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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.

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer. [ABSTRACT FROM AUTHOR]

Background: Although mRNA vaccines encapsulated in lipid nanoparticles (LNPs) have demonstrated a safety profile with minimal serious adverse events in clinical trials, there is opportunity to further reduce mRNA reactogenicity. The development of naked mRNA vaccines could improve vaccine tolerability. Naked nucleic acid delivery using the jet injection method may be a solution. Methods: In the first part of the study, the optimal conditions providing low traumatization and high expression of the model mRNA-GFP molecule in the tissues of laboratory animals were determined. Then, we used the selected protocol to immunize BALB/c mice with mRNA-RBD encoding the SARS-CoV-2 receptor-binding domain (RBD). It was demonstrated that mice vaccinated with naked mRNA-RBD developed a high level of specific antibodies with virus-neutralizing activity. The vaccine also induced a strong RBD-specific T-cell response and reduced the viral load in the lungs of the animals after infection with the SARS-CoV-2 virus. The level of immune response in mice immunized with mRNA-RBD using a spring-loaded jet injector was comparable to that in animals immunized with mRNA-RBD encapsulated in LNPs. Results: In this study, the efficacy of an inexpensive, simple, and safe method of mRNA delivery using a spring-loaded jet injector was evaluated and validated. Conclusions: Our findings suggest that the jet injection method may be a possible alternative to LNPs for delivering mRNA vaccines against SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis that causes high fetal and neonatal mortality rates in ruminants and sometimes severe to fatal complications like encephalitis and hemorrhagic fever in humans. There is no licensed RVF vaccine for human use while approved livestock vaccines have suboptimal safety or efficacy. We designed self-amplifying RNA (saRNA) RVF vaccines and assessed their humoral immunogenicity in mice. Plasmid DNA encoding the Rift Valley fever virus (RVFV) medium (M) segment consensus sequence (WT consensus) and its derivatives mutated to enhance cell membrane expression of the viral surface glycoproteins n (Gn) and c (Gc) were assessed for in vitro expression. The WT consensus and best-expressing derivative (furin-T2A) were cloned into a Venezuelan equine encephalitis virus (VEEV) plasmid DNA replicon and in vitro transcribed into saRNA. The saRNA was formulated in lipid nanoparticles and its humoral immunogenicity in BALB/c mice was assessed. High quantities of dose-dependent RVFV Gn IgG antibodies were detected in the serum of all mice immunized with either WT consensus or furin-T2A saRNA RVF vaccines. Significant RVFV pseudovirus-neutralizing activity was induced in mice immunized with 1 µg or 10 µg of the WT consensus saRNA vaccine. The WT consensus saRNA RVF vaccine warrants further development. [ABSTRACT FROM AUTHOR]

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer.

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.

Background: Although mRNA vaccines encapsulated in lipid nanoparticles (LNPs) have demonstrated a safety profile with minimal serious adverse events in clinical trials, there is opportunity to further reduce mRNA reactogenicity. The development of naked mRNA vaccines could improve vaccine tolerability. Naked nucleic acid delivery using the jet injection method may be a solution. Methods: In the first part of the study, the optimal conditions providing low traumatization and high expression of the model mRNA-GFP molecule in the tissues of laboratory animals were determined. Then, we used the selected protocol to immunize BALB/c mice with mRNA-RBD encoding the SARS-CoV-2 receptor-binding domain (RBD). It was demonstrated that mice vaccinated with naked mRNA-RBD developed a high level of specific antibodies with virus-neutralizing activity. The vaccine also induced a strong RBD-specific T-cell response and reduced the viral load in the lungs of the animals after infection with the SARS-CoV-2 virus. The level of immune response in mice immunized with mRNA-RBD using a spring-loaded jet injector was comparable to that in animals immunized with mRNA-RBD encapsulated in LNPs. Results: In this study, the efficacy of an inexpensive, simple, and safe method of mRNA delivery using a spring-loaded jet injector was evaluated and validated. Conclusions: Our findings suggest that the jet injection method may be a possible alternative to LNPs for delivering mRNA vaccines against SARS-CoV-2 infection.

The COVID-19 pandemic has raised the standard regarding the current vaccine development pace, as several messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines have proved their ability to induce strong immunogenicity and protective efficacy. We developed 1-methylpseudouridine-containing mRNA-LNP vaccines, expressing either the more conserved SARS-CoV-2 nucleoprotein (mRNA-N) or spike protein (mRNA-S), both based on the prototypic viral sequences. When combining both mRNA-S and mRNA-N together (mRNA-S+N), the vaccine showed high immunogenicity and broad protection against different SARS-CoV-2 variants, including wildtype, Delta, BA.1, BA.5, and BQ.1. To better understand the mechanisms behind this broad protection obtained by mRNA-S+N, we analyzed innate and adaptive immune parameters following vaccination in mice. Compared to either mRNA-S or mRNA-N alone, mice vaccinated with mRNA-S+N exhibited an increase in the innate immune response, as depicted by the higher cytokine (IL-6 and chemokine (MCP-1) levels. In addition, lymph node immunophenotyping showed the maturation and activation of dendritic cells and natural killer cells, respectively. To understand the adaptive immune response, RNA-Seq analyses of the lung and spleen samples of the vaccinated mice were performed in parallel and revealed a stronger immune gene-expression profile in the lung than that in the spleen. Compared to mRNA-S alone, mRNA-S+N vaccination elicited higher levels of expression for genes involved in multiple immune pathways, including T cells, cytokine signaling, antigen presentation, B cells, and innate immunity. Together, our studies provide immunological insights into the mechanisms of broad protection conferred by dual mRNA vaccination against SARS-CoV-2 variants. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 variants have evolved over time in recent years, demonstrating immune evasion of vaccine-induced neutralizing antibodies directed against the original S protein. Updated S-targeted vaccines provide a high level of protection against circulating variants of SARS-CoV-2, but this protection declines over time due to ongoing virus evolution. To achieve a broader protection, novel vaccine candidates involving additional antigens with low mutation rates are currently needed. Based on our recently studied mRNA lipid nanoparticle (mRNA-LNP) platform, we have generated mRNA-LNP encoding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combination in vivo. As a result, all mRNA-LNP vaccine compositions encoding the S and N proteins induced excellent titers of RBD- and N-specific binding antibodies. The T cell responses were mainly specific CD4+ T cell lymphocytes producing IL-2 and TNF-alpha. mRNA-LNP encoding the M protein did not show a high immunogenicity. High neutralizing activity was detected in the sera of mice vaccinated with mRNA-LNP encoding S protein (alone or in combinations) against closely related strains, but was undetectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to mRNA-N in the vaccine composition enhanced the immunogenicity of mRNA-N and induced a more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancing the immune response to the N protein when they are both encoded in the mRNA-LNP vaccine. [ABSTRACT FROM AUTHOR]

Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection. [ABSTRACT FROM AUTHOR]

The development of mRNA vaccines has increased rapidly since the COVID-19 pandemic. As one of the critical attributes, understanding mRNA lipid nanoparticle (LNP) stability is critical in the vaccine product development. However, the correlation between LNPs' physiochemical characteristics and their potency still remains unclear. The lack of regulatory guidance on the specifications for mRNA LNPs is also partially due to this underexplored relationship. In this study, we performed a three-month stability study of heat-stressed mRNA LNP samples. The mRNA LNP samples were analyzed for their mRNA degradation, LNP particle sizes, and mRNA encapsulation efficiency. In vitro cell potency was also evaluated and correlated with these above-mentioned physiochemical characterizations. The mRNA degradation–cell potency correlation data showed two distinct regions, indicating a critical cut-off size limit for mRNA degradation. The same temperature dependence was also observed in the LNP size–cell potency correlation. [ABSTRACT FROM AUTHOR]

The Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows for frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as nanocarriers for delivering DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. The intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal responses in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only 1 out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines. [ABSTRACT FROM AUTHOR]

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has raised the standard regarding the current vaccine development pace, as several messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines have proved their ability to induce strong immunogenicity and protective efficacy. We developed 1-methylpseudouridine-containing mRNA-LNP vaccines, expressing either the more conserved SARS-CoV-2 nucleoprotein (mRNA-N) or spike protein (mRNA-S), both based on the prototypic viral sequences. When combining both mRNA-S and mRNA-N together (mRNA-S+N), the vaccine showed high immunogenicity and broad protection against different SARS-CoV-2 variants, including wildtype, Delta, BA.1, BA.5, and BQ.1. To better understand the mechanisms behind this broad protection obtained by mRNA-S+N, we analyzed innate and adaptive immune parameters following vaccination in mice. Compared to either mRNA-S or mRNA-N alone, mice vaccinated with mRNA-S+N exhibited an increase in the innate immune response, as depicted by the higher cytokine (IL-6 and chemokine (MCP-1) levels. In addition, lymph node immunophenotyping showed the maturation and activation of dendritic cells and natural killer cells, respectively. To understand the adaptive immune response, RNA-Seq analyses of the lung and spleen samples of the vaccinated mice were performed in parallel and revealed a stronger immune gene-expression profile in the lung than that in the spleen. Compared to mRNA-S alone, mRNA-S+N vaccination elicited higher levels of expression for genes involved in multiple immune pathways, including T cells, cytokine signaling, antigen presentation, B cells, and innate immunity. Together, our studies provide immunological insights into the mechanisms of broad protection conferred by dual mRNA vaccination against SARS-CoV-2 variants.

SARS-CoV-2 variants have evolved over time in recent years, demonstrating immune evasion of vaccine-induced neutralizing antibodies directed against the original S protein. Updated S-targeted vaccines provide a high level of protection against circulating variants of SARS-CoV-2, but this protection declines over time due to ongoing virus evolution. To achieve a broader protection, novel vaccine candidates involving additional antigens with low mutation rates are currently needed. Based on our recently studied mRNA lipid nanoparticle (mRNA-LNP) platform, we have generated mRNA-LNP encoding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combination in vivo. As a result, all mRNA-LNP vaccine compositions encoding the S and N proteins induced excellent titers of RBD- and N-specific binding antibodies. The T cell responses were mainly specific CD4+ T cell lymphocytes producing IL-2 and TNF-alpha. mRNA-LNP encoding the M protein did not show a high immunogenicity. High neutralizing activity was detected in the sera of mice vaccinated with mRNA-LNP encoding S protein (alone or in combinations) against closely related strains, but was undetectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to mRNA-N in the vaccine composition enhanced the immunogenicity of mRNA-N and induced a more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancing the immune response to the N protein when they are both encoded in the mRNA-LNP vaccine.

Lipid nanoparticles (LNPs) are widely used as delivery systems for mRNA vaccines. The stability and bilayer fluidity of LNPs are determined by the properties and contents of the various lipids used in the formulation system, and the delivery efficiency of LNPs largely depends on the lipid composition. For the quality control of such vaccines, here we developed and validated an HPLC-CAD method to identify and determine the contents of four lipids in an LNP-encapsulated COVID-19 mRNA vaccine to support lipid analysis for the development of new drugs and vaccines. [ABSTRACT FROM AUTHOR]

The development of mRNA vaccines has increased rapidly since the COVID-19 pandemic. As one of the critical attributes, understanding mRNA lipid nanoparticle (LNP) stability is critical in the vaccine product development. However, the correlation between LNPs’ physiochemical characteristics and their potency still remains unclear. The lack of regulatory guidance on the specifications for mRNA LNPs is also partially due to this underexplored relationship. In this study, we performed a three-month stability study of heat-stressed mRNA LNP samples. The mRNA LNP samples were analyzed for their mRNA degradation, LNP particle sizes, and mRNA encapsulation efficiency. In vitro cell potency was also evaluated and correlated with these above-mentioned physiochemical characterizations. The mRNA degradation–cell potency correlation data showed two distinct regions, indicating a critical cut-off size limit for mRNA degradation. The same temperature dependence was also observed in the LNP size–cell potency correlation.

Chronic infection with hepatitis B virus (HBV) develops in millions of patients per year, despite the availability of effective prophylactic vaccines. Patients who resolve acute HBV infection develop HBV-specific polyfunctional T cells accompanied by neutralizing antibodies, while in patients with chronic hepatitis B (CHB), immune cells are dysfunctional and impaired. We describe a lipid nanoparticle (LNP)-formulated mRNA vaccine, optimized for the expression of HBV core, polymerase, and surface (preS2-S) antigens with the aim of inducing an effective immune response in patients with CHB. Prime and prime/boost vaccination with LNP-formulated mRNA encoding for core, pol, and/or preS2-S dosing strategies were compared in naive C57BL/6 and BALB/c mice. Immune responses were assessed by IFN-γ ELISpot, intracellular cytokine staining (ICS), and ELISA for antibody production, whereas anti-viral efficacy was evaluated in the AAV-HBV mouse model. The mRNA vaccine induced strong antigen-specific polyfunctional T cell responses in these mouse models, accompanied by the emergence of anti-HBs and anti-HBe antibodies. After three immunizations, the antigen-specific immune stimulation resulted in up to 1.7 log10 IU/mL reduction in systemic HBV surface antigen (HBsAg), accompanied by a transient drop in systemic HBeAg, and this was observed in 50% of the AAV-HBV-transduced mice in the absence of additional modalities such as adjuvants, HBsAg reducing agents, or checkpoint inhibitors. However, no treatment-related effect on viremia was observed in the liver. These results warrant further optimization and evaluation of this mRNA vaccine as a candidate in a multimodal therapeutic regimen for the treatment of chronic HBV infection.

Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced technologies for the highly efficient in vivo delivery of exogenous mRNA, particularly for COVID-19 vaccine delivery. LNPs comprise four different lipids: ionizable lipids, helper or neutral lipids, cholesterol, and lipids attached to polyethylene glycol (PEG). In this review, we present recent the advances and insights for the design of LNPs, as well as their composition and properties, with a subsequent discussion on the development of COVID-19 vaccines. In particular, as ionizable lipids are the most critical drivers for complexing the mRNA and in vivo delivery, the role of ionizable lipids in mRNA vaccines is discussed in detail. Furthermore, the use of LNPs as effective delivery vehicles for vaccination, genome editing, and protein replacement therapy is explained. Finally, expert opinion on LNPs for mRNA vaccines is discussed, which may address future challenges in developing mRNA vaccines using highly efficient LNPs based on a novel set of ionizable lipids. Developing highly efficient mRNA delivery systems for vaccines with improved safety against some severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains difficult. [ABSTRACT FROM AUTHOR]

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

The Influenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows for frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as nanocarriers for delivering DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. The intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal responses in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only 1 out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines.

We report a case of vasospastic angina (VSA) following COVID-19 mRNA vaccination. Despite the widespread occurrence of myocarditis, there have been few reports of post-vaccinal VSA. A 41-year-old male patient was referred for chest pain at rest following mRNA vaccination; he had never experienced chest pain prior to vaccination. He was diagnosed by an acetylcholine (Ach) provocation test that showed multivessel vasospasm. After the initiation of treatment with a calcium channel blocker and nitrate, no further exacerbation of chest pain was observed. To our knowledge, this constitutes the first reported case of VSA proven by Ach provocation test after COVID-19 vaccination. The vaccination may increase coronary artery spasticity. VSA should be ruled out in post-vaccine new onset resting chest pain. [ABSTRACT FROM AUTHOR]

COVID-19 vaccines have been used to counteract the global COVID-19 pandemic. While these are effective, adverse reactions have been reported, such as injection-site pain, muscle ache, fever, palpitation, and chest discomfort. The release of inflammatory cytokines, such as interleukin (IL)-6 and IL-1β, is a potential mechanism for post-vaccine side-effects. Chest discomfort after the vaccination, including myocarditis and acute coronary syndrome, is a particularly serious adverse reaction. It is important to be familiar with the differential diagnoses of chest discomfort and organ-specific diseases associated with COVID-19 vaccines as the preparation for booster shots and vaccinations among children aged 5–11 years begins. High-intensity exercise, alcohol, tobacco smoking, and baths promote inflammatory cytokines, such as IL-6, which may exacerbate the adverse reactions after vaccination. Japanese data show that deaths during baths are the most common for several days after mRNA vaccination. Additionally, alcohol and tobacco smoking were identified as predictive factors of lower antibody titers after vaccination. In this review, we aimed to provide a few recommendations to prevent vaccine-associated disease. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs) are widely used as delivery systems for mRNA vaccines. The stability and bilayer fluidity of LNPs are determined by the properties and contents of the various lipids used in the formulation system, and the delivery efficiency of LNPs largely depends on the lipid composition. For the quality control of such vaccines, here we developed and validated an HPLC-CAD method to identify and determine the contents of four lipids in an LNP-encapsulated COVID-19 mRNA vaccine to support lipid analysis for the development of new drugs and vaccines.

Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced technologies for the highly efficient in vivo delivery of exogenous mRNA, particularly for COVID-19 vaccine delivery. LNPs comprise four different lipids: ionizable lipids, helper or neutral lipids, cholesterol, and lipids attached to polyethylene glycol (PEG). In this review, we present recent the advances and insights for the design of LNPs, as well as their composition and properties, with a subsequent discussion on the development of COVID-19 vaccines. In particular, as ionizable lipids are the most critical drivers for complexing the mRNA and in vivo delivery, the role of ionizable lipids in mRNA vaccines is discussed in detail. Furthermore, the use of LNPs as effective delivery vehicles for vaccination, genome editing, and protein replacement therapy is explained. Finally, expert opinion on LNPs for mRNA vaccines is discussed, which may address future challenges in developing mRNA vaccines using highly efficient LNPs based on a novel set of ionizable lipids. Developing highly efficient mRNA delivery systems for vaccines with improved safety against some severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains difficult.

We report a case of vasospastic angina (VSA) following COVID-19 mRNA vaccination. Despite the widespread occurrence of myocarditis, there have been few reports of post-vaccinal VSA. A 41-year-old male patient was referred for chest pain at rest following mRNA vaccination; he had never experienced chest pain prior to vaccination. He was diagnosed by an acetylcholine (Ach) provocation test that showed multivessel vasospasm. After the initiation of treatment with a calcium channel blocker and nitrate, no further exacerbation of chest pain was observed. To our knowledge, this constitutes the first reported case of VSA proven by Ach provocation test after COVID-19 vaccination. The vaccination may increase coronary artery spasticity. VSA should be ruled out in post-vaccine new onset resting chest pain.

Zika virus (ZIKV), a mosquito-borne flavivirus, has recently triggered global concern due to severe health complications. In 2015, a large ZIKV outbreak occurred in the Americas and established a link between ZIKV and microcephaly in newborn babies, spontaneous abortion, persistent viremia, and Guillain–Barré syndrome. While antivirals are being developed and prevention strategies focus on vector control, a safe and effective Zika vaccine remains unavailable. Messenger RNA (mRNA) vaccine technology has arisen as a flexible, simplified, and fast vaccine production platform. Here, we report on an mRNA vaccine candidate that encodes the pre-membrane and envelope (prM–E) glycoproteins of ZIKV strain Brazil SPH2015 and is encapsulated in lipid nanoparticles (LNPs). Our ZIKV prM–E mRNA-LNP vaccine candidate induced antibody responses that protected in AG129 mice deficient in interferon (IFN) alpha/beta/gamma (IFN-α/β/γ) receptors. Notably, a single administration of ZIKV prM–E mRNA-LNP protected against a lethal dose of ZIKV, while a two-dose strategy induced strong protective immunity. E-specific double-positive IFN-γ and TNF-α T-cells were induced in BALB/c mice after immunizations with a two-dose strategy. With the success of mRNA vaccine technology in facing the coronavirus (COVID-19) pandemic, our data support the development of prM–E RNActive® as a promising mRNA vaccine against Zika to counter future epidemics.

COVID-19 vaccines have been developed with unprecedented speed which would not have been possible without decades of fundamental research on delivery nanotechnology. Lipid-based nanoparticles have played a pivotal role in the successes of COVID-19 vaccines and many other nanomedicines, such as Doxil® and Onpattro®, and have therefore been considered as the frontrunner in nanoscale drug delivery systems. In this review, we aim to highlight the progress in the development of these lipid nanoparticles for various applications, ranging from cancer nanomedicines to COVID-19 vaccines. The lipid-based nanoparticles discussed in this review are liposomes, niosomes, transfersomes, solid lipid nanoparticles, and nanostructured lipid carriers. We particularly focus on the innovations that have obtained regulatory approval or that are in clinical trials. We also discuss the physicochemical properties required for specific applications, highlight the differences in requirements for the delivery of different cargos, and introduce current challenges that need further development. This review serves as a useful guideline for designing new lipid nanoparticles for both preventative and therapeutic vaccines including immunotherapies.

Increases in the world’s population and population density promote the spread of emerging pathogens. Vaccines are the most cost-effective means of preventing this spread. Traditional methods used to identify and produce new vaccines are not adequate, in most instances, to ensure global protection. New technologies are urgently needed to expedite large scale vaccine development. mRNA-based vaccines promise to meet this need. mRNA-based vaccines exhibit a number of potential advantages relative to conventional vaccines, namely they (1) involve neither infectious elements nor a risk of stable integration into the host cell genome; (2) generate humoral and cell-mediated immunity; (3) are well-tolerated by healthy individuals; and (4) are less expensive and produced more rapidly by processes that are readily standardized and scaled-up, improving responsiveness to large emerging outbreaks. Multiple mRNA vaccine platforms have demonstrated efficacy in preventing infectious diseases and treating several types of cancers in humans as well as animal models. This review describes the factors that contribute to maximizing the production of effective mRNA vaccine transcripts and delivery systems, and the clinical applications are discussed in detail.

There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies.

A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered—both at the same time and in the same location—in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties.

Zika virus (ZIKV), a mosquito-borne flavivirus, has recently triggered global concern due to severe health complications. In 2015, a large ZIKV outbreak occurred in the Americas and established a link between ZIKV and microcephaly in newborn babies, spontaneous abortion, persistent viremia, and Guillain–Barré syndrome. While antivirals are being developed and prevention strategies focus on vector control, a safe and effective Zika vaccine remains unavailable. Messenger RNA (mRNA) vaccine technology has arisen as a flexible, simplified, and fast vaccine production platform. Here, we report on an mRNA vaccine candidate that encodes the pre-membrane and envelope (prM–E) glycoproteins of ZIKV strain Brazil SPH2015 and is encapsulated in lipid nanoparticles (LNPs). Our ZIKV prM–E mRNA-LNP vaccine candidate induced antibody responses that protected in AG129 mice deficient in interferon (IFN) alpha/beta/gamma (IFN-α/β/γ) receptors. Notably, a single administration of ZIKV prM–E mRNA-LNP protected against a lethal dose of ZIKV, while a two-dose strategy induced strong protective immunity. E-specific double-positive IFN-γ and TNF-α T-cells were induced in BALB/c mice after immunizations with a two-dose strategy. With the success of mRNA vaccine technology in facing the coronavirus (COVID-19) pandemic, our data support the development of prM–E RNActive® as a promising mRNA vaccine against Zika to counter future epidemics. [ABSTRACT FROM AUTHOR]

Increases in the world's population and population density promote the spread of emerging pathogens. Vaccines are the most cost-effective means of preventing this spread. Traditional methods used to identify and produce new vaccines are not adequate, in most instances, to ensure global protection. New technologies are urgently needed to expedite large scale vaccine development. mRNA-based vaccines promise to meet this need. mRNA-based vaccines exhibit a number of potential advantages relative to conventional vaccines, namely they (1) involve neither infectious elements nor a risk of stable integration into the host cell genome; (2) generate humoral and cell-mediated immunity; (3) are well-tolerated by healthy individuals; and (4) are less expensive and produced more rapidly by processes that are readily standardized and scaled-up, improving responsiveness to large emerging outbreaks. Multiple mRNA vaccine platforms have demonstrated efficacy in preventing infectious diseases and treating several types of cancers in humans as well as animal models. This review describes the factors that contribute to maximizing the production of effective mRNA vaccine transcripts and delivery systems, and the clinical applications are discussed in detail. [ABSTRACT FROM AUTHOR]

COVID-19 vaccines have been developed with unprecedented speed which would not have been possible without decades of fundamental research on delivery nanotechnology. Lipid-based nanoparticles have played a pivotal role in the successes of COVID-19 vaccines and many other nanomedicines, such as Doxil® and Onpattro®, and have therefore been considered as the frontrunner in nanoscale drug delivery systems. In this review, we aim to highlight the progress in the development of these lipid nanoparticles for various applications, ranging from cancer nanomedicines to COVID-19 vaccines. The lipid-based nanoparticles discussed in this review are liposomes, niosomes, transfersomes, solid lipid nanoparticles, and nanostructured lipid carriers. We particularly focus on the innovations that have obtained regulatory approval or that are in clinical trials. We also discuss the physicochemical properties required for specific applications, highlight the differences in requirements for the delivery of different cargos, and introduce current challenges that need further development. This review serves as a useful guideline for designing new lipid nanoparticles for both preventative and therapeutic vaccines including immunotherapies. [ABSTRACT FROM AUTHOR]

There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies. [ABSTRACT FROM AUTHOR]