Search Results (40)

Background/Objectives: It is challenging to develop efficient vaccines against intracellular pathogens such as viruses, and since viral infections are one of the main challenges for farmed salmon, a novel vaccine strategy is needed. mRNA vaccines are optimized and approved for humans, but for fish, the mRNA technology is new, and optimization is required to ensure efficient protein expression. We made an mRNA tailored to salmon and studied the effect of modified nucleosides and the length of the poly(A) tail on protein expression from in vitro-transcribed mRNA in CHSE-214 cells, using enhanced green fluorescent protein (EGFP) as a reporter. Methods: Different lengths of the poly(A) tail were tested, and various modified nucleotides were incorporated in the mRNA during in vitro transcription, including pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), N6-methyladenosine (m6A), 5-methyluridine (m5U), and 5-methylcytidine (m5C). Protein expression was observed in fluorescence microscopy and quantified using flow cytometry. Results: mRNA containing Ψ resulted in the strongest EGFP expression 1–3 days post-transfection (dpt), while EGFP expression from m5C mRNA was high throughout the experiment (<10 dpt). m5U-containing mRNA had low EGFP expression until 6 dpt, but reached the level of m5C mRNA at 10 dpt. The m5U mRNA, however, expressed EGFP at much higher intensity than all the other mRNAs at all time points. Poly(A) tails with lengths of 40, 100, and >100 were tested, and the one with >100 adenines showed the highest expression. The effects of phosphatase treatment and purification of the mRNA were also investigated. Furthermore, EGFP expression was observed in yolk-sac salmon larvae following micro-injection. Conclusions: Our study provides an important basis for the development of efficient mRNA-based vaccines in the future. Full article

While current influenza vaccines often lack broad protection against antigenically drifted strains, some modified hemagglutinin (HA) protein antigens have shown promise in eliciting broadly neutralizing antibodies against conserved epitopes. During infection, the mildly acidic environment of the late endosome triggers irreversible HA conformational changes resulting in a post-fusion structure with altered antigenicity. While enhancing the stability of other structural class I viral fusion protein antigens has been instrumental in improving the effectiveness of COVID-19 and RSV vaccines, the role of HA stability in influenza vaccine immunogenicity is relatively unclear. Here, we used the nucleoside-modified mRNA-LNP platform to test engineered HA antigens with specific acid-stabilizing mutations (E47K, K58I, R106K, and K153E) in the HA stalk. All mutations increased HA acid stability, but E47K and R106K did not increase immunogenicity. K153E and K58I, but not E47K and R106K, enhanced the cell-surface expression of the HA protein in vitro. In mice, K153E- and K58I-containing mRNA-LNP vaccines elicited increased neutralizing antibody titers against homologous virus. K153E conferred greater protection than wild-type vaccine against lethal heterologous A/PR/8/34 challenge at low doses (0.5–1.0 µg), despite the absence of neutralizing antibodies against the challenge strain. K153E also elicited greater expansion of antigen-specific antibody-secreting cells (ASCs) in the bone marrow, as well as cross-reactive T follicular helper (Tfh) cells in the spleen. For the vaccines studied, increased HA expression was a stronger correlate of mRNA-LNP enhancement than increased HA stability. Full article

Background/Objectives: Pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans. Machupo virus (MACV), a New World (NW) mammarenavirus, causes Bolivian hemorrhagic fever in humans, and there are no approved vaccines. Methods: Here, we describe and compare the immunogenicity of three vaccines expressing the MACV glycoprotein complex (GPC) in C57BL/6 mice: a recombinant vesicular stomatitis virus (rVSV) and two different lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA (mRNA-LNP) vaccines. The first mRNA-LNP vaccine, designated MACV mRNA, expresses the full-length MACV GPC. The second mRNA-LNP vaccine, called MACV VLP mRNA, encodes MACV GPC with appended sequences that induce the budding of virus-like particles (VLPs) with MACV GPC on the surface. This is the first description of any mRNA-LNP vaccine for MACV and the first comparison of mRNA and rVSVs as vaccine candidates for MACV. Results: We find that two doses of either MACV mRNA or MACV VLP mRNA are required for the induction of robust humoral and cellular immune responses including total MACV GPC IgG, neutralizing antibodies, cross-reactive antibodies that bind the related Junín virus GPC, and MACV-specific T-cell responses. To further investigate vaccination strategies for MACV, we also evaluated a heterologous prime-boost regimen involving the MACV mRNA vaccine coupled with the rVSV-based MACV vaccine. We find that the highest levels of MACV GPC-specific IgG and neutralizing titers were achieved when heterologous mRNA and rVSV prime-boost regimens were employed. Conclusions: These results elucidate differences in the immune response to different vaccine platforms for MACV and can inform future vaccine development for NW arenaviruses. Full article

Background/Objectives: Eye infection with herpes simplex virus type 1 (HSV-1) can result in keratitis, a leading cause of corneal blindness. We evaluated whether an experimental vaccine containing HSV-2 immunogens to prevent genital herpes also protects against HSV-1 eye infection and neuroinvasion. Methods: Mice were immunized twice, one month apart, with PBS or a nucleoside-modified lipid nanoparticle vaccine containing mRNA encoding for gC2, gD2, and gE2. One month later, 10⁶ plaque forming units (PFU) (10 lethal dose 50, LD₅₀) of the HSV-1 McKrae strain were added to the intact cornea of each eye. Results: The vaccine prevented death and markedly reduced eyelid and attached conjunctival inflammation (blepharoconjunctivitis) and weight loss compared with the PBS group. Tissues from the ocular conjunctiva and eye bulb, olfactory bulb/peduncle, trigeminal ganglia, and brain (brainstem, cerebrum, and cerebellum) were harvested 5 days post-infection from 5 mice each in the PBS and vaccine groups, and from another 10 mice in the vaccine group 7 weeks post-infection. At 5 days, HSV-1 was not detected in any tissue in the vaccine group, while viral titers were positive in 16 of 25 (64%), and HSV-1 DNA was detected in 22 of 25 (88%) individual tissues in the PBS group. Histopathological and immunohistochemical analysis at 5 days post-infection confirmed that the vaccine protected against inflammation; however, some animals experienced breakthrough blepharoconjunctivitis. At 7 weeks, 3 of 10 (30%) mice in the vaccine group had HSV-1 DNA detected in the eyes or trigeminal ganglia tissues, but no animal had HSV-1 DNA detected in brain tissues. The vaccine produced cross-reactive HSV-1 neutralizing antibodies and gD1 IgG binding antibodies, but low or undetectable cross-reactive binding antibodies to gC1 and gE1. Conclusions: Despite occasional mild, localized breakthrough infections, the vaccine provided disease-modifying immunity and was neuroprotective. The results suggest that a single herpes vaccine effective against genital HSV-2 may be neuroprotective against HSV-1 following eye infection. Full article

Background/Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the global COVID-19 pandemic, which led to hundreds of millions of human infections and more than seven million deaths worldwide. Major variants of concern, particularly the Omicron variant and its associated subvariants, can escape the vaccines developed so far to target previous strains/subvariants. Therefore, effective vaccines that broadly neutralize different Omicron subvariants and show good protective efficacy are needed to prevent further spread of Omicron. The spike (S) protein, including its receptor-binding domain (RBD), is a key vaccine target. Methods: Here, we designed a unique mRNA vaccine encoding Omicron-KP.3 RBD based on RBD-truncated S protein backbone of an earlier Omicron subvariant EG.5 (KP3 mRNA), and evaluated its stability, immunogenicity, neutralizing activity, and protective efficacy in a mouse model. Results: Our data showed that the nucleoside-modified, lipid nanoparticle-encapsulated mRNA vaccine was stable at various temperatures during the period of detection. In addition, the vaccine elicited potent antibody responses with broadly neutralizing activity against multiple Omicron subvariants, including KP.2, KP.3, XEC, and NB.1.8.1. This mRNA vaccine protected immunized transgenic mice from challenge with SARS-CoV-2 Omicron-KP.3. Immune serum also protected against subsequent virus challenge, with the level of protection associating positively with the serum neutralizing antibody titer. Conclusions: Taken together, the data presented herein suggest that this newly designed mRNA vaccine has potential against current and future Omicron subvariants. Full article

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus with a high case-fatality ratio for which there is no approved vaccine. Studies have assessed different vaccine technologies. However, few studies have yet assessed the immunogenicity of heterologous prime-boost regimens. Methods: Here, we compare a lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA-based vaccine encoding the SFTSV glycoproteins, Gn and Gc, to our recently described recombinant VSV SFTSV (rVSV-SFTSV) vaccine in single dose, homologous, and heterologous prime-boost regimens in mice. Results: We show that all regimens protect from pathogenic SFTSV challenge and elicit strong long-lasting antibody responses. Furthermore, strong cellular immunity is elicited by mRNA-LNP immunizations and by heterologous immunization with an rVSV-SFTSV prime and mRNA-LNP boost. Cellular responses robustly polarized towards a type 1 response, characterized by high levels of IFNγ, TNFα, and IL-2. Immunization with mRNA led to a mixed type 1/type 2 immune response, as determined by antibody isotypes IgG1 and IgG2c. We found that homologous immunization leads to stronger antibody responses while heterologous immunization drives a slightly stronger cellular response. Conclusions: Taken together, the vaccine platforms described here represent strong vaccine candidates for further development. Full article

Background/Objectives: Circulating influenza strains antigenically differing from vaccine antigens increase disease burden by decreasing vaccine efficacy. Nucleoside-modified mRNA (modRNA) influenza vaccines may facilitate rapid production allowing later antigen selection and improved antigenic similarity compared to circulating strains. We studied different influenza modRNA vaccine (IRV) formulations and dose levels. Methods: This phase 1/2 randomized study evaluated IRV safety/tolerability and immunogenicity in healthy 18- through 85-year-olds. Based on safety and immunogenicity for different IRV doses, schedules, and valencies versus the quadrivalent influenza vaccine (QIV; Fluzone High-Dose Quadrivalent, Sanofi Pasteur) in phase 1 (65–85-year-olds), quadrivalent IRV (qIRV) was further evaluated in 65- through 85-year-olds and 18- through 64-year-olds in phase 2, leading to phase 3 dose selection. Results: Phase 1 (65–85-year-olds) safety/tolerability and immunogenicity findings supported qIRV 30-µg and 60-µg phase 2 assessment (18–85-year-olds, N = 610). qIRV was well tolerated. Injection site pain was the most frequently reported local reaction. Reactogenicity event incidences ≤ 7 days postvaccination for qIRV were generally higher versus QIV, observed more frequently in 18- through 64-year-olds than 65- through 85-year-olds, and showed dose-related trends (60 μg > 30 μg). qIRV and QIV adverse event profiles in 65- through 85-year-olds were similar. There were higher postvaccination hemagglutination inhibition assay geometric mean titers and fold rises and seroconversion rates observed with qIRV versus QIV for A strains, with no consistent pattern for B strains. Cell-mediated immune responses to qIRV by Day 7 showed overall higher T-cell responses against all strains versus QIV. Antibody and cell-mediated immune responses showed comparable trends across qIRV doses in 18- through 85-year-olds; a dose-related pattern was observed in 65- through 85-year-olds (60 μg > 30 μg). Conclusions: Phase 3 investigations of qIRV 60 µg in older adults and qIRV 30 µg in younger adults are warranted (ClinicalTrials.gov Identifier: NCT05052697). Full article

Nucleoside-5′-triphosphates (5′-NTPs) are essential building blocks of nucleic acids in nature and play an important role in molecular biology, diagnostics, and mRNA therapeutic synthesis. Chemical synthesis has long been the standard method for producing modified 5′-NTPs. However, chemical routes face limitations, including low regio- and stereoselectivity, along with the need for protection/deprotection cycles, resulting in low yields, high costs, and lengthy processes. In contrast, enzymatic synthesis methods offer significant advantages, such as improved regio- and stereoselectivity and the use of mild reaction conditions, which often leads to higher product yields in “one-pot” reactions. Despite the extensive review of chemical synthesis routes for 5′-NTPs, there has not yet been any comprehensive analysis of enzymatic approaches. Initially, this review provides a brief overview of the enzymes involved in nucleotide metabolism, introducing valuable biocatalysts for 5’-NTP synthesis. Furthermore, the available enzymatic methods for efficient 5′-NTP synthesis using purified enzymes and starting from either nucleobases or nucleosides are examined, highlighting their respective advantages and disadvantages. Special attention is also given to the importance of ATP regeneration systems for 5′-NTP synthesis. We aim to demonstrate the remarkable potential of enzymatic in vitro cascade reactions, promoting their broader application in both basic research and industry. Full article

Background/Objectives: Chlamydia (C.) psittaci is an avian respiratory pathogen that regularly infects budgerigars (Melopsittacus undulatus) and is a known zoonosis. This study aimed to evaluate the efficacy of a nucleoside-modified mRNA vaccine formulated in lipid nanoparticles (LNPs), either with (mRNA Galsomes) or without (mRNA LNPs) the glycolipid antigen α-Galactosylceramide, in protecting budgerigars against C. psittaci genotype A infection. Methods: Three groups of eight budgerigars received two intramuscular vaccinations with PBS, mRNA LNPs or mRNA Galsomes, and were subsequently challenged via aerosol with the C. psittaci genotype A strain 90/1051. Vaccine efficacy was assessed over 14 days post challenge by monitoring clinical signs, macroscopic and microscopic lesions, pathogen excretion and chlamydial burden in organs. Antibody levels were evaluated at baseline, after vaccination and post challenge. Results: Both mRNA LNPs and mRNA Galsomes induced significant serum antibody responses post booster. Vaccination significantly reduced clinical signs, chlamydial burden in the lungs and macroscopic lesions in conjunctiva, conchae, lungs and thoracic airsacs, compared to controls. Additionally, mRNA Galsomes-treated birds showed a significantly reduced lung inflammation and fewer macroscopic lesions in abdominal airsacs and liver, compared to non-vaccinated animals. These animals also experienced a significantly lower chlamydial burden in the spleen, fewer clinical signs at day 11 and fewer fecal shedding at day 14 post challenge, compared to mRNA LNP-treated animals. Conclusions: This study demonstrated that mRNA vaccination confers partial protection against C. psittaci in budgerigars, with mRNA Galsomes appearing to provide enhanced efficacy. However, the absence of species-specific reagents for assessing cellular immunity in Psittaciformes limits a comprehensive understanding of vaccine-induced protection. The development of psittacine-specific T cell markers and cytokine assays is necessary to further elucidate immune mechanisms and optimize vaccine formulations. Full article

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4′-C-substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4′-C-(N-ethyl) aminoethyl (4′-EAE-T), 4′-C-(N-butyl) aminoethyl (4′-BAE-T), and 4′-C-(N-octyl) aminoethyl (4′-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4′-C-aminoethyl (4′-AE-T) and 4′-C-(N-methyl) aminoethyl (4′-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4′-EAE-T and 4′-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4′-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4′-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4′-EAE-T resulting in the highest stability, followed by 4′-BAE-T and 4′-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4′-OAE-modified nucleotide, which exhibited low activity. Full article

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. Full article

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is in its sixth year and is being maintained by the inability of current spike-alone-based COVID-19 vaccines to prevent transmission leading to the continuous emergence of variants and sub-variants of concern (VOCs). This underscores the critical need for next-generation broad-spectrum pan-Coronavirus vaccines (pan-CoV vaccine) to break this cycle and end the pandemic. The development of a pan-CoV vaccine offering protection against a wide array of VOCs requires two key elements: (1) identifying protective antigens that are highly conserved between passed, current, and future VOCs; and (2) developing a safe and efficient antigen delivery system for induction of broad-based and long-lasting B- and T-cell immunity. This review will (1) present the current state of antigen delivery platforms involving a multifaceted approach, including bioinformatics, molecular and structural biology, immunology, and advanced computational methods; (2) discuss the challenges facing the development of safe and effective antigen delivery platforms; and (3) highlight the potential of nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP) as the platform that is well suited to the needs of a next-generation pan-CoV vaccine, such as the ability to induce broad-based immunity and amenable to large-scale manufacturing to safely provide durable protective immunity against current and future Coronavirus threats. Full article

After the COVID-19 pandemic, mRNA-based vaccines have emerged as a revolutionary technology in immunization and vaccination. These vaccines have shown remarkable efficacy against the virus and opened up avenues for their possible application in other diseases. This has renewed interest and investment in mRNA vaccine research and development, attracting the scientific community to explore all its other applications beyond infectious diseases. Recently, researchers have focused on the possibility of adapting this vaccination approach to cancer immunotherapy. While there is a huge potential, challenges still remain in the design and optimization of the synthetic mRNA molecules and the lipid nanoparticle delivery system required to ensure the adequate elicitation of the immune response and the successful eradication of tumors. This review points out the basic mechanisms of mRNA-LNP vaccines in cancer immunotherapy and recent approaches in mRNA vaccine design. This review displays the current mRNA modifications and lipid nanoparticle components and how these factors affect vaccine efficacy. Furthermore, this review discusses the future directions and clinical applications of mRNA-LNP vaccines in cancer treatment. Full article

Therapeutic modified mRNAs are being developed for a broad range of human diseases. However, the impact of potential miscoding of modified mRNAs on self-tolerance remains unknown. Additionally, more studies are needed to explore the effects of nucleoside alkylation on translation. While all six tested modifications are tolerated as substrates by T7 RNA polymerase and inhibited mRNA immunogenicity, the translation efficiency varied significantly depending on the type of modification. In contrast to methylation, ethylation at the N1 position of pseudouridine (Ψ) hindered translation, suggesting that the C5-C1’ glycosidic bond alone is not a critical element for high translation. Inhibition of mRNA translation was also observed with 5-methoxyuridine modification. However, this inhibition was partially alleviated through the optimization of mRNA coding sequences. BALB/c mice immunized with syngeneic ψ-modified mRNA encoding for Wilms’ tumor antigen-1 (WT1) developed a low but significant level of anti-WT1 IgG antibodies compared to those immunized with either unmodified or N1-methyl ψ-modified mRNA. Overall, the data indicate that adding a simple ethyl group (-CH₂CH₃) at the N1 position of ψ has a major negative effect on translation despite its reduced immunogenicity. Additionally, mRNA containing Ψ may alter translation fidelity at certain codons, which could lead to a breakdown of immune tolerance to self-antigens. This concern should be taken into account during gene replacement therapies, although it could benefit mRNA-based vaccines by generating a diverse repertoire of antigens. Full article

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. Full article