Methicillin-resistant Staphylococcus aureus (MRSA) represents a major global health threat due to its resistance to conventional antibiotics. The commensal microbiota maintains a symbiotic relationship with the host, playing essential roles in metabolism, energy regulation, immune modulation, and pathogen control. Mammals harbor a wide range of commensal bacteria capable of producing unique metabolites with potential therapeutic properties. This study demonstrated that M28 family peptidase (M28), derived from commensal bacteria Peribacillus frigoritolerans (P. f), provided protective effects against MRSA-induced pneumonia. M28 enhanced the phagocytosis and bactericidal activity of macrophages by inducing trained immunity. RNA sequencing and metabolomic analyses identified the CFB-C3a-C3aR-HIF-1α axis-mediated phosphatidylcholine accumulation as the key mechanism for M28-induced trained immunity. Phosphatidylcholine, like M28, also induced trained immunity. To enhance M28-mediated therapeutic potential, it was encapsulated in liposomes (M28-LNPs), which exhibited superior immune-stimulating properties compared to M28 alone. In vivo experiments revealed that M28-LNPs significantly reduced bacterial loads and lung damage following MRSA infection, which also provided enhanced protection against Klebsiella pneumoniae and Candida albicans. We first confirmed a link between complement activation and trained immunity, offering valuable insights into the treatment and prevention of complement-related autoimmune diseases.

mRNA-loaded lipid nanoparticles (LNPs) have emerged as a potent and versatile platform that underpins the success of mRNA vaccines, but guidelines for designing safe and effective formulations with minimal off-target effects remain unclear. In this study, we focused on a rational design for a novel ionizable lipid library that is based on ionizable tri-oleoyl-tris (iTOT) compounds with a high yield via a simple 2-step synthesis. To further enhance the efficacy and safety of this potent library for vaccine applications, we identified the optimal composition for a vaccine by focusing on the molar ratio of specific lipid excipients in the formulation. This composition brought about a shift in delivery to the spleen, and the LNP formulation, which contained 15 mol% DSPC (15%DSPC-LNPs), was thoroughly taken up by both B cells and other splenic immune cells. This formulation requires neither additional lipid components nor targeting ligand modifications, and it is accompanied by antigen-specific cytotoxic T lymphocyte responses. The rigid, hydrophobic, and charge-neutral surface of 15%DSPC-LNPs minimizes apolipoprotein E-dependent hepatic uptake and maximizes complement receptor-mediated B-cell targeting. Furthermore, as an intramuscularly administered vaccine, 15%DSPC-LNPs induce antigen-specific immune responses and, importantly, results in significantly lower levels of hepatotoxicity compared with that of the mRNA vaccine formulations currently being marketed. In summary, this study demonstrated how the passive targeting of mRNA-LNPs to organs and cells could be regulated by designing novel ionizable lipids combined with adjusting the relative proportions of lipid components. The results of this study also emphasize how selective mRNA delivery to the spleen could avoid the liver, which highlights a promising strategy for the development of safe and effective vaccines.

It has been five years since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we are also approaching the five-year mark of the COVID-19 pandemic. The vaccine is a significant weapon in combating infectious diseases like SARS-CoV-2. Several vaccines were developed against SARS-CoV-2, and they demonstrated efficacy and safety during these five years. The rapid development of multiple next-generation vaccine candidates in different platforms with very little time is the success story of the vaccine development endeavor. This remarkable success of rapid vaccine development is a new paradigm for fast vaccine development that might help develop infectious diseases and fight against the pandemic. With the completion of five years since the beginning of SARS-CoV-2 origin, we are looking back on the five years and reviewing the milestones, vaccine platforms, animal models, clinical trials, successful collaborations, vaccine safety, real-world effectiveness, and challenges. Lessons learned during these five years will help us respond to public health emergencies and to fight the battle against future pandemics.

Human papillomavirus (HPV)-associated cancers remain a critical health challenge, prompting the development of effective therapeutic vaccines. This study presents a lipid nanoparticle (LNP)-based vaccine co-loading E7 antigen peptide and magnesium ions as the adjuvant. Microfluidic technology was employed to optimize LNP preparation and formulation, ensuring efficient co-delivery of antigen and adjuvant. Magnesium ions were chosen over conventional aluminum-based adjuvants, which often suffer from limited efficacy and adverse effects, particularly for cancer immunotherapy. Compared to aluminum, magnesium ions exhibited superior capabilities in enhancing T-cell activation and promoting cellular immune response. Mechanistic insights suggest that magnesium ions facilitate dendritic cell maturation and antigen presentation via a collagen-CD36 axis, contributing to the adjuvant activity of magnesium. Through design of experiments (DoE) optimization, the LNP formulation was tailored for enhanced encapsulation and stability, positioning it as a targeted system for immune activation. These findings support the promise of magnesium ions as effective and safer adjuvants in LNP-based vaccines, marking a potential advancement for therapeutic cancer vaccination.

Lipid nanoparticles (LNPs) have extensively been used in drug delivery over the years, and the perspective of their significance has been well established. Latest findings have demonstrated the inherent adjuvant capacity of some specific lipid components, especially in stimulating immune compartments, which prompted the rational use of lipid-based delivery vehicles in drug R&D. In this concise review, we summarize current knowledge on the adjuvant properties of LNP, with a particular focus on the key components that mediate such effects. Specifically, we describe the vital role of ionizable lipids in triggering innate immune activation and inflammation and highlight the importance of these lipids in determining vaccine effectiveness or safety. Furthermore, the mechanisms by which LNP enhance the immune response are discussed in detail, shedding light on their potential applications in next-generation vaccine design and development. We also present compelling pre-clinical studies that serve as strong evidence for the adjuvant properties of LNP components in enhancing vaccine immunogenicity.

The current influenza vaccine is based on immunity to hemagglutinin (HA) and provides poor cross-protection. Here, we generated mRNA vaccine encoding influenza B virus (IBV) neuraminidase (NA) conjugated to influenza A virus M2 ectodomain (M2e), encapsulated in lipid nanoparticles (LNP), capable of inducing cross-lineage IBV protection in a dose-dependent pattern. The combination of low-dose NA mRNA and inactivated split IBV vaccines was found to induce significantly higher levels of cross-reactive IgG responses, NA and HA inhibition titers, effector and memory cellular immune responses as well as cross-lineage protection than either NA mRNA or split vaccine alone. This study suggests that the NA mRNA vaccine not only provides cross-lineage protection with a high dose but also enhances the cross-protective efficacy of the combined low-dose NA mRNA and split vaccines. Our findings support a new strategy of using mRNA LNP-supplemented conventional vaccination to enhance cross-protection.IMPORTANCEThis study highlights a significant advancement in influenza vaccination strategies. To test a new vaccination strategy, we developed an influenza B virus (IBV) neuraminidase (NA) mRNA vaccine which could provide cross-lineage protection at a high dose. More importantly, the co-administration of NA mRNA and split IBV vaccine at low doses was found to significantly enhance the hemagglutinin and NA immunity as well as cross-lineage protection of seasonal IBV vaccines. This proof-of-concept study provides evidence for a novel strategy to enhance the immunogenicity and cross-protective efficacy of conventional vaccines by supplementing with new targets of mRNA vaccines.

The selection of an effective delivery carrier is crucial to assessing mRNA-based vaccines and therapeutics in vivo. Although lipid nanoparticles (LNPs) are commonly used for mRNA delivery, the LNP-mRNA formulation process is laborious and time-consuming and requires a high-cost microfluidic device. Instead, mixing with commercial reagents may simplify mRNA transfection into cells. However, their potential as in vivo carriers in intramuscular vaccination in mouse models remains unclear. In this study, we used three types of commercial RNA transfection reagents, MessengerMAX (MAX; liposome), TransIT-mRNA (IT; cationic polymer), and Invivofectamine (IVF; LNP), to produce nanoparticles directly by pipetting. The particle characteristics and mRNA delivery efficacy of the mRNA-transfection reagent mixtures were analyzed. Additionally, immune responses to vaccine efficacy and protective immunity of the mRNA mixtures as vaccine antigens were evaluated in a mouse model. Although MAX and IT showed high in vitro transfection efficiencies, their in vivo performances were limited. In contrast, IVF exhibited notable particle stability and homogeneity, making it a promising delivery carrier. Intramuscular IVF injection significantly enhanced both innate and adaptive immune responses with a robust systemic protein expression. Notably, when using SARS-CoV-2 Spike mRNA, IVF showed robust humoral immune responses, including production of IgG and neutralizing antibodies, thereby resulting in complete protection against SARS-CoV-2 infection. Therefore, these findings position IVF as an accessible and efficient mRNA carrier for evaluating mRNA vaccines and therapeutic efficacy in basic research.

Ionizable lipids (ILs) are critical components in mRNA vaccines, which have been instrumental in the global response to SARS-CoV-2. However, current commercialized ILs in mRNA vaccines are typically synthesized through multiple-step organic reactions, complicating quality control and driving up production costs. To address this, we have developed novel ILs by a one-pot Ugi four-component reaction (Ugi-4CR), significantly simplifying synthesis while maintaining high yields and reducing costs. Here, from a library of 161 ILs, we chose six ILs with high expressing luciferase and investigated their performance in delivering the mRNA vaccine of SARS-CoV-2. These ILs feature distinct ionizable heads, N,N-dimethylethyl (R1), N,N-dimethylpropyl (R2), and N,N-diethylpropyl (R3), paired with hydrophobic tails of varying unsaturation, cis-9-octadecenoic (U1) and (9Z,12Z)-9,12-octadecadienoic (U2), respectively. In murine models, R2-and R3-based mSpike-LNPs induce higher antibody titers and stronger cellular immune responses compared to the R1-based counterparts, suggesting their superior mRNA delivery and expression efficiency. Notably, R2U2- and R3U2-based mSpike-LNPs further enhance IFN-γ+ splenocyte responses and activation of TNF-α+CD4+/CD8+ T cells, coupled with improved dendritic cell activation and retention in lymph nodes. We confirm that the R2U2-based LNPs on different mRNA antigens exhibit immune responses and safety profiles comparable to the commercial ALC-0315-based LNPs. Moreover, intranasal and intratracheal administration of R2U2-based mSpike-LNPs enhances mucosal immunity, as evidenced by elevated sIgA levels in mice. Further evaluation in cynomolgus macaques proves the efficacy of this LNP system, highlighting its potential for developing cost-effective mRNA vaccines.

Developing effective vaccines against bacteria is critical given the growing threat of antimicrobial resistance (AMR). In this study, we developed mRNA vaccines targeting Pseudomonas aeruginosa (P. aeruginosa), a key AMR pathogen, using PureCap mRNA encapsulated in lipid nanoparticles (LNPs). The PureCap technology offers a facile method for removing immunostimulatory impurities from in vitro transcribed mRNA, such as uncapped RNA and double-stranded RNA (dsRNA). Following intramuscular vaccination of mice with mRNA encoding a model antigen, PureCap mRNA elicited antibody titers that were 26-fold higher than those induced by conventional ARCA-capped mRNA. Mechanistic analyses revealed that both uncapped RNA and dsRNA impurities in ARCA-capped mRNA were responsible for the reduced humoral immune responses. While PureCap mRNA enhanced protein expression efficiency and reduced pro-inflammatory responses compared to ARCA-capped mRNA, minimizing pro-inflammatory responses was particularly critical. When anti-interferon-α/β receptor antibodies were administered, antibody responses to ARCA-capped mRNA vaccination were restored to levels comparable to those achieved with PureCap mRNA vaccination, highlighting the negative impact of type I interferon responses on antibody responses following vaccination with ARCA-capped mRNA. In a vaccination targeting the PcrV protein of P. aeruginosa, PureCap mRNA, but not ARCA-capped mRNA, significantly prolonged the survival of mice following bacterial challenges, presumably due to enhanced antibody production. Furthermore, PureCap mRNA vaccination significantly reduced bacterial loads in the lungs and mitigated tissue damage, edema, and inflammatory responses. These findings underscore the potential of PureCap mRNA as a promising platform for bacterial vaccination, offering a valuable strategy to combat AMR.

The co-circulation of both influenza viruses and SARS-CoV-2 poses a significant health risk, especially for the elderly. While vaccination against both diseases remains an effective strategy to reduce the burden of symptomatic infections, the effect of administering COVID-19 mRNA and seasonal influenza vaccines (COV-Flu) on elicited antibody responses has not been explored.

Participants between 18 and 90 years old were vaccinated with COVID-19 mRNA vaccines (n = 67), seasonal influenza vaccines (n = 130), or both (n = 201) within a three-month period between 2021 and 2024. Serum hemagglutination-inhibition (HAI) titers against influenza A (H1N1, H3N2) and B (Yamagata, Victoria) strains were measured from the COV-Flu participants or the participants vaccinated with influenza vaccines only (mono-Flu). SARS-CoV-2 neutralization assays were performed on sera collected from the COV-Flu participants and the participants receiving the mRNA vaccine only (mono-COVID-19).

The administration of influenza virus vaccines and COVID-19 mRNA vaccines within a three-month period significantly enhanced the post-vaccination HAI titers against both influenza A and B vaccine components, particularly in the elderly (65-90) participants. There were no significant differences in SARS-CoV-2 neutralization titers in COV-Flu participants compared to mono-COVID-19 participants.

Vaccination with both the COVID-19 mRNA and influenza vaccines enhances influenza-specific HAI titers without compromising the neutralization titers elicited by COVID-19 mRNA vaccination against SARS-CoV-2, especially in the elderly. These findings indicate the potential benefits of this approach, particularly for older adults, by boosting influenza virus vaccine-induced serum HAI activity while maintaining COVID-19 protective immunity.

For more than two centuries, the field of vaccine development has progressed through the adaptation of novel platforms in parallel with technological developments. Building off the advantages and shortcomings of first and second-generation vaccine platforms, the advent of third-generation nucleic acid vaccines has enabled new approaches to tackle emerging infectious diseases, cancers, and pathogens where vaccines remain unavailable. Unlike traditional vaccine platforms, nucleic acid vaccines offer several new advantages, including their lower cost and rapid production, which was widely demonstrated during the COVID-19 pandemic. Beyond production, DNA and mRNA vaccines can elicit unique and targeted responses through specialized design and delivery approaches. Considering the growth of nucleic acid vaccine research over the past two decades, the evaluation of their efficacy in at-risk populations is paramount for refining and improving vaccine design. Importantly, the aging population represents a significant portion of individuals highly susceptible to infection and disease. This review seeks to outline the major impairments in vaccine-induced responses due to aging that may be targeted for improvement with design and delivery components encompassing mRNA and DNA vaccine formulations. Results of pre-clinical and clinical applications of these vaccines in aged animal models and humans will also be evaluated to outline current successes and limitations observed in these platforms.

Mucosal delivery of vaccine boosters induces robust local protective immune responses even without any adjuvants. Yet, the mechanisms by which antigen alone induces mucosal immunity in the respiratory tract remain unclear. Here we show that an intranasal booster with an unadjuvanted recombinant SARS-CoV-2 spike protein, after intramuscular immunization with 1 μg of mRNA-LNP vaccine encoding the full-length SARS-CoV-2 spike protein (Pfizer/BioNTech BNT162b2), elicits protective mucosal immunity by retooling the lymph node-resident immune cells. On intranasal boosting, peripheral lymph node-primed B cells rapidly migrated to the lung through CXCR3-CXCL9 and CXCR3-CXCL10 signaling and differentiated into antigen-specific IgA-secreting plasma cells. Memory CD4+ T cells in the lung served as a natural adjuvant for developing mucosal IgA by inducing the expression of chemokines CXCL9 and CXCL10 for memory B cell recruitment. Furthermore, CD40 and TGFβ signaling had important roles in mucosal IgA development. Repeated mucosal boosting with an unadjuvanted protein amplified anamnestic IgA responses in both the upper and the lower respiratory tracts. These findings help explain why nasal boosters do not require an adjuvant to induce robust mucosal immunity at the respiratory mucosa and can be used to design safe and effective vaccines against respiratory pathogens.

The efficient induction of antigen-specific CD8+ T cell activation is crucial in the development of mRNA tumor vaccines. Endogenous antigens are primarily degraded through the ubiquitin-proteasome system, followed by antigen presentation via major histocompatibility complex class I (MHC-I) molecules, leading to the activation of CD8+ T cells. Therefore, in this study, a novel mRNA vaccine was developed by fusing the mRNA sequence encoding the antigen with a proteasome-targeting peptide (PTP), aiming to enhance proteasomal targeting of the antigen and facilitate its degradation through the ubiquitin-proteasome system, thereby inducing a stronger CD8+ T cell immune response. This study confirmed a significant increase in antigen expression of the antigen-PTP fused mRNA vaccine upon treatment with a VHL inhibitor, as well as notable upregulation of genes associated with the MHC-I antigen-presenting pathway following treatment with the antigen-PTP fused mRNA vaccine. The intramuscular administration of the antigen-PTP fused mRNA vaccine significantly promoted the activation of dendritic cells, macrophages, and T cells in draining lymph nodes and spleens. Additionally, in TC-1 tumor-bearing mice, it markedly suppressed tumor growth, facilitated infiltration of intratumoral antigen-specific CD8+ T cells, and induced immune memory.

Developing vaccines that promote CD8+ T cell memory is a challenge for infectious disease and cancer immunotherapy. TCF-1+ stem cell-like memory CD8+ T (TSCM) cells are important determinants of long-lived memory. Yet, the developmental requirements for TSCM cell formation are unclear. Here, we identify the temporal window for type I interferon receptor (IFNAR) blockade to drive TSCM cell generation following viral infection and mRNA-lipid nanoparticle vaccination. We reveal a reversible developmental trajectory where transcriptionally distinct TSCM cells emerged from a transitional precursor of exhausted T cellular state concomitant with viral clearance. TSCM cell differentiation correlated with T cell retention within the lymph node paracortex due to disrupted CXCR3 chemokine gradient formation. These effects were linked to increased antigen load and a counterintuitive increase in IFNγ, which controlled cell location. Vaccination with the IFNAR blockade promoted TSCM cell differentiation and enhanced protection against chronic infection. These findings propose an approach to vaccine design whereby modulation of inflammation promotes memory formation and function.

mRNA vaccines are a potent tool for immunization against viral diseases and cancer. However, the lack of a vaccine adjuvant limits the efficacy of these treatments. In this study, we used cGAS mRNA, which encodes the DNA innate immune sensor, complexed with lipid nanoparticles (LNP), to boost the immune response. By introducing specific mutations in human cGAS mRNA (hcGASK187N/L195R), we significantly enhanced cGAS activity, resulting in a more potent and sustained stimulator of interferon gene (STING)-mediated IFN response. cGAS mRNA-LNPs exhibited stimulatory effects on maturation, antigen engulfment, and antigen presentation by antigen-presenting cells, both in vitro and in vivo. Moreover, the hcGASK187N/L195R mRNA-LNP combination demonstrated a robust adjuvant effect and amplified the potency of mRNA and protein vaccines, which was a result of strong humoral and cell-mediated responses. Remarkably, the hcGASK187N/L195R mRNA-LNP complex, either alone or in combination with antigens, demonstrated exceptional efficacy in eliciting antitumor immunity. In addition to its immune-boosting properties, hcGASK187N/L195R mRNA-LNP exerted antitumor effects with IFNγ directly on tumor cells, further promoting tumor restriction. In conclusion, we developed a cGAS mRNA-based immunostimulatory adjuvant compatible with various vaccine forms to boost the adaptive immune response and cancer immunotherapies.

Lackluster results from recently completed gene therapy clinical trials of VEGF-A delivered by viral vectors have heightened the need to develop alternative delivery strategies. This study aims to demonstrate the pre-clinical efficacy and safety of extracellular vesicles (EVs) loaded with VEGF-A mRNA for the treatment of ischaemic vascular disease.

After encapsulation of full-length VEGF-A mRNA into fibroblast-derived EVs via cellular nanoporation (CNP), collected VEGF-A EVs were delivered into mouse models of ischaemic injury. Target tissue delivery was verified by in situ analysis of protein and gene expression. Functional rescue was confirmed by in vivo imaging and histology. The safety of single and serial delivery was demonstrated using immune-based assays.

VEGF-A EVs were generated with high mRNA content using a CNP methodology. VEGF-A EV administration demonstrated expression of exogenous VEGF-A mRNA by in situ RNA hybridization and elevated protein expression by western blot, microscopy, and enzyme-linked immunosorbent assay. Mice treated with human VEGF-A EVs after femoral or coronary artery ligation exhibited heightened neovascularization in ischaemic tissues with increased arterial perfusion and improvement in left ventricular function, respectively. Serial delivery of VEGF-EVs in injured skin showed improved wound healing with repeat administration. Importantly, as compared with adeno-associated viral and lipid nanoparticle VEGF-A gene therapy modalities, murine VEGF-A EV delivery did not trigger innate or adaptive immune responses at the injection site or systemically.

This study demonstrated that VEGF-A EV therapy offers efficient, dose-dependent VEGF-A protein formation with low immunogenicity, resulting in new vessel formation in murine models of ischaemic vascular disease.

The development of ionizable mRNA-lipid nanoparticle (mRNA-LNP) nucleic acid carriers facilitated the clinical translation of the Coronavirus 2019 (COVID-19) mRNA vaccines BNT162b2 and mRNA-1273. Here, we discuss insights into rational improvements to mRNA vaccines, focusing on LNP modifications for mRNA-LNP biodistribution control, miRNA-based biodistribution control of encoded transcripts, and precision adjuvantation strategies.

Nucleic acid drug delivery with lipid nanoparticle (LNP) formulations has enabled the development of novel therapeutics and vaccines. LNP formulations are composed of both naturally occurring and synthetic lipid excipients. This perspective shares current practices in the nonclinical safety assessment of novel lipid excipients contained in LNP formulations and identifies gaps in current regulatory guidance on this topic. There is no globally harmonized regulatory guidance for the nonclinical safety assessment of novel excipients or guidance specific to safety testing of novel excipients in LNPs. Given the complexity of these LNP formulations, most nonclinical safety studies to support development are conducted with the drug product or with a LNP that contains non-active cargo. Three case studies (Onpattro®, Comirnaty®, and SpikeVax®) highlight that specific assessments may differ depending on the encapsulated modality, the intended use (e.g., therapeutic versus preventative vaccine), dose, and frequency of dosing. These case studies also suggest that regulatory agencies are open to scientific rationale to justify why certain tests should or should not be performed. As more products are approved, it will be important to understand how precedents set for approved products can be leveraged and what additional unique strategies may be applied to ensure nonclinical safety assessments are predictive, relevant, and meaningful for human safety. Proactive alignment with regulatory authorities will be critical in this context, especially as new approaches are proposed. Guidance documents may need to be revised or created as more experience is acquired to reflect the unique considerations for these novel excipients.

Since the beginning of the COVID-19 pandemic, the development of messenger RNA (mRNA) vaccines has made significant progress in the pharmaceutical industry. The two COVID-19 mRNA vaccines from Moderna and Pfizer/BioNTech have been approved for marketing and have made significant contributions to preventing the spread of SARS-CoV-2. In addition, mRNA therapy has brought hope to some diseases that do not have specific treatment methods or are difficult to treat, such as the Zika virus and influenza virus infections, as well as the prevention and treatment of tumors. With the rapid development of in vitro transcription (IVT) technology, delivery systems, and adjuvants, mRNA therapy has also been applied to hereditary diseases such as Fabry's disease. This article reviews the recent development of mRNA vaccines for structural modification, treatment and prevention of different diseases; delivery carriers and adjuvants; and routes of administration to promote the clinical application of mRNA therapies.

Immunocompetent cells of B lineage function in the humoral immunity system in the adaptive immune responses. B cells differentiate into plasmacytes upon antigen-induced activation and produce different subclasses of immunoglobulins/antibodies. Secreted immunoglobulins not only interact with pathogens to inactivate and neutralize them, but also involve the complement system to exert antibacterial activities and trigger opsonization. Endometrium is a mucosal tissue that lines the mammalian uterus and is indispensable for the establishment of a successful pregnancy. The lymphocytes of B cell lineage are a minority in the human cycling endometrium. Human endometrial B cells have therefore been understudied so far. However, the disorders of the female reproductive tract, including chronic endometritis and endometriosis, have highlighted the importance of further research on the endometrial B cell lineage. This review aims to revisit lymphopoiesis, maturation, commitment, and survival of B cells, shedding light on their physiological and pathological implications in the human endometrium.

Self-amplifying RNA (saRNA)-based vaccines have emerged as a potent and durable RNA vaccine platform relative to first generation mRNA vaccines. However, RNA vaccine platforms trigger undesirable side effects at protective doses, underscoring the need for improved tolerability. To address this, we leveraged the Cardiovirus leader protein, which is well-characterized to dampen host innate signaling by modulating nucleocytoplasmic transport (NCT). Co-administration of a leader-protein-encoding mRNA (which we have named "RNAx") delivered alongside vaccine cargo saRNA reduced interferon production while enhancing Influenza hemagglutinin (HA) expression in human primary cells and murine models. RNAx potently decreased serum biomarkers of reactogenicity after immunizations with an HA-expressing saRNA-LNP vaccine while maintaining the magnitude of the antibody and cellular response. RNAx also consistently enhanced binding antibody titers after a single injection and in some conditions enhanced binding antibody and neutralization titers post-boost. These findings support RNAx as a promising platform approach for improving tolerability of saRNA-LNP vaccines while preserving or enhancing immunogenicity.

Lung cancer is the most lethal malignancy worldwide, having the highest incidence rate. This is a heterogeneous disease classified according to its histological and molecular characteristics. Depending on these, different therapeutic approaches have already been approved for lung cancer treatment targeting genetic alterations or even the immune system. Nonetheless, other therapies are being studied to continuously improve the care and survival of lung cancer patients. Among them, immunotherapies are one of the main targets of investigation to try and combat the ability of some malignant cells to evade anti-tumor responses mediated by the immune system. Cancer vaccine development has emerged as a promising approach to strengthen the patient's immune system and combat the disease, especially mRNA vaccines. Currently, there are several ongoing studies investigating the therapeutic efficacy of mRNA vaccines in lung cancer treatment alone or combined with other therapeutic drugs. This review aims to highlight the importance of immunotherapy in lung cancer treatment, presenting the most recent advances particularly in mRNA-based vaccines as well as the challenges and future perspectives.

The clinical progress of tumor nucleotide vaccines is limited due to insufficient recognition and killing of tumor cells with low antigen expression by cytotoxic T lymphocytes (CTL). Here, natural cholesterol analogs are screened to assemble self-adjuvant lipid nanoparticles (LNPs) for antigens tagging tumor cells and dendritic cells (DC) activation. First, a library of ginsenosides are collected, and then screened according to their anti-tumor immunity. Then, ginsenoside-Rg3 based-LNPs loaded with antigens (Rg3-LNPs) are identified as the optimal formulation by investigating the physicochemical and biological properties. Finally, Rg3-LNPs and granulocyte-macrophage colony-stimulating factor (GM-CSF) are co-loaded into a macroporous hydrogel for long-term immune response. Rg3-LNPs could accumulate into both tumors and LNs. Rg3-LNPs targeted tumor cells with high glucose transporter-1 expression via the targeting ligand Rg3, and anchored antigens on the tumor cell surface, thus promoting the recognition of CTL to tumor cells; Rg3-LNPs can accumulate into the LNs to promote DC activation and antigen presentation, thus stimulating CTL activation. Besides, Rg3, as an adjuvant, cooperated with GM-CSF to remodel the tumor microenvironment, thus promoting the killing of CTL to tumor cells. Collectively, this work highlights the importance of tagging antigens to tumor cells in tumor vaccine and has great clinical value for immune-escaping tumors.

Ionizable lipid nanoparticles (LNPs) are clinically relevant non-viral vectors that allow intracellular delivery of mRNA vaccines to immune cells. To fight against notorious pathogens and cancer, mRNA vaccines necessitate the addition of an adjuvant to induce strong and durable cell-mediated immune responses. Adjuvants that stimulate Toll-like receptor 7 (TLR7) induce the secretion of type I interferons and proinflammatory cytokines, vital for generating strong immune responses. However, the intracellular delivery of TLR7 adjuvants to precisely stimulate the endosomal TLR7 receptor remains a huge challenge. This issue can be addressed by exploiting ionizable LNP platforms, which can encapsulate and carry mRNA vaccines and small molecule hydrophobic adjuvants to immune cells. CL347 is a potent lipid-based adjuvant that selectively stimulates the TLR7 receptor. In this study, we developed ionizable LNPs incorporating SM102 and CL347 adjuvant as the ionizable lipid and TLR7 adjuvant, respectively. CL347-SM102 LNPs exhibited particle sizes of less than 150 nm with spherical morphology and mRNA encapsulation efficiency of greater than 95%. In vivo studies showed a two-fold increase in IFN-γ producing CD4 and CD8 T cells in the lymphoid organs of the mice immunized with adjuvanted LNPs compared to the non-adjuvanted LNPs. Human PBMCs treated with adjuvanted LNPs exhibited significantly higher CD40 expression and pro-inflammatory cytokine (IL-6 and IFN-γ) secretion than non-adjuvanted LNPs. Together, these results suggest the potential of ionizable LNPs as a platform for concurrent delivery of mRNA and adjuvants for prophylactic and therapeutic vaccine applications.

Adjuvants, as a critical component of vaccines, are capable of eliciting more robust and sustained immune responses. Nanomaterials have shown unique advantages and broad application prospects in adjuvant development due to their high adjustability and distinctive physicochemical properties. This review focuses on nanoadjuvants and their immunological mechanisms. First, various types of adjuvants are introduced with an emphasis on metal and metal oxide nanoparticles, coordination polymers, liposomes, polymer nanoparticles, and other inorganic nanoparticles that can serve as vaccine adjuvants. Second, this review describes the current status of the clinical applications of nanoadjuvants. Next, the mechanisms of action for nanoadjuvants have been thoroughly elucidated, including the depot effect, NLRP3 inflammasome activation, targeting C-type lectin receptors, activation of toll-like receptors, and activation of the cGAS-STING signaling pathway. Finally, the challenges and opportunities associated with the development of nanoadjuvants have also been addressed.

Post-infection sequela of several viruses have been linked with Parkinson's disease (PD). Here, we investigated whether mice infected with SARS-CoV-2 alone or in combination with two putative Parkinsonian toxins, MPTP and paraquat, increased the susceptibility to develop Parkinsonian pathology. We also examined if G2019S LRRK2 mice had any change in sensitivity to SARS-CoV-2 as well as if vaccination against this virus altered any neuropathology. Infection with WA-1/2020 or Omicron B1.1.529 strains sensitized both WT and G2019S LRRK2 mice to the neuropathological effects of a subtoxic exposure to MPTP, but not paraquat. These neuropathologies were rescued in WT mice vaccinated with mRNA- or protein-based SARS-CoV-2 vaccines. However, G2019S LRRK2 mutant mice were only protected with the protein-based vaccine. These results highlight the role of both environmental exposures and familial background on the development of Parkinsonian pathology secondary to viral infection and the benefit of vaccines in reducing these risks.

Ionizable lipid nanoparticles (LNP) that have enabled the success of messenger RNA (mRNA) vaccines have been shown to be immunostimulatory in the absence of mRNA. However, the mechanisms through which they activate innate immune cells is incompletely understood. Using a monocyte cell line, we compared the ability of three LNP formulations to activate transcription factors Nuclear Factor-kappa B (NF-κB) and Interferon Regulatory Factor (IRF). Comparison of signaling in knockout cell lines illustrated a role for Toll-like receptor (TLR) 4 in initiation of this signaling cascade and the contribution of the ionizable lipid component. Activation induced by empty LNPs was similar to that induced by LNPs containing mRNA, indicating that LNPs may provide the majority of innate stimulation for the mRNA vaccine platform. Our findings demonstrate that ionizable lipids within LNPs signal through TLR4 to activate NF-κB and IRF, identifying a mechanism for innate activation that can be optimized for adjuvant design.

Nucleic acid vaccines have grown in importance over the past several years, with the development of new approaches remaining a focus. We describe a lipid nanoparticle-formulated DNA (DNA-LNP) formulation which induces robust innate and adaptive immunity with similar serological potency to mRNA-LNPs and adjuvanted protein. Using an influenza hemagglutinin (HA)-encoding construct, we show that priming with our HA DNA-LNP demonstrated stimulator of interferon genes (STING)-dependent upregulation and activation of migratory dendritic cell (DC) subpopulations. HA DNA-LNP induced superior antigen-specific CD8+ T cell responses relative to mRNA-LNPs or adjuvanted protein, with memory responses persisting beyond one year. In rabbits immunized with HA DNA-LNP, we observed immune responses comparable or superior to mRNA-LNPs at the same dose. In an additional model, a SARS-CoV-2 spike-encoding DNA-LNP elicited protective efficacy comparable to spike mRNA-LNPs. Our study identifies a platform-specific priming mechanism for DNA-LNPs divergent from mRNA-LNPs or adjuvanted protein, suggesting avenues for this approach in prophylactic and therapeutic vaccine development.

Lipid nanoparticles (LNPs) are used to encapsulate messenger ribonucleic acids (mRNAs) and enhance mRNA vaccine efficacy by producing inflammatory mediators. However, the overproduction of inflammatory mediators via LNP injection causes severe side effects, presenting a potential limitation. To resolve this issue, we developed pH-responsive dipeptide-conjugated lipid (DPL)-based LNPs (DPL-LNPs) for efficient small interfering RNA delivery with excellent biocompatibility. In detail, we optimized the dipeptide sequence and lipid-tail length of DPL, the helper-lipid compositions, and the molecular weight and lipid-tail length of the polyethylene glycol (PEG)-lipid to achieve highly efficient and safe mRNA delivery. Our results revealed that the LNPs prepared using glutamic acid (E)- and arginine (R)-conjugated DPL (DPL-ER) displayed higher protein-expression efficacy than DPL-threonine-R- and DPL-aspartic acid-R-based LNPs. Additionally, the lipid-tail length of the C22-bearing DPL-ER (DPL-ER-C22)-based LNPs displayed higher protein-expression efficacies than their C18 (DPL-ER-C18)- and C24 (DPL-ER-C24)-based LNPs. Moreover, the DPL-ER-C22-based LNPs incorporating low-lipid-tail-length phospholipids and PEG-lipids exhibited efficient protein expression. Most importantly, the injection of optimized DPL-LNPs exhibited comparable antigen-specific antibody production levels, with significantly lower inflammatory-mediator production compared with those of the commercially available LNPs. These results indicate that DPL-based LNPs (DPL-LNPs) can be deployed as highly efficient, safe carriers for mRNA delivery for developing mRNA vaccine formulations.

Psoriasis is a chronic inflammatory autoimmune disease, yet it affects hundreds of millions of people. Long-term effective intervention of the disease by targeting the causative genes via RNAi (RNA interference) has become a reality. However, its further application is hindered by inflammatory side effects caused by delivery systems such as LNP (lipid nanoparticles).

This study aimed to develop a novel anti-inflammatory LNP rationally tailored for topical application in psoriasis and to validate its potential to deliver Stat3 (signal transducer and activator of transcription 3) siRNA for the treatment of psoriasis.

To assess the transfection efficiency, anti-inflammatory capacity of LNPs. The therapeutic effect of modified anti-inflammatory LNP delivery of Stat3 siRNA on psoriasis was evaluated both in vitro and in an imiquimod-induced mice.

LNPs exhibit both superior transfection efficiency and significant anti-inflammatory effects. In vitro functional studies showed that in an inflammatory DC model, anti-inflammatory LNP (C8B2) inhibited inflammatory mediators much better than classical LNPs by delivering Stat3 siRNA; in pathological HaCat cells, Stat3 siRNA reduced cell proliferation and promoted apoptosis. In the imiquimod-induced mouse model, the C8B2-si-Stat3 group demonstrated a clear reduction in psoriasis progression, whereas the C8B2 carrier group also exhibited a notable decrease in inflammation.

In this study, we successfully developed a novel anti-inflammatory LNP, which demonstrated notable advantages in delivery capacity, anti-inflammatory effect, and targeting therapy against STAT3, providing new ideas and strategies for nucleic acid therapy of psoriasis. This LNP platform could be broadly applicable to various inflammatory conditions, offering a versatile tool for targeted gene modulation and inflammation control.

In the previously published version of the paper, the term "AS03" was used to describe the AddaS03 adjuvant used in animal experiments. This could lead to confusion among the trade and public as to a connection between the AddaS03 adjuvant and GSK's AS03. Upon request by GSK, the authors clarify that no AS03 from GSK was used in this study, and the results obtained with AddaS03 are not transposable to the GSK's AS03 adjuvant. The article has now been corrected, and the conclusions of this paper remain unchanged. Corrections highlighted in bold. The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a glycoprotein, expressed on the virion surface, that mediates infection of host cells by directly interacting with host receptors. As such, it is a reasonable target to neutralize the infectivity of the virus. Here we found that a recombinant S protein vaccine adjuvanted with Alhydrogel or the QS-21-like adjuvant Quil-A effectively induced anti-S receptor binding domain (RBD) serum IgG and neutralizing antibody titers in the Syrian hamster model, resulting in significantly low SARS-CoV-2 replication in respiratory organs and reduced body weight loss upon virus challenge. Severe lung inflammation upon virus challenge was also strongly suppressed by vaccination. We also found that the S protein vaccine adjuvanted with Alhydrogel, Quil-A, or AddaS03 elicited significantly higher neutralizing antibody titers in mice than did unadjuvanted vaccine. Although the neutralizing antibody titers against the variant viruses B.1.351 and B.1.617.2 declined markedly in mice immunized with wild-type S protein, the binding antibody levels against the variant S proteins were equivalent to those against wild-type S. When splenocytes from the immunized mice were re-stimulated with the S protein in vitro, the induced Th1 or Th2 cytokine levels were not significantly different upon re-stimulation with wild-type S or variant S, suggesting that the T-cell responses against the variants were the same as those against the wild-type virus. Upon Omicron XBB-challenge in hamsters, wild-type S-vaccination with Alhydrogel or AddaS03 reduced lung virus titers on Day 3, and the Quil-A adjuvanted group showed less body weight loss, although serum neutralizing antibody titers against XBB were barely detected in vitro. Collectively, recombinant vaccines coupled with different adjuvants may be promising modalities to combat new variant viruses by inducing various arms of the immune response.

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract infections, especially in infants and the elderly. Developing an RSV vaccine that promotes a robust mucosal immune response is necessary to successfully prevent viral transmission and the development of severe disease. We previously reported that crosslinked carbon dots (CCD) may be an excellent adjuvant candidate for intranasal (IN) protein subunit vaccines. Considering the strong immunogenicity of RSV prefused F protein (preF), we prepared an IN RSV vaccine composed of the CCD adjuvant and the preF protein as antigen (CCD/preF) and evaluated the induced antigen-specific humoral and cellular immunity. We found that IN immunization with the CCD/preF vaccine elicited strong serum IgG responses and mucosal immunity, including secreted IgA antibodies, tissue-resident memory T (TRM) cells, and antigen-specific B cells, which lasted for at least 1 year. In addition, a combination of intramuscular and IN immunization with CCD/preF vaccine induced stronger systemic and mucosal immunity. Together, this study proved the high immunogenicity of the CCD/preF vaccines and supported the university of the mucosal CCD adjuvant, supporting further development of the CCD/preF vaccine in larger animal models and clinical studies.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, leading to the absence of dystrophin and progressive muscle degeneration. Current therapeutic strategies, such as exon-skipping and gene therapy, face limitations including truncated dystrophin production and safety concerns. To address these issues, a novel mRNA-based therapy is explored using polyplex nanomicelles to deliver mRNA encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha isoform 4 (PGC-1α4) via hydrodynamic limb vein (HLV) administration. Using an in vivo muscle torque measurement technique, it is observed that nanomicelle-delivered Pgc-1α4 mRNA significantly improved muscle damage resistance and mitochondrial activity in mdx mice. Specifically, HLV administration of Pgc-1α4 mRNA in dystrophic muscles significantly relieved the torque reduction and myofiber injury induced by eccentric contraction (ECC), boosted metabolic gene expression, and enhanced muscle oxidative capacity. In comparison, lipid nanoparticles (LNPs), a widely used mRNA delivery system, does not achieve similar protective effects, likely due to their intrinsic immunogenicity. This foundational proof-of-concept study highlights the potential of mRNA-based therapeutics for the treatment of neuromuscular diseases such as DMD and demonstrates the capability of polyplex nanomicelles as a safe and efficient mRNA delivery system for therapeutic applications.

Extracellular vesicles (EVs), including exosomes, mediate intercellular communication by transporting functional molecules between donor cells and recipient cells, thereby regulating biological processes, such as immune responses. miR-451a, an immune regulatory microRNA, is highly abundant in circulating EVs; however, its precise physiological significance remains to be fully elucidated. Here, we demonstrate that miR-451a deficiency exacerbates delayed-type hypersensitivity (DTH) in mice. Notably, miR-451a knockout resulted in a significant increase in the number of interleukin (IL)-17A-expressing T helper 17 and γδ T cells infiltrating DTH-induced ear lesions. miR-451a deficiency also increased the number of γδ T cells in the secondary lymphoid tissues. Comprehensive analyses revealed that miR-451 deficiency promoted the expression of Rorc and γδ T cell-related genes following sensitization with allergens. Moreover, intravenous administration of wild-type EVs to miR-451a knockout mice increased cellular miR-451a levels in tissues and significantly attenuated the severity of DTH. Furthermore, synthetic lipid nanoparticles encapsulating miR-451a effectively mitigated DTH. Our findings indicate the importance of circulating miR-451a in the proliferation of γδ T cells and highlight the therapeutic potential of lipid nanoparticle-based microRNA delivery platforms for interventions in immune-related diseases.