Search Results (48)

Fritillaria unibracteata is a rare and endangered medicinal plant in the Liliaceae family, whose bulbs have been used in traditional Chinese traditional medicine for over 2000 years. The mevalonate (MVA) pathway is involved in the growth, development, response to environmental stress, and active ingredient production of plants; however, the functional characterization of MVA-pathway genes in the Liliaceae family remains poorly documented. In this study, an Acetyl-CoA C-acetyltransferase gene (FuAACT) was first cloned from F. unibracteata. It exhibited structural features of the thiolase family and showed the highest sequence identity with the Dioscorea cayenensis homolog. The K_m, V_max, and K_cat of the recombinant FuAACT were determined to be 3.035 ± 0.215 μM, 0.128 ± 0.0058 μmol/(min·mg), and 1.275 ± 0.0575 min⁻¹, respectively. The optimal catalytic conditions for FuAACT were ascertained to be 30 °C and pH 8.9. It was stable below 50 °C. His361 was confirmed to be a key amino acid residue to enzymatic catalysis by site-directed mutagenesis. Subsequent subcellular localization experiments demonstrated that FuAACT was localized in chloroplasts and cytoplasm. FuAACT-overexpressing transgenic Arabidopsis thaliana plants showed higher drought tolerance than wild-type plants. This phenotypic difference was corroborated by significant differences in seed germination rate, lateral root number, plant height, and leaf number (p < 0.05). Furthermore, the FuAACT transgenic plants resulted in the formation of a more developed fibrous root system. These results indicated that the FuAACT gene revealed substantial biological activity in vitro and in vivo, hopefully providing the basis for its further research and application in liliaceous ornamental and medicinal plants. Full article

Retinoids, including retinol, retinal, and retinoic acid, are a group of vitamin A derivatives with skin-improving effects. Retinoic acid is highly effective for skin anti-aging but can cause irritation, requiring a prescription. Retinol, a less irritating alternative, needs conversion to retinal and then retinoic acid in the skin, whereas direct absorption of retinal enhances efficacy by bypassing this conversion process. This study aimed to produce retinal through precision fermentation using metabolically engineered Saccharomyces cerevisiae. The introduction of heterologous retinal biosynthetic genes and overexpression of the truncated HMG-CoA reductase (tHMG1) and acetyl-CoA acetyltransferase (ERG10) genes in the mevalonate (MVA) pathway increased retinal production up to 10.2 mg/L. At the same time, ethanol was produced as a major byproduct in S. cerevisiae. To address this, a pyruvate decarboxylase (Pdc)-deficient S. cerevisiae strain, incapable of producing ethanol, was employed. Overexpression of ERG10 and tHMG1 in the Pdc-deficient S. cerevisiae harboring the retinal biosynthetic plasmids achieved a retinal production up to 117.4 mg/L in the dodecane layer without ethanol through a two-phase in situ fermentation and extraction. This study demonstrates that eliminating pyruvate decarboxylase activity effectively redirects carbon flux toward retinal biosynthesis in the recombinant S. cerevisiae, offering a promising approach for sustainable retinal production through precision fermentation. Full article

Background: The Epstein-Barr virus (EBV) is intricately linked to a range of human malignancies, with EBV latent membrane protein 2A (LMP2A) emerging as a potential target antigen for immunotherapeutic strategies in the treatment of nasopharyngeal carcinoma (NPC). Methods: The modified vaccinia virus Ankara (MVA) is universally used in vector vaccine research because of its excellent safety profile and highly efficient recombinant gene expression. Here, we constructed a novel MVA-LMP2A recombinant virus and investigated its specific immune response induction and oncolytic effect. Results: An immunization dose of 2 × 10⁷ PFU induced the highest specific immune response, which was no longer increased by boost injections after four doses. Three weeks post-final immunization, the specific immune response reached its peak. The MVA-LMP2A vaccine-induced LMP2A-specific cytotoxic T lymphocytes (CTLs), which exhibited substantial efficacy against target cells and effectively inhibited tumor growth. Conclusions: Thus, the MVA-LMP2A recombinant virus effectively induces strong LMP2A-specific cellular and humoral immune responses and anti-tumor activity. This work provides a promising therapeutic strategy for developing NPC candidate vaccines, as well as a reference for the treatment of EBV LMP2-associated malignancies. Full article

Background/Objectives: Poxviruses are large DNA viruses that replicate in the host cytoplasm without a nuclear phase. As vaccine vectors, they can package and express large recombinant cassettes from different positions of their genomic core region. We present a comparison between wildtype modified vaccinia Ankara (MVA) and isolate CR19, which has significantly expanded inverted terminal repeats (ITRs). With this expansion, a site in wildtype MVA, called deletion site (DS) IV, has been duplicated at both ends of the genome and now occupies an almost central position in the newly formed ITRs. Methods: We inserted various reporter genes into this site and found that the ITRs can be used for transgene expression. However, ITRs are genomic structures that can rapidly adapt to selective pressure through transient duplication and contraction. To test the potential utility of insertions into viral telomers, we inserted a factor from the cellular innate immune system that interferes with viral replication as an example of a difficult transgene. Results: A site almost in the centre of the ITRs can be used for transgene expression, and both sides are mirrored into identical copies. The example of a challenging transgene, tetherin, proved to be surprisingly efficient in selecting candidate vectors against the large background of parental viruses. Conclusions: Insertion of transgenes into ITRs automatically doubles the gene doses. The functionalisation of viruses with tetherin may accelerate the identification and generation of recombinant vectors for personalised medicine and pandemic preparedness. Full article

Background/Objectives: Marburg virus (MARV) is the etiological agent of Marburg Virus Disease (MVD), a rare but severe hemorrhagic fever disease with high case fatality rates in humans. Smaller outbreaks have frequently been reported in countries in Africa over the last few years, and confirmed human cases outside Africa are, so far, exclusively imported by returning travelers. Over the previous years, MARV has also spread to non-endemic African countries, demonstrating its potential to cause epidemics. Although MARV-specific vaccines are evaluated in preclinical and clinical research, none have been approved for human use. Modified Vaccinia virus Ankara (MVA), a well-established viral vector used to generate vaccines against emerging pathogens, can deliver multiple antigens and has a remarkable clinical safety and immunogenicity record, further supporting its evaluation as a vaccine against MARV. The rapid availability of safe and effective MVA-MARV vaccine candidates would expand the possibilities of multi-factored intervention strategies in endemic countries. Methods: We have used an optimized methodology to rapidly generate and characterize recombinant MVA candidate vaccines that meet the quality requirements to proceed to human clinical trials. As a proof-of-concept for the optimized methodology, we generated two recombinant MVAs that deliver either the MARV glycoprotein (MVA-MARV-GP) or the MARV nucleoprotein (MVA-MARV-NP). Results: Infections of human cell cultures with recombinant MVA-MARV-GP and MVA-MARV-NP confirmed the efficient synthesis of MARV-GP and MARV-NP proteins in mammalian cells, which are non-permissive for MVA replication. Prime-boost immunizations in C57BL/6J mice readily induced circulating serum antibodies binding to recombinant MARV-GP and MARV-NP proteins. Moreover, the MVA-MARV-candidate vaccines elicited MARV-specific T-cell responses in C57BL/6J mice. Conclusions: We confirmed the suitability of our two backbone viruses MVA-mCherry and MVA-GFP in a proof-of-concept study to rapidly generate candidate vaccines against MARV. However, further studies are warranted to characterize the protective efficacy of these recombinant MVA-MARV vaccines in other preclinical models and to evaluate them as vaccine candidates in humans. Full article

The emergence of hitherto unknown viral pathogens presents a great challenge for researchers to develop effective therapeutics and vaccines within a short time to avoid an uncontrolled global spread, as seen during the coronavirus disease 2019 (COVID-19) pandemic. Therefore, rapid and simple methods to identify immunogenic antigens as potential therapeutical targets are urgently needed for a better pandemic preparedness. To address this problem, we chose the well-characterized Modified Vaccinia virus Ankara (MVA)-T7pol expression system to establish a workflow to identify immunogens when a new pathogen emerges, generate candidate vaccines, and test their immunogenicity in an animal model. By using this system, we detected severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) nucleoprotein (N)-, and spike (S)-specific antibodies in COVID-19 patient sera, which is in line with the current literature and our observations from previous immunogenicity studies. Furthermore, we detected antibodies directed against the SARS-CoV-2-membrane (M) and -ORF3a proteins in COVID-19 patient sera and aimed to generate recombinant MVA candidate vaccines expressing either the M or ORF3a protein. When testing our candidate vaccines in a prime-boost immunization regimen in humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice, we were able to demonstrate M- and ORF3a-specific cellular and humoral immune responses. Hence, the established workflow using the MVA-T7pol expression system represents a rapid and efficient tool to identify potential immunogenic antigens and provides a basis for future development of candidate vaccines. Full article

Chlamydia trachomatis remains a major global health problem with increasing infection rates, requiring innovative vaccine solutions. Modified Vaccinia Virus Ankara (MVA) is a well-established, safe and highly immunogenic vaccine vector, making it a promising candidate for C. trachomatis vaccine development. In this study, we evaluated two novel MVA-based recombinant vaccines expressing spCTH522 and CTH522:B7 antigens. Our results show that while both vaccines induced CD4⁺ T-cell responses in C57BL/6J mice, they failed to generate antigen-specific systemic CD8⁺ T cells. Only the membrane-anchored CTH522 elicited strong IgG2b and IgG2c antibody responses. In an HLA transgenic mouse model, both recombinant MVAs induced Th1-directed CD4⁺ T cell and multifunctional CD8⁺ T cells, while only the CTH522:B7 vaccine generated antibody responses, underscoring the importance of antigen localization. Collectively, our data indicate that distinct antigen formulations can induce different immune responses depending on the mouse strain used. This research contributes to the development of effective vaccines by highlighting the importance of careful antigen design and the selection of appropriate animal models to study specific vaccine-induced immune responses. Future studies should investigate whether these immune responses provide protection in humans and should explore different routes of immunization, including mucosal and systemic immunization. Full article

The sudden emergence of SARS-CoV-2 demonstrates the need for new vaccines that rapidly protect in the case of an emergency. In this study, we developed a recombinant MVA vaccine co-expressing SARS-CoV-2 prefusion-stabilized spike protein (ST) and SARS-CoV-2 nucleoprotein (N, MVA-SARS-2-ST/N) as an approach to further improve vaccine-induced immunogenicity and efficacy. Single MVA-SARS-2-ST/N vaccination in K18-hACE2 mice induced robust protection against lethal respiratory SARS-CoV-2 challenge infection 28 days later. The protective outcome of MVA-SARS-2-ST/N vaccination correlated with the activation of SARS-CoV-2-neutralizing antibodies (nABs) and substantial amounts of SARS-CoV-2-specific T cells especially in the lung of MVA-SARS-2-ST/N-vaccinated mice. Emergency vaccination with MVA-SARS-2-ST/N just 2 days before lethal SARS-CoV-2 challenge infection resulted in a delayed onset of clinical disease outcome in these mice and increased titers of nAB or SARS-CoV-2-specific T cells in the spleen and lung. These data highlight the potential of a multivalent COVID-19 vaccine co-expressing S- and N-protein, which further contributes to the development of rapidly protective vaccination strategies against emerging pathogens. Full article

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model. Full article

Aging is associated with a decline in immune system functionality. So-called immunosenescence may impair the successful vaccination of elderly people. Thus, improved vaccination strategies also suitable for an aged immune system are required. Modified Vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus that has been established as a multipurpose viral vector for vaccine development against various infections. We characterized a recombinant MVA expressing a prefusion-stabilized version of SARS-CoV-2 S protein (MVA-ST) in an aged-hamster model for COVID-19. Intramuscular MVA-ST immunization resulted in protection from disease and severe lung pathology. Importantly, this protection was correlated with a potent activation of SARS-CoV-2 specific T-cells and neutralizing antibodies. Our results suggest that MVA vector vaccines merit further evaluation in preclinical models to contribute to future clinical development as candidate vaccines in elderly people to overcome the limitations of age-dependent immunosenescence. Full article

Antrodia cinnamomea (AC), a rare fungus endemic to Taiwan, contains high levels of various secondary metabolites, notably triterpenoids, having useful medicinal and pharmacological properties. Techniques for increasing the production of AC triterpenoids (ACT) for medicinal purposes are a high research priority. We measured and compared the biomass and ACT content of AC mycelia under various liquid fermentation culture conditions. Relative gene expression levels of ten enzymes involved in the mevalonate (MVA) pathway and “subsequent group modification pathway” were determined, and correlation analysis was performed to evaluate the roles of these enzyme genes in ACT synthesis. Two representative genes encoding the enzymes lanosterol synthase (AcLSS) and sterol C-24 reductase (AcERG4), whose activity is closely associated with ACT content, were selected for homologous expression. AcLSS and AcERG4 were separately linked to plasmid pCT74, and transformed into prepared AC protoplasts to obtain two recombinant strains, termed RpLSS and RpERG4, by polyethylene glycol (PEG)-CaCl2-mediated protoplast transformation. Upregulated expression levels of AcLSS and AcERG4 (1.78- and 1.41-fold, respectively) were associated with significantly higher (1.82- and 1.37-fold, respectively) ACT content in the recombinant strains in comparison with the wild-type. Our findings provide a theoretical and practical basis for the enhancement of ACT production using homologous expression techniques. Full article

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society’s health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies’ binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail. Full article

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell–cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell–cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine. Full article

The combination of mosaic Gag and CAP256 envelope in an HIV vaccine regimen comprising DNA prime and modified vaccinia Ankara (MVA) boost followed by protein boost has previously been shown to generate robust autologous Tier 2 neutralizing antibodies (nAbs) in rabbits. Further refinements of this strategy have been investigated to improve antibody responses. The delivery of both DNA and recombinant MVA vaccines with a needle-free device was compared to delivery by injection, and the effect of formulating the DNA vaccine with adjuvant CpG ODN 1826 was determined. The Pharmajet Stratis^® needle-free injection device (PharmaJet, Golden, CO, USA) improved binding antibody responses to the DNA vaccine as well as both binding and neutralizing antibody responses to the MVA vaccines. Formulation of the DNA vaccines with CpG adjuvant further improved the antibody responses. A shortened vaccination regimen of a single DNA inoculation followed by a single MVA inoculation did not elicit Tier 1B nor Tier 2 neutralization responses as produced by the two DNA, followed by two MVA vaccination regimen. This study showed the immunogenicity of HIV DNA and MVA vaccines administered in a DDMM regimen could be improved using the PharmaJet Stratis needle-free injection device and formulation of the DNA vaccines with CpG adjuvant. Full article

Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy. Full article