Namit Chaudhary

Lipid nanoparticles (LNPs) are clinically proven mRNA delivery vehicles, and repeated administration is often required to maintain therapeutic or prophylactic benefit. However, a single dose can provoke adaptive immune responses against polyethylene glycol (PEG), a polymeric excipient on the LNP surface. In this event, resultant anti-PEG antibodies neutralize subsequent LNP doses, causing efficacy loss and limiting therapeutic utility. Here, we investigated whether altering the LNP formulation between initial and subsequent doses could mitigate PEG-associated efficacy loss. Following the administration of an LNP known to elicit anti-PEG antibodies, we re-dosed with LNPs formulated with varied PEG-lipid chemistry, PEG concentration, mRNA cargo, and administration route. None of these adjustments prevented efficacy loss, which consistently correlated with elevated anti-PEG immunoglobulin M (IgM) and, in some cases, immunoglobulin G (IgG). Reduced PEG concentration diminished immunogenicity but also potency, and intraperitoneal dosing elicited the strongest responses upon repeat injection. Further, LNPs that elicit anti-PEG antibodies also impaired the subsequent delivery of an FDA-approved MC3 formulation, suggesting that anti-PEG immune responses can compromise any PEGylated LNP formulation. Together, these findings demonstrate that PEG-associated efficacy loss cannot be resolved through formulation optimization alone, when the ionizable lipid fails to engage the innate immune receptors TLR4 and CD1d. Instead, they suggest that the ionizable lipid’s engagement of innate immune receptors is the primary determinant of repeat dosing success. Graphical Abstract

Genital herpes, caused by herpes simplex virus 2 (HSV-2), remains a prevalent sexually transmitted infection with no available vaccine. Effective local immunity, mediated in part by tissue-resident memory T cells (T _RM cells) and luminal antibodies, provides immediate viral control. Here, we developed bioactive enhanced adjuvant chemokine oligonucleotide nanoparticles (BEACONs) formed via electrostatic interactions between CpG oligodeoxynucleotides (CpG ODNs) and the chemokine CXCL9. This adjuvant enhances antigen-presenting cell engagement and innate immune signaling, promotes CD8 T cell recruitment, and reduces local neutrophilic inflammation relative to CpG ODNs. After intramuscular priming with HSV-2 glycoprotein-encoding messenger RNA–lipid nanoparticles, vaginal boosting with cognate recombinant glycoprotein and BEACONs improved protection against HSV-2 by increasing local CD8 T _RM cell populations and mucosal antibody responses. Vaccine-mediated protection required local delivery of both antigen and adjuvant and was reduced by CD8 T cell or B cell depletion. These findings support engineered mucosal adjuvants for vaccines targeting genital herpes and other sexually transmitted infections.

Technologies that simplify complex dosing regimens as single-shot immunizations may be important for vaccines against difficult to neutralize pathogens. Here we characterized delivery mechanism of clinically-relevant HIV vaccine immunogens using core-shell microparticles comprising immunogen formulations spray-dried to form solid spherical microparticles and subsequently coated with a nanoscopic alumina shell using atomic layer deposition (ALD). ALD particles exhibited a time delay in antigen release programmed by the alumina shell followed by prolonged antigen release, which steadily accumulated in antigen-presenting cells at the injection site and draining lymph nodes and accumulated on follicular dendritic cells in B cell follicles. ALD vaccines elicited continuous expansion of antigen-specific germinal center B cells over 8 weeks, serum antibody responses with >10-fold slower antigen-binding off-rates, and 2-fold more long-lived plasma cells compared to traditional bolus vaccination with potent adjuvants. Single administration ALD technology thus promotes key events in the primary immune response important for vaccines against HIV and other challenging pathogens.

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due to scalability, adaptability, and potency. Yet, there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen-presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel technology, which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single-cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available severe acute respiratory syndrome coronavirus 2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

Milk is a multifaceted biofluid that is essential for infant nutrition and development, yet its cellular and bioactive components, particularly maternal milk cells, remain understudied. Early research on milk cells indicated that they cross the infant's intestinal barrier and accumulate within systemic organs. However, due to the absence of modern analytical techniques, these studies were limited in scope and mechanistic analysis. To overcome this knowledge gap, we have investigated the transintestinal transport of milk cells and components in pups over a 21‐day period. Studies employed a mT / mG foster nursing model in which milk cells express a membrane‐bound fluorophore, tdTomato . Using flow cytometry, we tracked the transport of milk cell‐derived components across local and systemic tissues, including the intestines, blood, thymus, mesenteric lymph nodes, and liver. These experiments identified milk‐derived fluorescent signals in intestinal epithelial and immune cells as well as liver macrophages in 7‐day‐old pups. However, the minute numbers of macrophages in mouse milk suggest that maternal cells are not systemically accumulating in the infant; instead, pup macrophages are consuming milk cell membrane components, such as apoptotic bodies or extracellular vesicles ( EVs ). Ex vivo experiments using primary macrophages support this hypothesis, showing that immune cells preferentially consumed EVs over milk cells. Together, these data suggest a more complex interplay between milk cells and the infant's immune and digestive systems than previously recognized and highlight the need for future research on the role of milk cells in infant health.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicle for RNA therapeutics, partly because of established lipid structure–activity relationships focused on formulation potency. Yet such knowledge has not extended to LNP immunogenicity. Here we show that the innate and adaptive immune responses elicited by LNPs are linked to their ionizable lipid chemistry. Specifically, we show that the amine headgroups in ionizable lipids drive LNP immunogenicity by binding to Toll-like receptor 4 and CD1d and by promoting lipid-raft formation. Immunogenic LNPs favour a type-1 T-helper-cell-biased immune response marked by increases in the immunoglobulins IgG2c and IgG1 and in the pro-inflammatory cytokines tumour necrosis factor, interferon γ and the interleukins IL-6 and IL-2. Notably, the inflammatory signals originating from these receptors inhibit the production of anti-poly(ethylene glycol) IgM antibodies, preventing the often-observed loss of efficacy in the LNP-mediated delivery of siRNA and mRNA. Moreover, we identified computational methods for the prediction of the structure-dependent innate and adaptive responses of LNPs. Our findings may help accelerate the discovery of well-tolerated ionizable lipids suitable for repeated dosing.

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due in part to scalability, adaptability, and potency. Yet there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel depot technology which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available SARS-CoV-2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.

During pregnancy, the risk of maternal and fetal adversities increases due to physiological changes, genetic predispositions, environmental factors, and infections. Unfortunately, treatment options are severely limited because many essential interventions are unsafe, inaccessible, or lacking in sufficient scientific data to support their use. One potential solution to this challenge may lie in emerging RNA therapeutics for gene therapy, protein replacement, maternal vaccination, fetal gene editing, and other prenatal treatment applications. In this review, the current landscape of RNA platforms and non‐viral RNA delivery technologies that are under active development for administration during pregnancy is explored. Advancements of pregnancy‐specific RNA drugs against SARS‐CoV‐2, Zika, influenza, preeclampsia, and for in‐utero gene editing are discussed. Finally, this study highlights bottlenecks that are impeding translation efforts of RNA therapies, including the lack of accurate cell‐based and animal models of human pregnancy and concerns related to toxicity and immunogenicity during pregnancy. Overcoming these challenges will facilitate the rapid development of this new class of pregnancy‐safe drugs.