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mRNA technologies have the potential to transform areas of medicine, including the prophylaxis of infectious diseases. The advantages for vaccines range from the acceleration of immunogen discovery to rapid response and multiple disease target manufacturing. A greater understanding of quality attributes that dictate translation efficiency, as well as a comprehensive appreciation of the importance of mRNA delivery, are influencing a new era of investment in development activities. The application of translational sciences and growing early-phase clinical experience continue to inform candidate vaccine selection. Here we review the state of the art for the prevention of infectious diseases by using mRNA and pertinent topics to the biotechnology and pharmaceutical industries.

Pulmonary delivery of mRNA via inhalation is a very attractive approach for RNA-based therapy for treatment of lung diseases. In this work, we have demonstrated successful development of an mRNA-lipid nanoparticle (LNP) dry powder product (DPP), wherein the LNPs were spray dried using hydroalcoholic solvent along with mannitol and leucine as excipients. The desired critical attributes for the DPP were accomplished by varying the excipients, lipid composition, concentration of LNPs, and weight percentage of mRNA. Leucine alone or in combination with mannitol improved the formulation by increasing the mRNA yield as well as decreasing the particle size. Intratracheal administration of the DPP in mice resulted in luciferase expression in the trachea and lungs indicating successful delivery of functional mRNA. Our results show formulation optimization of mRNA LNPs administered in the form of DPP results in an efficacious functional delivery with great promise for future development of mRNA therapeutics for lung diseases. Graphical Abstract

Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.

Background: Although mRNA vaccines have the potential to be developed and deployed rapidly to combat infectious diseases, the ideal method of administration and boosting schedule strategy for generating optimal immunogenicity is an area of active research. We compared the immune responses resulting from different schedules for prime–boost and boosting either ipsilaterally or contralaterally in relation to the initial vaccine dose. Methods: Influenza hemagglutinin (HA) was used as a model antigen for different vaccination regimens in mice using both mRNA lipid nanoparticles (mRNA-LNP) and AF03-adjuvanted recombinant protein (rHA-AF03) vaccines. Results: Increasing the prime–boost interval resulted in higher levels of serum anti-HA IgG and functional antibody hemagglutination inhibition (HAI) responses in mRNA-LNP-vaccinated animals, which correlated with an induction of germinal center (GC) B cells and follicular helper T (Tfh) cells in lymph nodes. In addition, longer prime–boost intervals resulted in higher levels of IL-2 and TNF-α producing CD4+ T cells two weeks after boosting. The number of Ig-secreting long-lived plasma cells increased with the length of prime–boost intervals. Contralateral boosting resulted in an increase in HAI titers and GC B cells compared to an ipsilateral boost. However, significantly higher numbers of GC B cells were induced in the draining lymph nodes following ipsilateral boosting than in the non-draining lymph nodes. Conclusions: Overall, our data provides insights into the immune mechanisms of action of mRNA-LNP to develop the optimal vaccine regimen for mRNA vaccine platforms.

Lipid nanoparticle (LNP) components can impact the safety and immunogenicity of mRNA vaccines. Here we examine the mechanisms contributing to the performance of mRNA-LNP vaccines by exploring the impact of nucleoside modifications and LNP components on translational efficiency, innate immune activation, and immunogenicity. Our data reveals several molecular and immunological parameters affected by nucleoside modification including a synergistic effect of the mRNA and ionizable lipid composition on the immune activation triggered by the mRNA-LNP formulation. Our results indicate changes in the LNP composition, independent from whether the mRNA is modified or unmodified, caused differential expression of genes associated with innate and antiviral immunity. We believe these findings offer valuable insights into mRNA vaccine function and offer strategies for enhancing vaccine efficacy and reducing the reactogenicity of next generation mRNA vaccines.

Background Dynein axonemal intermediate chain 1 protein (DNAI1) plays an essential role in cilia structure and function, while its mutations lead to primary ciliary dyskinesia (PCD). Accurate quantitation of DNAI1 in lung tissue is crucial for comprehensive understanding of its involvement in PCD, as well as for developing the potential PCD therapies. However, the current protein quantitation method is not sensitive enough to detect the endogenous level of DNAI1 in complex biological matrix such as lung tissue. Methods In this study, a quantitative method combining immunoprecipitation with nanoLC-MS/MS was developed to measure the expression level of human wild-type (WT) DNAI1 protein in lung tissue. To our understanding, it is the first immunoprecipitation (IP)-MS based method for absolute quantitation of DNAI1 protein in lung tissue. The DNAI1 quantitation was achieved through constructing a standard curve with recombinant human WT DNAI1 protein spiked into lung tissue matrix. Results This method was qualified with high sensitivity and accuracy. The lower limit of quantitation of human DNAI1 was 4 pg/mg tissue. This assay was successfully applied to determine the endogenous level of WT DNAI1 in human lung tissue. Conclusions The results clearly demonstrate that the developed assay can accurately quantitate low-abundance WT DNAI1 protein in human lung tissue with high sensitivity, indicating its high potential use in the drug development for DNAI1 mutation-caused PCD therapy.

•No breadth against VoC attained with heterologous sequences in primary vaccination.•Boosting with original vaccine results in breadth against VoC and SARS-CoV-1.•Neutralizing breadth after boost is independent of vaccine sequence or modality.•Pan-corona neutralizing effect is achieved with third dose of the original vaccine. The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500β, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited T H 1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with T H 2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.

mRNA technologies have the potential to transform areas of medicine, including the prophylaxis of infectious diseases. The advantages for vaccines range from the acceleration of immunogen discovery to rapid response and multiple disease target manufacturing. A greater understanding of quality attributes that dictate translation efficiency, as well as a comprehensive appreciation of the importance of mRNA delivery, are influencing a new era of investment in development activities. The application of translational sciences and growing early-phase clinical experience continue to inform candidate vaccine selection. Here we review the state of the art for the prevention of infectious diseases by using mRNA and pertinent topics to the biotechnology and pharmaceutical industries.