mRNA vaccine development and applications: A special focus on tumors (Review).

Cancer is characterized by unlimited proliferation and metastasis, and traditional therapeutic strategies usually result in the acquisition of drug resistance, thus highlighting the need for more personalized treatment. mRNA vaccines transfer the gene sequences of exogenous target antigens into human cells through transcription and translation to stimulate the body to produce specific immune responses against the encoded proteins, so as to enable the body to obtain immune protection against said antigens; this approach may be adopted for personalized cancer therapy. Since the recent coronavirus pandemic, the development of mRNA vaccines has seen substantial progress and widespread adoption. In the present review, the development of mRNA vaccines, their mechanisms of action, factors influencing their function and the current clinical applications of the vaccine are discussed. A focus is placed on the application of mRNA vaccines in cancer, with the aim of highlighting unique advances and the remaining challenges of this novel and promising therapeutic approach.

Enhancing cross-protection against influenza by heterologous sequential immunization with mRNA LNP and protein nanoparticle vaccines.

Enhancing influenza vaccine cross-protection is imperative to alleviate the significant public health burden of influenza. Heterologous sequential immunization may synergize diverse vaccine formulations and routes to improve vaccine potency and breadth. Here we investigate the effects of immunization strategies on the generation of cross-protective immune responses in female Balb/c mice, utilizing mRNA lipid nanoparticle (LNP) and protein-based PHC nanoparticle vaccines targeting influenza hemagglutinin. Our findings emphasize the crucial role of priming vaccination in shaping Th bias and immunodominance hierarchies. mRNA LNP prime favors Th1-leaning responses, while PHC prime elicits Th2-skewing responses. We demonstrate that cellular and mucosal immune responses are pivotal correlates of cross-protection against influenza. Notably, intranasal PHC immunization outperforms its intramuscular counterpart in inducing mucosal immunity and conferring cross-protection. Sequential mRNA LNP prime and intranasal PHC boost demonstrate optimal cross-protection against antigenically drifted and shifted influenza strains. Our study offers valuable insights into tailoring immunization strategies to optimize influenza vaccine effectiveness.

An mRNA vaccine for pancreatic cancer designed by applying in silico immunoinformatics and reverse vaccinology approaches.

Pancreatic ductal adenocarcinoma is the most prevalent pancreatic cancer, which is considered a significant global health concern. Chemotherapy and surgery are the mainstays of current pancreatic cancer treatments; however, a few cases are suitable for surgery, and most of the cases will experience recurrent episodes. Compared to DNA or peptide vaccines, mRNA vaccines for pancreatic cancer have more promise because of their delivery, enhanced immune responses, and lower proneness to mutation. We constructed an mRNA vaccine by analyzing S100 family proteins, which are all major activators of receptors for advanced glycation end products. We applied immunoinformatic approaches, including physicochemical properties analysis, structural prediction and validation, molecular docking study, in silico cloning, and immune simulations. The designed mRNA vaccine was estimated to have a molecular weight of 165023.50 Da and was highly soluble (grand average of hydropathicity of -0.440). In the structural assessment, the vaccine seemed to be a well-stable and functioning protein (Z score of -8.94). Also, the docking analysis suggested that the vaccine had a high affinity for TLR-2 and TLR-4 receptors. Additionally, the molecular mechanics with generalized Born and surface area solvation analysis of the "Vaccine-TLR-2" (-141.07 kcal/mol) and "Vaccine-TLR-4" (-271.72 kcal/mol) complexes also suggests a strong binding affinity for the receptors. Codon optimization also provided a high expression level with a GC content of 47.04% and a codon adaptation index score 1.0. The appearance of memory B-cells and T-cells was also observed over a while, with an increased level of helper T-cells and immunoglobulins (IgM and IgG). Moreover, the minimum free energy of the mRNA vaccine was predicted at -1760.00 kcal/mol, indicating the stability of the vaccine following its entry, transcription, and expression. This hypothetical vaccine offers a groundbreaking tool for future research and therapeutic development of pancreatic cancer.

Potential of mRNA-based vaccines for the control of tick-borne pathogens in one health perspective.

The One Health approach, which integrates the health of humans, animals, plants, and ecosystems at various levels, is crucial for addressing interconnected health threats. This is complemented by the advent of mRNA vaccines, which have revolutionized disease prevention. They offer broad-spectrum effectiveness and can be rapidly customized to target specific pathogens. Their utility extends beyond human medicine, showing potential in veterinary practices to control diseases and reduce the risk of zoonotic transmissions. This review place mRNA vaccines and One Health in the context of tick-borne diseases. The potential of these vaccines to confer cross-species immunity is significant, potentially disrupting zoonotic disease transmission cycles and protecting the health of both humans and animals, while reducing tick populations, infestations and circulation of pathogens. The development and application of mRNA vaccines for tick and tick-borne pathogens represent a comprehensive strategy in global health, fostering a healthier ecosystem for all species in our interconnected world.

Delivery of Experimental mRNA Vaccine Encoding the RBD of SARS-CoV-2 by Jet Injection.

We studied a needle-free jet injection delivery of an experimental mRNA vaccine encoding the receptor-binding domain of the SARS-CoV-2 S protein (mRNA-RBD). Immunization of BALB/c mice with mRNA-RBD by a needle-free jet injector induced high levels of antibodies with virus-neutralizing activity and a virus-specific T-cell response. The immune response was low in the group of mice that received intramuscular injection of mRNA-RBD. The effectiveness of this simple and safe method of mRNA delivering has been demonstrated. Thus, jet injection of mRNA vaccine can be a good alternative to lipid nanoparticles.

Design and evaluation of mRNA encoding recombinant neutralizing antibodies for botulinum neurotoxin type B intoxication prophylaxis.

Among all natural and synthetic toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botulinum in an anaerobic environment, are the most toxic polymer proteins. Currently, the most effective modalities for botulism prevention and treatment are vaccination and antitoxin use, respectively. However, these modalities are associated with long response time for active immunization, side effects, and donor limitations. As such, the development of more promising botulism prevention and treatment modalities is warranted. Here, we designed an mRNA encoding B9-hFc - a heavy-chain antibody fused to VHH and human Fc that can neutralize BoNT serotype B (BoNT/B) effectively - and assessed its expression in vitro and in vivo. The results confirmed that our mRNA demonstrates good expression in vitro and in vivo. Moreover, a single mRNA lipid nanoparticle injection effectively prevents BoNT/B intoxication in vivo, with effects comparable to those of protein antibodies. In conclusion, we explored and clarified whether mRNA drugs encoding neutralizing antibodies prevent BoNT/B intoxication. Our results provide an efficient strategy for further research on the prevention and treatment of intoxication by botulinum toxin.

Constructing a Ready-to-Use mRNA Vaccine Delivery System for the Prevention of Influenza A virus, Utilizing FDA-Approved Raw Materials.

Messenger ribonucleic acid (mRNA) vaccines, serving as a rapid and easily scalable emergency preventive measure, have played a pivotal role in preventing infectious diseases. The effectiveness of mRNA vaccines heavily relies on the delivery carrier, but the current market options are predominantly lipid nanoparticles. Their intricate preparation process and high transportation costs pose challenges for widespread use in remote areas. In this study, we harnessed FDA-approved polymer PLGA and lipid components widely employed in clinical experiments to craft a ready-to-use mRNA vaccine delivery system known as lipid-polymer hybrid nanoparticles (LPP). Following formulation optimization, the PDCD nanoparticles emerged as the most effective, showcasing exceptional mRNA delivery capabilities both in vitro and in vivo. Loading PDCD nanoparticles with mRNA encoding the H1N1 influenza virus HA antigen-fused M2e peptide enabled the successful induction of M2e-specific antibodies and T cell immune responses in immunized mice. After three rounds of vaccine immunization, the mice demonstrated weight recovery to normal levels and maintained a survival rate exceeding 80% following an encounter with the H1N1 influenza virus. The innovative mRNA delivery system that we designed demonstrates outstanding effectiveness in preventing infectious diseases, with the potential to play an even more significant role in future clinical applications.

Evaluation of the immunogenicity of an mRNA vectored Nipah virus vaccine candidate in pigs.

Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 µg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.

[Application of mRNA nano-delivery system in CAR-T tumor immunotherapy].

Chimeric antigen receptor T cells (CAR-T) immunotherapy, which activates immunity specific to the system in order to achieve antitumor effects, has experienced exciting progress in recent years. mRNA nano-delivery systems, which encapsulate tumor immunotherapy-related antigen mRNA with nanoparticles, have shown great potential in CAR-T tumor immunotherapy. On one hand, these systems can directly target T cells to generate CAR-T cells that directly act upon the corresponding tumor cells. On the other hand, they can be delivered to antigen-presenting cells through targeting, thereby enhancing the function of CAR-T cells and further inducing specific immune responses against tumor cells. This review summarizes the synthesis of mRNA nano-delivery systems and their application in CAR-T tumor immunotherapy.

Development of an mRNA-based therapeutic vaccine mHTV-03E2 for high-risk HPV-related malignancies.

Human papillomavirus (HPV) 16 and 18 infections are related to many human cancers. Despite several preventive vaccines for high-risk (hr) HPVs, there is still an urgent need to develop therapeutic HPV vaccines for targeting pre-existing hrHPV infections and lesions. In this study, we developed a lipid nanoparticle (LNP)-formulated mRNA-based HPV therapeutic vaccine (mHTV)-03E2, simultaneously targeting the E2/E6/E7 of both HPV16 and HPV18. mHTV-03E2 dramatically induced antigen-specific cellular immune responses, leading to significant CD8+ T cell infiltration and cytotoxicity in TC-1 tumors derived from primary lung epithelial cells of C57BL/6 mice expressing HPV E6/E7 antigens, mediated significant tumor regression, and prolonged animal survival, in a dose-dependent manner. We further demonstrated significant T cell immunity against HPV16/18 E6/E7 antigens for up to 4 months post-vaccination in immunological and distant tumor rechallenging experiments, suggesting robust memory T cell immunity against relapse. Finally, mHTV-03E2 synergized with immune checkpoint blockade to inhibit tumor growth and extend animal survival, indicating the potential in combination therapy. We conclude that mHTV-03E2 is an excellent candidate therapeutic mRNA vaccine for treating malignancies caused by HPV16 or HPV18 infections.

Advancing vaccine development: Evaluation of a mannose-modified lipid nanoparticle-based candidate for African swine fever p30 mRNA vaccine eliciting robust immune response in mice.

The African swine fever virus (ASFV) continues to pose significant economic and pandemic risks. Consequently, discovering new, efficient vaccines is crucial. Messenger RNA (mRNA) vaccines have emerged as promising candidates, providing minimal risk of insertional mutagenesis, high safety profiles, effectiveness, rapid scalability in production, and cost-effectiveness. In this study, we have developed an ASF p30 mRNA vaccine candidate (mRNA/Man-LNP) employing mannose-modified lipid nanoparticles (LNPs). The mRNA/Man-LNP exhibited effective antigen presentation and facilitated dendritic cells (DCs) maturation. Notably, it elicited strong IgG titers and activated CD4+ and CD8+ T-cells in immunized mice, all while adhering to stringent biosafety standards. This investigation demonstrates that mRNA/Man-LNP can trigger both humoral and cellular immune responses, suggesting its potential as a potent and promising vaccine candidate for controlling African swine fever (ASF).

Cancer mRNA vaccines: clinical advances and future opportunities.

mRNA vaccines have been revolutionary in terms of their rapid development and prevention of SARS-CoV-2 infections during the COVID-19 pandemic, and this technology has considerable potential for application to the treatment of cancer. Compared with traditional cancer vaccines based on proteins or peptides, mRNA vaccines reconcile the needs for both personalization and commercialization in a manner that is unique to each patient but not beholden to their HLA haplotype. A further advantage of mRNA vaccines is the availability of engineering strategies to improve their stability while retaining immunogenicity, enabling the induction of complementary innate and adaptive immune responses. Thus far, no mRNA-based cancer vaccines have received regulatory approval, although several phase I-II trials have yielded promising results, including in historically poorly immunogenic tumours. Furthermore, many early phase trials testing a wide range of vaccine designs are currently ongoing. In this Review, we describe the advantages of cancer mRNA vaccines and advances in clinical trials using both cell-based and nanoparticle-based delivery methods, with discussions of future combinations and iterations that might optimize the activity of these agents.

Long-term stability and immunogenicity of lipid nanoparticle COVID-19 mRNA vaccine is affected by particle size.

Messenger ribonucleic acid (mRNA) technology has been rapidly applied for the development of the COVID-19 vaccine. However, naked mRNA itself is inherently unstable. Lipid nanoparticles (LNPs) protect mRNAs from extracellular ribonucleases and facilitate mRNA trafficking. For mRNA vaccines, antigen-presenting cells utilize LNPs through uptake to elicit antigen-specific immunity. There are reports on the impact of various physical characteristics of LNPs, particularly those with sizes less than 200 nm, especially 50 to 150 nm, on the overall stability and protective efficacy of mRNA vaccines. To address this, a single change in the size of LNPs using the same mRNA stock solution was assessed for the physicochemical characterization of the resulting mRNA-LNPs vaccine, along with the evaluation of their protective efficacy. Particles of smaller sizes generally disperse more effectively in solutions, with minimized occurrence of particle precipitation and aggregation. Here, we demonstrate that the vaccine containing 80-100 nm mRNA-LNPs showed the best stability and protection at 4°C and -20°C. Furthermore, we can conclude that freezing the vaccine at -20°C is more appropriate for maintaining stability over the long term. This effort is poised to provide a scientific basis for improving the quality of ongoing mRNA vaccine endeavors and providing information on the development of novel products.

An mRNA-LNP-based Lassa virus vaccine induces protective immunity in mice.

The mammarenavirus Lassa virus (LASV) causes the life-threatening hemorrhagic fever disease, Lassa fever. The lack of licensed medical countermeasures against LASV underscores the urgent need for the development of novel LASV vaccines, which has been hampered by the requirement for a biosafety level 4 facility to handle live LASV. Here, we investigated the efficacy of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of the prototypic mammarenavirus, lymphocytic choriomeningitis virus (LCMV), in mice. Two doses of LASgpc- or LCMnp-mRNA-LNP administered intravenously (i.v.) protected C57BL/6 mice from a lethal challenge with a recombinant (r) LCMV expressing a modified LASgpc (rLCMV/LASgpc2m) inoculated intracranially. Intramuscular (i.m.) immunization with two doses of LASgpc- or LCMnp-mRNA-LNP significantly reduced the viral load in C57BL/6 mice inoculated i.v. with rLCMV/LASgpc2m. High levels of viremia and lethality were observed in CBA mice inoculated i.v. with rLCMV/LASgpc2m, which were abrogated by i.m. immunization with two doses of LASgpc-mRNA-LNP. The protective efficacy of two i.m. doses of LCMnp-mRNA-LNP was confirmed in a lethal hemorrhagic disease model of FVB mice i.v. inoculated with wild-type rLCMV. In all conditions tested, negligible and high levels of LASgpc- and LCMnp-specific antibodies were detected in mRNA-LNP-immunized mice, respectively, but robust LASgpc- and LCMnp-specific CD8+ T cell responses were induced. Accordingly, plasma from LASgpc-mRNA-LNP-immunized mice did not exhibit neutralizing activity. Our findings and surrogate mouse models of LASV infection, which can be studied at a reduced biocontainment level, provide a critical foundation for the rapid development of mRNA-LNP-based LASV vaccines.IMPORTANCELassa virus (LASV) is a highly pathogenic mammarenavirus responsible for several hundred thousand infections annually in West African countries, causing a high number of lethal Lassa fever (LF) cases. Despite its significant impact on human health, clinically approved, safe, and effective medical countermeasures against LF are not available. The requirement of a biosafety level 4 facility to handle live LASV has been one of the main obstacles to the research and development of LASV countermeasures. Here, we report that two doses of mRNA-lipid nanoparticle-based vaccines expressing the LASV glycoprotein precursor (LASgpc) or nucleoprotein (LCMnp) of lymphocytic choriomeningitis virus (LCMV), a mammarenavirus genetically closely related to LASV, conferred protection to recombinant LCMV-based surrogate mouse models of lethal LASV infection. Notably, robust LASgpc- and LCMnp-specific CD8+ T cell responses were detected in mRNA-LNP-immunized mice, whereas no virus-neutralizing activity was observed.

Polyvalent mpox mRNA vaccines elicit robust immune responses and confer potent protection against vaccinia virus.

The 2022 mpox outbreak led the World Health Organization (WHO) to declare it a public health emergency of international concern (PHEIC). There is a need to develop more effective and safer mpox virus (MPXV)-specific vaccines in response to the mpox epidemic. The mRNA vaccine is a promising platform to protect against MPXV infection. In this study, we construct two bivalent MPXV mRNA vaccines, designated LBA (B6R-A29L) and LAM (A35R-M1R), and a quadrivalent mRNA vaccine, LBAAM (B6R-A35R-A29L-M1R). The immunogenicity and protective efficacy of these vaccines alone or in combination were evaluated in a lethal mouse model. All mRNA vaccine candidates could elicit potential antigen-specific humoral and cellular immune responses and provide protection against vaccinia virus (VACV) infection. The protective effect of the combination of two bivalent mRNA vaccines and the quadrivalent vaccine was superior to that of the individual bivalent mRNA vaccine. Our study provides valuable insights for the development of more efficient and safer mRNA vaccines against mpox.

mRNA vaccines against infectious diseases and future direction.

Vaccines are used for the control of infectious diseases of animals. Over other types of vaccinations like live attenuated or killed vaccines, mRNA-based vaccines have significant advantages. As only a small portion of the pathogen's genetic material is employed and the dose rate of mRNA-based vaccines is low, there is the least possibility that the pathogen will reverse itself. A carrier or vehicle that shields mRNA-based vaccines from the host's cellular RNases is necessary for their delivery. mRNA vaccines have been shown to be effective and to induce both a cell-mediated immune response and a humoral immune response in clinical trials against various infectious diseases (viral and parasitic) affecting the animals, including rabies, foot and mouth disease, toxoplasmosis, Zikavirus, leishmaniasis, and COVID-19. The current review aims to highlight the use of mRNA-based vaccines both in viral and parasitic diseases of animals.

mRNA vaccines contribute to innate and adaptive immunity to enhance immune response in vivo.

Messenger RNA (mRNA) therapeutics have been widely employed as strategies for the treatment and prevention of diseases. Amid the global outbreak of COVID-19, mRNA vaccines have witnessed rapid development. Generally, in the case of mRNA vaccines, the initiation of the innate immune system serves as a prerequisite for triggering subsequent adaptive immune responses. Critical cells, cytokines, and chemokines within the innate immune system play crucial and beneficial roles in coordinating tailored immune reactions towards mRNA vaccines. Furthermore, immunostimulators and delivery systems play a significant role in augmenting the immune potency of mRNA vaccines. In this comprehensive review, we systematically delineate the latest advancements in mRNA vaccine research, present an in-depth exploration of strategies aimed at amplifying the immune effectiveness of mRNA vaccines, and offer some perspectives and recommendations regarding the future advancements in mRNA vaccine development.

Development of a nucleoside-modified mRNA vaccine against clade 2.3.4.4b H5 highly pathogenic avian influenza virus.

mRNA lipid nanoparticle (LNP) vaccines would be useful during an influenza virus pandemic since they can be produced rapidly and do not require the generation of egg-adapted vaccine seed stocks. Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. Here, we generate an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. The H5 mRNA-LNP vaccine elicits strong T cell and antibody responses in female mice, including neutralizing antibodies and broadly-reactive anti-HA stalk antibodies. The H5 mRNA-LNP vaccine elicits antibodies at similar levels compared to whole inactivated vaccines in female mice with and without prior H1N1 exposures. Finally, we find that the H5 mRNA-LNP vaccine is immunogenic in male ferrets and prevents morbidity and mortality of animals following 2.3.4.4b H5N1 challenge. Together, our data demonstrate that a monovalent mRNA-LNP vaccine expressing 2.3.4.4b H5 is immunogenic and protective in pre-clinical animal models.

Sequence-Optimized mRNA Vaccines Against Infectious Disease.

Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA vaccines (RNActive® technology) with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by formulation into lipid nanoparticles. Methods described here include the synthesis, purification, and formulation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.

Production and Evaluation of Nucleoside-Modified mRNA Vaccines for Infectious Diseases.

Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have demonstrated potency in multiple preclinical models against various pathogens and have recently received considerable attention due to the success of the two safe and effective COVID-19 mRNA vaccines developed by Moderna and Pfizer-BioNTech. The use of nucleoside modification in mRNA vaccines seems to be critical to achieve a sufficient level of safety and immunogenicity in humans, as illustrated by the results of clinical trials using either nucleoside-modified or unmodified mRNA-based vaccine platforms. It is well documented that the incorporation of modified nucleosides in the mRNA and stringent mRNA purification after in vitro transcription render it less inflammatory and highly translatable; these two features are likely key for mRNA vaccine safety and potency. Formulation of the mRNA into LNPs is important because LNPs protect mRNA from rapid degradation, enabling efficient delivery and high levels of protein production for extended periods of time. Additionally, recent studies have provided evidence that certain LNPs with ionizable cationic lipids (iLNPs) possess adjuvant activity that fosters the induction of strong humoral and cellular immune responses by mRNA-iLNP vaccines.In this chapter we describe the production of iLNP-encapsulated, nucleoside-modified, and purified mRNA and the evaluation of antigen-specific T cell and antibody responses elicited by this vaccine form.