Nucleoside-modified mRNA Research Articles

Effect of The Pfizer-Biontech Vaccine on Ifn-Γ Serum Levels and its Genetic Variations in Response to Vaccination

Background: Pfizer vaccine, which is a nucleoside-modified mRNA vaccine against coronavirus disease 2019 (COVID-19) is one of the first mRNA products to be approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA). Aim of the study: The aim of this study is to study the impact of Pfizer vaccine on the levels of interferon gamma (IFN-γ) serum levels and its gene polymorphism after 3 months and 6 months of vaccination with Pfizer vaccine . Materials and methods: This case-control study involved (150) subjects who were divided into three groups (each group included 50 subjects). The first group included (50) vaccinated group 3 months after second dose of Pfizer vaccine, and the second group included (50) vaccinated group 6 month after second dose of Pfizer vaccine. The third group was the control group which included (50) healthy unvaccinated subjects. The study was done during the period from May /2022 to September / 2022). Serum levels of IFN-γ were estimated by ELISA technique. The PCR technique was used to study gene polymorphism for IFN-γ. Results: The results in figure (1) showed that the mean levels of INF-γ among the male participants (after 3 months of vaccination, after 6 months of vaccinations, and the control group) were (292.54, 231.48, and 15.71), respectively, with highly significant differences (HS) between the three groups (p<0.01). The results also showed that the mean levels of INF-γ among female participants (after 3 months of vaccination, after 6 months of vaccinations, and the control group) were (317.31, 228.14, and 15.74), respectively, with highly significant differences (HS) between the three groups (p<0.01). Analysis of rs2430561 SNP of (IFN-γ) gene using Sanger sequencing. Single “T” peak indicative of a T homozygous allele showed that Single “A” peak indicative of a A homozygous allele. Presence of the “T” and “A” peak indicative of T/A heterozygous allele. Conclusion: Pfizer vaccine was shown to have significant impacts on IFN-γ levels and presence of single T/A alleles is indicative of heterogeneous T/A allele on analysis of rs2430561 SNP of (IFN-γ) gene among vaccinated individuals.

MRNA vaccines for infectious diseases - advances, challenges and opportunities.

The concept of mRNA-based vaccines emerged more than three decades ago. Groundbreaking discoveries and technological advancements over the past 20 years have resolved the major roadblocks that initially delayed application of this new vaccine modality. The rapid development of nucleoside-modified COVID-19 mRNA vaccines demonstrated that this immunization platform is easy to develop, has an acceptable safety profile and can be produced at a large scale. The flexibility and ease of antigen design have enabled mRNA vaccines to enter development for a wide range of viruses as well as for various bacteria and parasites. However, gaps in our knowledge limit the development of next-generation mRNA vaccines with increased potency and safety. A deeper understanding of the mechanisms of action of mRNA vaccines, application of novel technologies enabling rational antigen design, andinnovative vaccine delivery strategies and vaccination regimens will likely yield potent novel vaccines against a wide range of pathogens.

Comparison of the immunogenicity of mRNA-encoded and protein HIV-1 Env-ferritin nanoparticle designs.

Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env-ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env-ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose in vivo and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env-NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression in vitro and immunogenicity at low doses in vivo are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.IMPORTANCEAn effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher in vivo protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.

Broad protection and respiratory immunity of dual mRNA vaccination against SARS-CoV-2 variants

While first-generation, spike (S)-based COVID-19 vaccines were effective against early SARS-CoV-2 strains, the rapid evolution of novel Omicron subvariants have substantially reduced vaccine efficacy. As such, broadly protective vaccines against SARS-CoV-2 are needed to prevent future viral emergence. In addition, it remains less clear whether peripheral immunization, especially with mRNA vaccines, elicits effective respiratory immunity. Our group has developed a nucleoside-modified mRNA vaccine expressing the nucleocapsid (N) protein of the ancestral SARS-CoV-2 virus and has tested its use in combination with the S-based mRNA vaccine (mRNA-S). In this study, we examined efficacy of mRNA-N alone or in combination with mRNA-S (mRNA-S+N) against more immune evasive Omicron variants in hamsters. Our data show that mRNA-N alone induces a modest but significant protection against BA.5 and that dual mRNA-S+N vaccination confers complete protection against both BA.5 and BQ.1, preventing detection of virus in the hamster lungs. Analysis of respiratory immune response in mice shows that intramuscular mRNA-S+N immunization effectively induces respiratory S- and N-specific T cell responses in the lungs and in bronchoalveolar lavage (BAL), as well as antigen-specific binding IgG in BAL. Together, our data further support mRNA-S+N as a potential pan-COVID-19 vaccine for broad protection against current and emerging SARS-CoV-2 variants.

A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice

ABSTRACT Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Transient growth factor expression via mRNA in lipid nanoparticles promotes hepatocyte cell therapy in mice

Primary human hepatocyte (PHH) transplantation is a promising alternative to liver transplantation, whereby liver function could be restored by partial repopulation of the diseased organ with healthy cells. However, currently PHH engraftment efficiency is low and benefits are not maintained long-term. Here we refine two male mouse models of human chronic and acute liver diseases to recapitulate compromised hepatocyte proliferation observed in nearly all human liver diseases by overexpression of p21 in hepatocytes. In these clinically relevant contexts, we demonstrate that transient, yet robust expression of human hepatocyte growth factor and epidermal growth factor in the liver via nucleoside-modified mRNA in lipid nanoparticles, whose safety was validated with mRNA-based COVID-19 vaccines, drastically improves PHH engraftment, reduces disease burden, and improves overall liver function. This strategy may overcome the critical barriers to clinical translation of cell therapies with primary or stem cell-derived hepatocytes for the treatment of liver diseases.

High-Throughput In Vivo Screening Identifies Differential Influences on mRNA Lipid Nanoparticle Immune Cell Delivery by Administration Route.

Immune modulation through the intracellular delivery of nucleoside-modified mRNA to immune cells is an attractive approach for in vivo immunoengineering, with applications in infectious disease, cancer immunotherapy, and beyond. Lipid nanoparticles (LNPs) have come to the fore as a promising nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process. In this study, we employed high-throughput in vivo LNP screening based on molecular barcoding to investigate the influence of LNP composition on immune tropism with applications in vaccines and systemic immunotherapies. Screening a large LNP library under both intramuscular (i.m.) and intravenous (i.v.) injection, we observed differential influences on LNP uptake by immune populations across the two administration routes, gleaning insight into LNP design criteria for in vivo immunoengineering. In validation studies, the lead LNP formulation for i.m. administration demonstrated substantial mRNA translation in the spleen and draining lymph nodes with a more favorable biodistribution profile than LNPs formulated with the clinical standard ionizable lipid DLin-MC3-DMA (MC3). The lead LNP formulations for i.v. administration displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes. Altogether, the immunotropic LNPs identified by high-throughput in vivo screening demonstrated significant promise for both locally- and systemically-delivered mRNA and confirmed the value of the LNP design criteria gleaned from our screening process, which could potentially inform future endeavors in mRNA vaccine and immunotherapy applications.

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.

Synthetic BZLF1-targeted transcriptional activator for efficient lytic induction therapy against EBV-associated epithelial cancers

The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.

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.

The mRNACalc webserver accounts for the N1-methylpseudouridine hypochromicity to enable precise nucleoside-modified mRNA quantification

Nucleoside-modified messenger RNA (mRNA) technologies necessarily incorporate N1-methylpseudouridine into the mRNA molecules to prevent the over-stimulation of cytoplasmic RNA sensors. Despite this modification, mRNA concentrations remain mostly determined through the measurement of UV absorbance at 260nm wavelength (A260). Herein, we report that the N1-methylpseudouridine absorbs approximately 40% less UV light at 260nm than uridine, and its incorporation into mRNAs leads to the under-estimation of nucleoside-modified mRNA concentrations, with 5%-15% error, in an mRNA-sequence-dependent manner. We therefore examined the RNA quantification methods and developed the mRNACalc webserver. It accounts for the molar absorption coefficient of modified nucleotides at 260nm wavelength, the RNA composition of the mRNA, and the A260 of the mRNA sample to enable accurate quantification of nucleoside-modified mRNAs.

A mRNA Vaccine for Crimean-Congo Hemorrhagic Fever Virus Expressing Non-Fusion GnGc Using NSm Linker Elicits Unexpected Immune Responses in Mice.

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo Hemorrhagic virus (CCHFV), is listed in the World Health Organization's list of priority diseases. The high fatality rate in humans, the widespread distribution of CCHFV, and the lack of approved specific vaccines are the primary concerns regarding this disease. We used microfluidic technology to optimize the mRNA vaccine delivery system and demonstrated that vaccination with nucleoside-modified CCHFV mRNA vaccines encoding GnNSmGc (vLMs), Gn (vLMn), or Gc (vLMc) induced different immune responses. We found that both T-cell and B-cell immune responses induced by vLMc were better than those induced by vLMn. Interestingly, immune responses were found to be lower for vLMs, which employed NSm to link Gn and Gc for non-fusion expression, compared to those for vLMc. In conclusion, our results indicated that NSm could be a factor that leads to decreased specific immune responses in the host and should be avoided in the development of CCHFV vaccine antigens.

Safety, Immunogenicity, and Mechanism of a Rotavirus mRNA-LNP Vaccine in Mice.

Rotaviruses (RVs) are a major cause of diarrhea in young children worldwide. The currently available and licensed vaccines contain live attenuated RVs. Optimization of live attenuated RV vaccines or developing non-replicating RV (e.g., mRNA) vaccines is crucial for reducing the morbidity and mortality from RV infections. Herein, a nucleoside-modified mRNA vaccine encapsulated in lipid nanoparticles (LNP) and encoding the VP7 protein from the G1 type of RV was developed. The 5' untranslated region of an isolated human RV was utilized for the mRNA vaccine. After undergoing quality inspection, the VP7-mRNA vaccine was injected by subcutaneous or intramuscular routes into mice. Mice received three injections in 21 d intervals. IgG antibodies, neutralizing antibodies, cellular immunity, and gene expression from peripheral blood mononuclear cells were evaluated. Significant differences in levels of IgG antibodies were not observed in groups with adjuvant but were observed in groups without adjuvant. The vaccine without adjuvant induced the highest antibody titers after intramuscular injection. The vaccine elicited a potent antiviral immune response characterized by antiviral clusters of differentiation CD8+ T cells. VP7-mRNA induced interferon-γ secretion to mediate cellular immune responses. Chemokine-mediated signaling pathways and immune response were activated by VP7-mRNA vaccine injection. The mRNA LNP vaccine will require testing for protective efficacy, and it is an option for preventing rotavirus infection.

Trans-Amplifying RNA Vaccines Against Infectious Diseases: A Comparison with Non-Replicating and Self-Amplifying RNA.

The recent COVID-19 pandemic as well as other past and recent outbreaks of newly or re-emerging viruses show the urgent need to develop potent new vaccine approaches, that enable a quick response to prevent global spread of infectious diseases. The breakthrough of first messenger RNA (mRNA)-based vaccines 2019 approved only months after identification of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), opens a big new field for vaccine engineering. Currently, two major types of mRNA are being pursued as vaccines for the prevention of infectious diseases. One is non-replicating mRNA, including nucleoside-modified mRNA, used in the current COVID-19 vaccines of Moderna and BioNTech (Sahin et al., Nat Rev Drug Discov 13(10):759-780, 2014; Baden et al., N Engl J Med 384(5):403-416, 2021; Polack et al., N Engl J Med 383(27):2603-2615, 2020), the other is self-amplifying RNA (saRNA) derived from RNA viruses. Recently, trans-amplifying RNA, a split vector system, has been described as a third class of mRNA (Spuul et al., J Virol 85(10):4739-4751, 2011; Blakney et al., Front Mol Biosci 5:71, 2018; Beissert et al., Mol Ther 28(1):119-128, 2020). In this chapter we review the different types of mRNA currently used for vaccine development with focus on trans-amplifying RNA.

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.

A Pvs25 mRNA vaccine induces complete and durable transmission-blocking immunity to Plasmodium vivax

Plasmodium vivax (P. vivax) is the major malaria parasite outside of Africa and no vaccine is available against it. A vaccine that interrupts parasite transmission (transmission-blocking vaccine, TBV) is considered highly desirable to reduce the spread of P. vivax and to accelerate its elimination. However, the development of a TBV against this pathogen has been hampered by the inability to culture the parasite as well as the low immunogenicity of the vaccines developed to date. Pvs25 is the most advanced TBV antigen candidate for P. vivax. However, in previous phase I clinical trials, TBV vaccines based on Pvs25 yielded low antibody responses or had unacceptable safety profiles. As the nucleoside-modified mRNA–lipid nanoparticle (mRNA–LNP) vaccine platform proved to be safe and effective in humans, we generated and tested mRNA–LNP vaccines encoding several versions of Pvs25 in mice. We found that in a prime-boost vaccination schedule, all Pvs25 mRNA–LNP vaccines elicited robust antigen-specific antibody responses. Furthermore, when compared with a Pvs25 recombinant protein vaccine formulated with Montanide ISA-51 adjuvant, the full-length Pvs25 mRNA–LNP vaccine induced a stronger and longer-lasting functional immunity. Seven months after the second vaccination, vaccine-induced antibodies retained the ability to fully block P. vivax transmission in direct membrane feeding assays, whereas the blocking activity induced by the protein/ISA-51 vaccine dropped significantly. Taken together, we report on mRNA vaccines targeting P. vivax and demonstrate that Pvs25 mRNA–LNP outperformed an adjuvanted Pvs25 protein vaccine suggesting that it is a promising candidate for further testing in non-human primates.

VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial-cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via nonintegrative and safe nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNPs) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and elimination of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This work defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals unexpected therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.

Cationic LNP-formulated mRNA expressing Tie2-agonist in the lung endothelium prevents pulmonary vascular leakage

Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies invitro and invivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model.

Effect of mRNA-LNP components of two globally-marketed COVID-19 vaccines on efficacy and stability

During the COVID-19 pandemic, Pfizer-BioNTech and Moderna successfully developed nucleoside-modified mRNA lipid nanoparticle (LNP) vaccines. SARS-CoV-2 spike protein expressed by those vaccines are identical in amino acid sequence, but several key components are distinct. Here, we compared the effect of ionizable lipids, untranslated regions (UTRs), and nucleotide composition of the two vaccines, focusing on mRNA delivery, antibody generation, and long-term stability. We found that the ionizable lipid, SM-102, in Moderna’s vaccine performs better than ALC-0315 in Pfizer-BioNTech’s vaccine for intramuscular delivery of mRNA and antibody production in mice and long-term stability at 4 °C. Moreover, Pfizer-BioNTech’s 5′ UTR and Moderna’s 3′ UTR outperform their counterparts in their contribution to transgene expression in mice. We further found that varying N1-methylpseudouridine content at the wobble position of mRNA has little effect on vaccine efficacy. These findings may contribute to the further improvement of nucleoside-modified mRNA-LNP vaccines and therapeutics.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.