Search Results (410)

Goose astrovirus (GoAstV) infection has become prevalent in major goose-producing regions, causing substantial economic losses to the industry. In this study, an indirect competitive ELISA (ic-ELISA) was developed based on a monoclonal antibody (mAb) targeting the GoAstV VP27 protein. The recombinant VP27 protein was expressed in E. coli and purified, followed by the generation of murine mAbs using the purified antigen. Through screening with GoAstV particles, mAb 3G11 exhibited strong immunoreactivity, which was further confirmed by Western blot and immunofluorescence assay (IFA). The ic-ELISA conditions were optimized as follows: GoAstV particle coating concentration of 10⁴ TCID₅₀ per well, 3G11 mAb dilution of 1:8000, and incubation times of 120 min for coating, 60 min for serum samples, and 60 min for mAb binding. The assay exhibited satisfactory performance in terms of sensitivity, specificity, and reproducibility. Using this method, serum samples collected from major goose farming areas in Shandong province were tested and showed an overall seropositivity rate of 11.7%. This study provided a reliable serological tool for detecting GoAstV-specific antibodies and would support future vaccine evaluation efforts. Full article

Background: Adoptive cell therapy using genetically engineered recombinant T cell receptors (rTCRs) expressed in T cells (TCR-T cell therapy) provides precision targeting of cancer cells expressing tumor-associated or tumor-specific antigens recognized by the rTCRs. Standardized analytical tools are lacking to easily quantify receptor expression. Methods: To overcome this hindrance, a universal tagging system (UniTope & TraCR) was designed consisting of a minimal peptide epitope (UniTope) inserted into the constant region of the rTCR α or β chain and a high-affinity monoclonal antibody (TraCR) specific to this tag. Detailed biophysical, biochemical, and functional assays were performed to evaluate rTCR expression, folding, pairing, and antigen recognition, as well as antibody performance, using the UniTope & TraCR System. Results: Tagged rTCRs were stably expressed in human T cells with surface densities comparable to untagged rTCRs. The TraCR antibody bound UniTope with nanomolar affinity and no detectable cross-reactivity was observed for endogenous proteins expressed by human cells of diverse origin, importantly, including T cells of the natural T cell repertoires of multiple human donors. Functional assays confirmed that UniTope-tagged rTCRs preserved their antigen-specific cytokine secretion and cytolytic activity upon antigen-specific stimulation. The UniTope & TraCR System enabled robust detection of rTCR-expressing T cells by flow cytometry, and rTCR protein expression by Western blot or immunoprecipitation, supporting the quantitative assessment of receptor copy number and structural integrity. Conclusions: The UniTope & TraCR System provides a modular, construct-agnostic platform for monitoring engineered rTCRs, integrated into TCR-T cell therapies currently in development. Full article

The cellular prion protein (PrP^C) displays a functional repertoire that extends well beyond its classical link to transmissible spongiform encephalopathies. Abundant in the nervous system and localized within lipid raft microdomains, PrP^C has emerged as a multifunctional signaling platform that regulates cell differentiation, neurogenesis, neuroprotection, and synaptic plasticity. Recent evidence highlights its dynamic expression in stem cell populations, where it participates in multimolecular complexes that control lineage commitment, particularly during neuronal differentiation. PrP^C expression tightly correlates with stem cell status, making it a promising biomarker of stemness and developmental progression. Through interactions with growth factors, extracellular matrix components, and synaptic proteins, PrP^C functions as a molecular integrator of signals essential for tissue repair and regeneration. Preclinical studies demonstrate that recombinant PrP^C can stimulate neurogenesis and tissue repair, while monoclonal antibodies modulate its physiological and pathological functions. Likewise, cell-based therapies leveraging PrP^C-enriched stem cells or PrP^C-dependent signaling profiles have shown promise in models of neurodegeneration and ischemia. Conversely, dysregulated PrP^C expression has also been observed in solid tumors, where it contributes to cancer cell survival, proliferation, metastasis, and therapy resistance, reinforcing its role as a regulator of cell fate and an oncological target. This review integrates stem cell biology, tissue regeneration, and oncology into a unified framework, offering a novel perspective in which PrP^C emerges as a shared molecular hub governing both physiological repair and pathological tumor behavior, opening previously unrecognized conceptual and translational opportunities. Full article

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections. Full article

West Nile fever is an infectious disease caused by the West Nile virus (WNV), which is transmitted by mosquitoes. Epidemiological surveillance confirms the potential risk of WNV infection in human populations. The lack of specific antiviral therapeutics and vaccines against WNV underscores the urgent need to develop effective therapeutic approaches. In this study, a recombinant chimeric monoclonal antibody (mAb) 900 was generated based on the broadly neutralizing and protective murine mAb 9E2. The antigen-binding regions of the murine mAb were fused with the constant domains (CH2–CH3) of human IgG1. Two key amino acid clusters, M252/S254/T256 and H433/N434, were introduced into the CH2–CH3 domains to enhance the affinity of mAb 900 for the neonatal Fc receptor (FcRn). The engineered mAb 900 was produced in CHO cells and purified to high homogeneity. Biophysical characterization confirmed its stability and correct dimeric assembly. Comparative analysis demonstrated that mAb 900 retained the high antigen-binding affinity and potent virus-neutralizing activity of its murine predecessor. Most importantly, mAb 900 demonstrated significant protective efficacy in a lethal mouse model of WNV infection. These results establish the proof of concept for mAb 900 as a promising candidate for further preclinical development against WNV infection. Full article

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV) is a dominant arthropod-borne disease in Asian countries. However, effective antiviral treatment for JEV has not yet been established. 2H4 is a previously identified mouse monoclonal antibody (mAb) which exhibited neutralizing activity against JEV infection. Herein, we designed a novel mAb F(ab’)₂ 2A10-2H4-CDR by transplanting the complementarity-determining regions (CDRs) of 2H4 into the corresponding regions of a murine mAb 2A10 which has high homology with human mAb. We further expressed the recombinant human–mouse chimeric mAb 2A10-2H4-CDR-hFc by linking 2A10-2H4-CDR with CH2 and CH3 domains of one human mAb. The results of indirect immunofluorescence assay and ELISA show that 2A10-2H4-CDR-hFc can recognize the E proteins of JEV and Zika virus (ZIKV), similar to its original form 2H4. Moreover, 2A10-2H4-CDR-hFc displayed neutralizing activities against JEV and ZIKV equivalent to that of 2H4 in vitro (NT₅₀ value against JEV = 0.079 μg/mL versus 0.022 μg/mL, respectively; NT₅₀ value against ZIKV = 1.584 μg/mL versus 0.446 μg/mL, respectively). Both 2H4 and 2A10-2H4-CDR-hFc significantly increased the survival and reduced the serum viral burden of mice challenged by JEV or ZIKV. This study successfully validates an anti-JEV and ZIKV human–mouse chimeric mAb and establishes a basis for future application of this Ab in preventing or/and treating of both JEV and ZIKV infections. Full article

Enzyme-Linked-Immunosorbent-Spot (ELISpot) is a highly sensitive technique capable of detecting low-level immune responses, offering critical insights into therapy-induced immune activation. Our mouse interferon-gamma (mIFN-γ) ELISpot assay was originally based on a monoclonal capture antibody and a rabbit polyclonal detection antibody. The objective of our study was to replace the polyclonal detection antibody with a monoclonal alternative, using a llama immune library and phage display technology. A llama was immunized with recombinant mIFN-γ, and an immune VHH library was constructed. The library underwent two rounds of panning using the recombinant antigen. Subsequently, 190 clones were screened by Enzyme-Linked-Immunosorbent Assay (ELISA), yielding 27 specific binders to mIFN-γ. Sequence analysis revealed 24 unique clones grouped into four families based on their CDR3-VH sequences. One representative clone from each family was reformatted as VHH-Human Fragment Crystallizable (VHH-hFc) fusion and produced recombinantly for testing in the ELISpot assay. The purified candidates were evaluated in pairs on native mIFN-γ from mouse splenocytes. Two candidates, H3 and G4, were selected for further trial. Comparative analysis of ELISpot performance showed that G4 is a promising substitute for the original rabbit polyclonal antibody, enhancing the overall performance of the mIFN-γ ELISpot assay. This study highlights the potential of VHH antibodies in ELISpot applications and supports their use as a robust, reproducible alternative to polyclonal antibodies. Full article

Background: Rapid and high-throughput diagnostic methods are essential for controlling the spread of infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Lateral flow immunoassay (LFIA) strips provide a cost-effective and user-friendly platform for point-of-care testing. However, the sensitivity of conventional LFIA kits is often limited by the performance of their detection probes. This study reports a highly sensitive LFIA strip for detecting the SARS-CoV-2 nucleocapsid (NP) protein using platinum-decorated poly(2-vinylpyridine) nanoparticles (Pt-P2VPs) as probes. Methods: Monoclonal antibodies against SARS-CoV-2 NP were conjugated with Pt-P2VPs and incorporated into LFIA strips. The test line was coated with anti–SARS-CoV-2 NP monoclonal antibody, and the control line with goat anti-mouse IgG. Recombinant proteins, viral strains, and nasopharyngeal swab specimens from patients were used to evaluate assay performance, with reverse transcription polymerase chain reaction (RT-PCR) as the reference standard. Diagnostic accuracy was assessed using nonparametric statistical tests. Results: Pt-P2VP-based LFIA strips enabled sensitive detection of recombinant NP and inactivated SARS-CoV-2, with minimal cross-reactivity. In 200 clinical specimens (100 PCR-negative and 100 PCR-positive), the assay achieved 74% sensitivity and 100% specificity, with strong correlation to viral RNA load. Compared with conventional LFIA kits, Pt-P2VP strips demonstrated superior sensitivity at lower viral loads. Conclusions: Pt-P2VPs represent a promising probe material for enhancing LFIA performance and may facilitate the development of rapid, sensitive, and scalable immunoassays for infectious disease diagnostics in biomedical applications. Full article

Somatic embryogenesis (SE) is a morphogenetic pathway widely employed in the commercial micropropagation of plants. This route enables the generation of somatic embryos from somatic tissues, which give rise to complete (bipolar) plants that develop like zygotic embryos. SE can proceed via direct or indirect pathways, and both approaches have been adapted not only for large-scale clonal propagation but also for the regeneration of genetically modified plants. In this context, SE can be harnessed as a versatile platform for recombinant protein production, including vaccine antigens and therapeutic proteins, by combining plant tissue culture with genetic transformation strategies. Successful examples include non-model plants, as Daucus carota and Eleutherococcus senticosus expressing the cholera and heat-labile enterotoxin B subunits, respectively; Oryza sativa, Nicotiana tabacum, and Medicago sativa producing complex proteins such as human serum albumin (HSA), α₁-antitrypsin (AAT), and monoclonal antibodies. However, challenges remain in optimizing transformation efficiency, scaling up bioreactor-based suspension cultures, and ensuring proper post-translational modifications under Good Manufacturing Practice (GMP) standards. Recent advances in synthetic biology, modular vector design, and glycoengineering have begun to address these limitations, improving control over transcriptional regulation and protein quality. This review highlights the application of SE as a biotechnological route for recombinant protein production, discusses current challenges, and presents innovative strategies and perspectives for the development of sustainable plant-derived biopharmaceutical systems. Full article

The Japanese encephalitis virus (JEV) remains a major cause of viral encephalitis in Asia, and recent epidemiological shifts driven by the predominance of genotype I and the re-emergence of genotype V have renewed concerns regarding control efforts. Licensed vaccines have a reduced incidence of more than 90% in several endemic regions; however, evidence of reduced cross-neutralization against heterologous genotypes indicates that vaccines derived from genotype III strains may not fully match the evolving antigenic landscape. This review synthesizes current knowledge on vaccine performance, genotype-driven antigenic variation, and implications for future strain alignment. Emerging platforms, including mRNA, DNA, virus-like particles, and structure-guided recombinant antigens, have been evaluated for their potential to enhance cross-genotype breadth, scalability, and thermostability. We also summarize the progress in antiviral discovery targeting viral nonstructural proteins, host pathways, and monoclonal antibody development, along with immunomodulatory and neuroprotective strategies. Translational challenges, such as blood–brain barrier penetration, therapeutic timing, and durability of immunity, have been highlighted as key barriers to clinical application. By integrating molecular, immunological, and epidemiological evidence, this review outlines strategic directions for developing broad-spectrum vaccines and therapeutics capable of addressing the evolving genetic and ecological landscape of JEV. Full article

African swine fever virus (ASFV) is a highly contagious pathogen causing African swine fever in wild boars, warthogs and domestic pigs. The disease leads tosubstantial economic losses to the global pork industry and poses a grave threat to biodiversity. The early-encoded structural protein p22, owing to its immunodominant characteristics and high conservation across most genotypes, represents a promising diagnostic target and subunit vaccine candidate. In this study, the soluble extracellular domain of p22 protein (aa 30–177) was successfully expressed and purified, yielding 1.220 g/L. Eleven strains of monoclonal antibodies against p22 were generated, with four selected for B-cell epitope screening. Bioinformatic prediction-guided design was employed to generate overlapping truncations and peptides for epitope mapping. Based on those strategies, three novel linear B-cell epitopes were identified to be ³⁰KKQQPPKK³⁷, ¹³⁰WGTDDCTG¹³⁷ and ¹⁵⁰YVYNNPHH¹⁵⁷ by monoclonal antibodies. Sequence alignment across ASFV isolates revealed 100% evolutionary conservation in genotypes I/II, with minor variation in genotypes IV/VIII/XX/XXII. This study provided valuable data for broadening the ASFV antigen spectrum and identifying immunological targets for subunit vaccine formulation strategies. Full article

Protein engineering is a rapidly evolving field that plays a critical role in transforming drug discovery and development. This innovative field harnesses the unique structural and functional properties of engineered proteins, such as monoclonal antibodies, nanobodies, therapeutic enzymes, and cytokines, to address complex diseases more effectively than traditional small-molecule drugs. These biologics not only enhance therapeutic specificity but also minimize adverse effects, marking a significant advancement in patient care. However, the journey of protein engineering is not without challenges. Issues related to protein folding, stability, and potential immunogenicity pose significant complications. Additionally, navigating the complex regulatory landscape can delay the transition from laboratory to clinical application. Addressing these hurdles requires the integration of cutting-edge technologies, including phage and yeast display technology, CRISPR, and advanced computational modeling, which enhance the predictability and efficiency of protein design. In this review, we explore the multifaceted impact of protein engineering on modern medicine, highlighting its potential to transform treatment paradigms, methodologies, challenges, and the successful development and approval of recombinant protein-based therapies. By navigating the complexities and leveraging technological advancements, the field is poised to unlock new therapeutic possibilities, ultimately improving patient outcomes and transforming healthcare. Full article

Biopharmaceuticals are medical drugs produced by means of biotechnology. In contrast with small-molecule (traditional) drugs, which are usually synthesized chemically, biopharmaceuticals are derived from biological sources, including tissues, cells, or microorganisms. Biopharmaceuticals comprise a wide extent of therapies, such as vaccines, monoclonal antibodies, cell therapies, recombinant proteins, and gene therapies, as well as biosimilars. These products are designed to become important treatment options for different diseases, including cancer, autoimmune pathological disorders, andinfectious diseases. The development of biopharmaceuticals often includes multifaceted processes, involving genetic engineering and cellular culture techniques, to guarantee efficacy and safety. Accordingly, biopharmaceuticals’ legislation is a key component for ensuring the highest quality of medical products, as well as protecting public health. As a rapidly developing area inside the pharmaceutical industry, biopharmaceuticals represent a significant advancing stage in modern medicine, offering targeted therapies that can improve patient outcomes. Accordingly, this paper seeks to provide current state-of-the-art didactic information, including better insight into various challenges related to biopharmaceuticals’ development, classification, medical use, legislation and ethics. Full article

African swine fever virus (ASFV) is the only member of the family Asfarviridae and can cause African swine fever, a disease with a consistently high mortality rate. The pE248R protein, a myristoylated integral membrane protein of ASFV, is required for virus infectivity and some early postentry event, making it a key target for studying the prevention and treatment of ASFV. In this study, BALB/c mice were immunized with purified recombinant pE248R protein, leading to the generation of five monoclonal antibodies (mAbs). Selected mAbs were subsequently subjected to further characterization. By identifying the reactivity of different pE248R protein peptide segments with these monoclonal antibodies, we screened and identified a linear B cell epitope (⁸⁷QEVALTQWMDAG⁹⁸) on the pE248R protein. These results provide a new theoretical basis for analyzing the structure and function of pE248R protein, particularly contributing to the construction of a comprehensive B-cell epitope map for ASFV immunogens. Full article

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology. Full article