Search Results (159)

Avian influenza A viruses (IAVs) pose a persistent threat to the poultry industry, causing substantial economic losses. Although traditional vaccines have helped reduce the disease burden, they typically rely on multivalent antigens, emphasize humoral immunity, and require intensive production. This study aimed to establish a genetically matched host–cell system to evaluate antigen-specific immune responses and identify conserved CD8+ T cell epitopes in avian influenza viruses. To this end, we developed an MHC class I genotype (B21)-matched host (Lohmann VALO SPF chicken) and cell vector (DF-1 cell line) model. DF-1 cells were engineered to express the hemagglutinin (HA) gene of clade 2.3.4.4b H5N1 either transiently or stably, and to stably express the matrix 1 (M1) and nucleoprotein (NP) genes of A/chicken/South Korea/SL20/2020 (H9N2, Y280-lineage). Following prime-boost immunization with HA-expressing DF-1 cells, only live cells induced strong hemagglutination inhibition (HI) and virus-neutralizing (VN) antibody titers in haplotype-matched chickens. Importantly, immunization with DF-1 cells transiently expressing NP induced stronger IFN-γ production than those expressing M1, demonstrating the platform’s potential for differentiating antigen-specific cellular responses. CD8+ T cell epitope mapping by mass spectrometry identified one distinct MHC class I-bound peptide from each of the HA-, M1-, and NP-expressing DF-1 cell lines. Notably, the identified HA epitope was conserved in 97.6% of H5-subtype IAVs, and the NP epitope in 98.5% of pan-subtype IAVs. These findings highlight the platform’s utility for antigen dissection and rational vaccine design. While limited by MHC compatibility, this approach enables identification of naturally presented epitopes and provides insight into conserved, functionally constrained viral targets. Full article

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Ensuring sustainable food systems is an urgent global priority as populations grow and environmental pressures mount. Technological innovations such as genetic engineering (GE) and nanotechnology (nano) have been promoted as promising pathways for achieving greater sustainability in agriculture and food production. Yet, the sustainability of these technologies is not defined by technical performance alone; it hinges on how they are perceived by key stakeholders and how well they align with broader societal values. This study addresses the critical question of how expert stakeholders evaluate the sustainability of GE and nano-based food and agriculture (agrifood) products. Using a multi-method online platform, we engaged 42 experts across academia, government, industry, and NGOs in the United States to assess six real-world case studies—three using GE and three using nano—across ten different dimensions of sustainability. We show that nano-based products were consistently rated more favorably than their GE counterparts in terms of environmental, economic, and social sustainability, as well as across ethical and societal dimensions. Like prior studies, our results reveal that stakeholders see meaningful distinctions between nanotechnology and biotechnology, likely due to underlying value-based concerns about animal welfare, perceived naturalness, or corporate control of agrifood systems. The fruit coating and flu vaccine—both nano-enabled—received the most positive ratings, while GE mustard greens and salmon were the most polarizing. These results underscore the importance of incorporating stakeholder perspectives in technology assessment and innovation governance. These results also suggest that responsible innovation efforts in agrifood systems should prioritize communication, addressing meaningful societal needs, and the contextual understanding of societal values to build trust and legitimacy. Full article

Outer-membrane vesicles (OMVs), naturally secreted by Gram-negative bacteria, have gained recognition as a versatile platform for the development of next-generation vaccines. OMVs are essential contributors to bacterial pathogenesis, horizontal gene transfer, cellular communication, the maintenance of bacterial fitness, and quorum sensing. Their intrinsic immunogenicity, adjuvant properties, and scalability establish OMVs as potent tools for combating infectious diseases and cancer. Recent advancements in genetic engineering and biotechnology have further expanded the utility of OMVs, enabling the incorporation of multiple epitopes and antigens from diverse pathogens. These developments address critical challenges such as antigenic variability and co-infections, offering broader immune coverage and cost-effective solutions. This review explores the unique structural and immunological properties of OMVs, emphasizing their capacity to elicit robust immune responses. It critically examines established and emerging engineering strategies, including the genetic engineering of surface-displayed antigens, surface conjugation, glycoengineering, nanoparticle-based OMV engineering, hybrid OMVs, and in situ OMV production, among others. Furthermore, recent advancements in preclinical research on OMV-based vaccines, including synthetic OMVs, OMV-based nanorobots, and nanodiscs, as well as emerging isolation and purification methods, are discussed. Lastly, future directions are proposed, highlighting the potential integration of synthetic biology techniques to accelerate research on OMV engineering. Full article

Uveal melanoma (UM), the most common primary intraocular malignancy in adults, poses a unique clinical challenge due to its high propensity for liver metastasis and poor responsiveness to conventional therapies. Despite the expanding landscape of immunotherapy in oncology, progress in managing metastatic uveal melanoma (mUM) remains limited, and no universally accepted standard of care has been established. In this review, we examine the current state and evolving strategies in immunotherapy for mUM, focusing on immune checkpoint inhibitors (ICIs), T cell receptor (TCR)-engineered therapies, and tumor-targeted vaccines. We also present a meta-analytical comparison of clinical outcomes between ICI monotherapy and combination regimens, alongside the recently FDA-approved T cell engager tebentafusp. Our analysis indicates that the triple combination of Ipilimumab, anti-PD-1 agents, and tebentafusp significantly enhances objective response rates, disease control rates, 1-year overall survival rates, and median overall survival (mOS) compared to ICI monotherapy alone. However, this enhanced efficacy is accompanied by increased toxicity due to broader immune activation. In contrast, tebentafusp offers superior tumor specificity and a more favorable safety profile in HLA-A*02:01-positive patients, positioning it as a preferred therapeutic option for this genetically defined subset of UM. Additionally, early-phase studies involving dendritic cell-based immunotherapies and peptide vaccines has shown encouraging signs of tumor-specific immune activation, along with improved tolerability. Collectively, this review underscores the urgent need for more precise and effective immunotherapeutic approaches tailored to the unique biology of mUM. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Background: Modern viral vector production needs to consider process intensification for higher yields from smaller production volumes. However, innate antiviral immunity triggered in the producer cell may limit virus replication. While commonly used cell lines (e.g., Vero or E1A-immortalised cells) are already compromised in antiviral pathways, the redundancy of innate signaling complicates host cell optimization by genetic engineering. Small molecules that are hypothesized to target antiviral pathways (Viral Sensitizers, VSEs) added to the culture media offer a versatile alternative to genetic modifications to increase permissiveness and, thus, viral yields across multiple cell lines. Methods: To explore how the yield for a chimeric Zika vaccine candidate (YF-ZIK) could be further be increased in an intensified bioprocess, we used spin tubes or an Ambr15 high-throughput microbioreactor system as scale-down models to optimize the dosing for eight VSEs in three host cell lines (AGE1.CR.pIX, BHK-21, and HEK293-F) based on their tolerability. Results: Addition of VSEs to an already optimized infection process significantly increased infectious titers by up to sevenfold for all three cell lines tested. The development of multi-component VSE formulations using a design of experiments approach allowed further synergistic titer increases in AGE1.CR.pIX cells. Scale-up to 1 L stirred-tank bioreactors and 3D-printed mimics of 200 or 2000 L reactors resulted in up to threefold and eightfold increases, respectively. Conclusions: Addition of single VSEs or combinations thereof allowed a further increase in YF-ZIK titers beyond the yield of an already optimized, highly intensified process. The described approach validates the use of VSEs and can be instructive for optimizing other virus production processes. Full article

Background/Objectives: Pancreatic cancer remains the fourth leading cause of cancer-related deaths. While peripheral blood-derived mature dendritic cell (mDC) vaccines have shown potential in eliciting anti-tumor immune responses, clinical efficacy has been limited. This study aimed to enhance the potency and scalability of DC-based immunotherapy by developing an allogeneic DC platform derived from CD34⁺ hematopoietic stem cells (HSCs), genetically engineered to overexpress CD93, CD40L, and CXCL13, followed by maturation and tumor antigen pulsing. Methods: Engineered DCs were generated from CD34⁺ HSCs and matured in vitro after lentiviral transduction of CD93, CD40L, and CXCL13. Tumor lysates were used for antigen pulsing. A scrambled-sequence control DC was used for comparison. In vitro assays were performed to assess T cell activation and tumor cell killing. In vivo efficacy was evaluated using orthotopic pancreatic tumors in BLT and PBMC-humanized NSG mice established with the MiaPaca-2 (MP2) cell line. Results: Engineered DCs significantly enhanced T cell activation and tumor-specific cytotoxicity in vitro compared to control DCs. Antigen pulsing further amplified immune activation. In vivo, treated humanized mice showed increased CD4⁺, CD8⁺, and NK cell frequencies in peripheral blood and within tumors, correlating with reduced tumor burden. Conclusions: Our data shows that the antigen-pulsed, engineered DCs have the potency to activate immune cells, which leads to a significant reduction in pancreatic tumors and therefore could potentially provide an effective therapeutic opportunity for the treatment of pancreatic cancer and other solid tumors. Full article

A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines. Full article

Mucosal immunization represents a promising strategy for preventing enteric infections. Rotavirus (RV), a leading gastrointestinal pathogen distinguished by its remarkable stability and segmented double-stranded RNA genome, has been engineered into a versatile oral vaccine vector through advanced reverse genetics systems. The clinical efficacy of live-attenuated RV vaccines highlights their unique capacity to concurrently induce mucosal IgA responses and systemic neutralizing antibodies, positioning them as a multiple action vector for multiple immune protection. In this review, we summarize the RV colonization of the intestine and stimulation of intestinal immunity, as well as recent advancements in RV reverse genetics, and focus on their application in the rational design of a multivalent mucosal vaccine vector targeting enteric pathogens considering the advantages and challenges of RV as a vector. We further propose molecular strategies to overcome genetic instability in recombinant RV vectors, including the codon optimization of heterologous inserts. These insights provide a theoretical foundation for developing next-generation mucosal immunization platforms with enhanced safety, stability, and cross-protective efficacy. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

Over the past several decades, viral vector-based vaccines have emerged as some of the most versatile and potent platforms in modern vaccinology. Their capacity to deliver genetic material encoding target antigens directly into host cells enables strong cellular and humoral immune responses, often superior to what traditional inactivated or subunit vaccines can achieve. This has accelerated their application to a wide array of pathogens and disease targets, from well-established threats like HIV and malaria to emerging infections such as Ebola, Zika, and SARS-CoV-2. The COVID-19 pandemic further highlighted the agility of viral vector platforms, with several adenovirus-based vaccines quickly authorized and deployed on a global scale. Despite these advances, significant challenges remain. One major hurdle is pre-existing immunity against commonly used vector backbones, which can blunt vaccine immunogenicity. Rare but serious adverse events, including vector-associated inflammatory responses and conditions like vaccine-induced immune thrombotic thrombocytopenia (VITT), have raised important safety considerations. Additionally, scaling up manufacturing, ensuring consistency in large-scale production, meeting rigorous regulatory standards, and maintaining equitable global access to these vaccines present profound logistical and ethical dilemmas. In response to these challenges, the field is evolving rapidly. Sophisticated engineering strategies, such as integrase-defective lentiviral vectors, insect-specific flaviviruses, chimeric capsids to evade neutralizing antibodies, and plug-and-play self-amplifying RNA approaches, seek to bolster safety, enhance immunogenicity, circumvent pre-existing immunity, and streamline production. Lessons learned from the COVID-19 pandemic and prior outbreaks are guiding the development of platform-based approaches designed for rapid deployment during future public health emergencies. This review provides an exhaustive, in-depth examination of the historical evolution, immunobiological principles, current platforms, manufacturing complexities, regulatory frameworks, known safety issues, and future directions for viral vector-based vaccines. Full article

Salmonella enterica serovar Typhi (S. Typhi) produces outer membrane vesicles (OMVs) that remain comparatively underexplored as potential biotechnological tools. Here, we investigated how hypervesiculating S. Typhi mutants (ΔtolR and ΔdegS) can be engineered to load and deliver the fluorescent reporter protein mCherry, targeting human epithelial cells and the murine immune system. Deletions in tolR and degS led to distinct OMV phenotypes characterized by higher vesicle production and altered cargo composition, underscoring the impact of disrupted membrane integrity and envelope stress on OMV biogenesis. By fusing mCherry with the S. Typhi OmpA signal peptide (SP_ompA), we achieved robust and functionally intact intravesicular packaging in all strains. Flow cytometry and confocal microscopy revealed that the ΔtolR mutant exhibited particularly high cargo loading in the OMV fraction and pronounced mCherry delivery to epithelial cells, highlighting the potential of hypervesiculation to enhance OMV-based protein transport. However, immunization studies in mice showed that wild-type OMVs, despite carrying less mCherry than their hypervesiculating counterparts, induced the strongest anti-mCherry IgG responses. These findings indicate that, at least under these conditions, antigen loading alone is not sufficient to fully determine immunogenicity. Instead, the intrinsic composition or adjuvant-like properties of OMVs play a pivotal role in driving robust immune activation. Our results establish S. Typhi OMVs, especially when genetically modified with a Sec-dependent targeting signal (SP_ompA), as versatile platforms for heterologous protein delivery. Although hypervesiculation facilitates increased protein encapsulation and delivery to epithelial cells, native OMVs appear to better preserve and/or present antigens for effective immunogenic responses in vivo. These insights set the stage for further optimization of S. Typhi OMVs in vaccine development and protein therapeutics, where balancing cargo loading with immunostimulatory features may be key to achieving maximal efficacy. Full article

Microalgae represent a promising platform for the synthesis of recombinant proteins, particularly in the context of biopharmaceutical applications. Their unique combination of eukaryotic cellular machinery and prokaryotic-like simplicity offers several advantages, including the ability to perform complex post-translational modifications, rapid growth rates, and cost-effective culture conditions. Advances in genome sequencing, genetic engineering tools, and omics technologies have significantly enhanced the feasibility and efficiency of using microalgae for therapeutic protein production. These advancements, coupled with the development of well-established transformation methods and optimized vectors, have enabled the successful expression of various biopharmaceuticals, ranging from vaccines to enzymes. Here, the main stages and current status of the production of exogenic recombinant proteins dedicated to human therapy are presented. Full article

Background/Objectives: Porcine epidemic diarrhea (PED) is a highly contagious enteric infectious disease that causes severe morbidity and mortality in piglets, posing significant economic losses to the swine industry worldwide. Oral vaccines based on Lactococcus lactis offer a promising approach due to their safety and genetic manipulability. This study aims to develop and evaluate an oral L. lactis-based vaccine expressing the full-length PEDV S protein. Methods: A recombinant L. lactis strain expressing the PEDV S protein was constructed and encapsulated in alginate–chitosan microcapsules. Vaccine stability was tested in simulated digestive fluids, and mice were orally immunized. Immune responses were evaluated by measuring specific antibodies, cytokines, and lymphocyte proliferation. Results: The recombinant L. lactis NZ3900/pNZ8149-S strain successfully expressed the full-length PEDV S protein and maintained stable plasmid inheritance. Oral immunization in mice induced detectable PEDV-specific immune responses. Both encapsulated and non-encapsulated vaccines stimulated the production of IgG and sIgA antibodies, as well as cytokines associated with Th1 and Th2 responses. Notably, encapsulation with alginate–chitosan significantly enhanced bacterial survival in digestive conditions and further amplified immune responses, including higher antibody titers, elevated levels of IFN-γ, IL-4, and IL-10, and greater lymphocyte proliferation, indicating improved immune memory. Conclusions: The oral L. lactis NZ3900/pNZ8149-S vaccine expressing the PEDV S protein effectively induced systemic and mucosal immunity in mice. Encapsulation with alginate–chitosan further enhanced its immunogenicity and stability in gastrointestinal conditions. These results suggest that both the engineered L. lactis strain and the encapsulation strategy contribute to the development of a promising oral vaccine platform for controlling PEDV in swine populations. Full article