their surface. In all cases, G614 Spike pseudovirions were moderately more susceptible to neutralization, indicating this is not an escape mutation that would impede current vaccines. Rather, the gain in infectivity provided by D614G came at the cost of making the virus more vulnerable to neutralizing antibodies.

A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.