Search Results (20)

CD38 is a multifunctional enzyme that plays a pivotal role in NAD⁺ metabolism and calcium signaling, and its abnormal activity is closely associated with multiple myeloma, age-related metabolic decline, neurodegenerative diseases, and other disorders. Although monoclonal antibodies such as daratumumab have been approved for clinical application, their inherent limitations necessitate the development of novel small-molecule CD38 inhibitors. In this study, we employed DNA-encoded library (DEL) technology for the high-throughput screening of CD38 inhibitors, using a DEL library containing more than 100,000 unique compounds to screen against recombinant human CD38. A total of 1043 enriched compounds were initially identified, and after rigorous validation and screening to exclude non-specific binding and previously reported active compounds, eight hit compounds with diverse chemical scaffolds were obtained, among which Fenbendazole—a clinically approved antiparasitic drug—was included. Surface plasmon resonance (SPR) assays confirmed the direct binding of these hit compounds to CD38, with dissociation constants (KD) ranging from 7.74 × 10⁻⁵ M to 2.15 × 10⁻⁴ M. Fluorescence-based enzymatic activity assays demonstrated that these compounds exert dose-dependent inhibitory effects on both the hydrolase (with ε-NAD as substrate) and cyclase (with NGD as substrate) activities of CD38. Further structure–activity relationship (SAR) analysis of Fenbendazole analogues revealed the critical structural features that regulate CD38 inhibitory potency, and Flubendazole was found to exhibit excellent inhibitory activity, with an IC₅₀ of 14.78 ± 4.21 μM against CD38 hydrolase and 26.31 ± 3.40 μM against cyclase. Molecular docking and 100 ns molecular dynamics (MD) simulations further elucidated the molecular mechanism of CD38 inhibition by lead compounds, confirming that van der Waals interactions are the main driving force for the binding of small-molecule ligands to CD38, with conserved aromatic residues in the active site mediating ligand recognition. This study validates DEL technology as an efficient and reliable platform for the discovery of CD38 inhibitors, and the identified lead compounds—especially Fenbendazole and its analog Flubendazole—provide valuable molecular scaffolds for the further structural optimization of CD38 inhibitors. These findings lay a solid foundation for the development of novel therapeutic agents for the treatment of CD38-associated diseases. Full article

Deoxyribonucleic acid (DNA) vaccines have re-emerged as a versatile and scalable platform by advances in synthetic biology and delivery systems, positioning them as powerful tools in the post-mRNA vaccine era. Historically considered less potent than viral or mRNA-based platforms, recent breakthroughs have dramatically improved their immunogenicity, safety, and precision. These innovations include synthetic gene circuits, self-amplifying DNA (saDNA), and DNA-encoded monoclonal antibodies (DMAbs), which enable programmable expression and robust immune activation. Clinically, DNA vaccines are expanding into diverse applications, from infectious disease prevention to therapeutic cancer immunotherapy and treatment of immune-mediated conditions. Compared to mRNA vaccines, DNA vaccines offer compelling advantages in terms of thermal stability, ease of manufacturing, and long-term storage. Furthermore, novel adjuvants, electroporation methods, and formulation strategies such as lyophilization and encapsulation continue to broaden their clinical potential. This review explores the full scope of DNA vaccine technology and its engineering foundations, emerging disease applications, and interdisciplinary innovations, while evaluating its comparative performance and future role in global vaccine strategy. With an emphasis on both mechanistic insights and translational feasibility, we propose a roadmap to integrate DNA vaccines into the next generation of precision immunotherapy. Full article

Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for full-length cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the C_H1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression. Full article

Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare thrombotic disorder first identified in 2021 as a catastrophic syndrome associated with anti-SARS-CoV-2 adenoviral vector (AdV)-vaccine administration. It is characterized by the presence of oligo- or monoclonal anti-PF4 antibodies able to induce in vitro platelet activation in the presence of PF4. In addition to this immune-based pathomechanism, random splicing events of the Adv-vector DNA encoding for SARS-CoV-2 spike protein resulting in the secretion of soluble spike variants have been postulated as a possible pathophysiological mechanism. More recently, some novel clinical-pathological anti-PF4-associated entities also characterized by thrombosis, thrombocytopenia, and VITT-like antibodies but independent from heparin or AdV-vaccine administration have been identified. To date, these VITT-like disorders have been reported following the administration of vaccines different from anti-SARS-CoV-2 AdV-vaccines, like human papillomavirus (HPV) and mRNA-based COVID-19 vaccines, following a bacterial or viral respiratory infection, and in patients with a monoclonal gammopathy of undetermined significance. The purpose of this review is to provide an update on the knowledge on VITT pathogenesis, focusing on recent findings on anti-PF4 antibodies, on a possible genetic predisposition to VITT, on VITT-antibody intracellular activated pathways, on lipid metabolism alterations, and on new VITT-like disorders. Full article

Staphylococcal enterotoxins (SEs) are major contributors to foodborne intoxications. Reliable detection methods for SEs are essential to maintain food safety and protect public health. Since the heat-stable toxins also exert their toxic effect in the absence of the bacterium, reliance on DNA detection alone can be misleading: it does not allow for determining which specific toxins encoded by a given strain are produced and epidemiologically linked with a given outbreak. Commercially available diagnostic assays for SE detection are so far limited in sensitivity and specificity as well as in the range of targeted toxins (SEA–SEE), thus non-targeted SEs linked to foodborne illness remain undetected at the protein level. This study aimed to develop a highly sensitive and specific multiplex suspension immunoassay (SIA) for SEA to SEI. To this end, high-affinity monoclonal antibodies (mAbs) for the specific detection of the individual SEs were generated. When implemented in sandwich ELISAs and multiplex SIA, these mAbs demonstrated exceptional sensitivity with detection limits in the low picogram per millilitre range. When applied for the analysis of SE production in liquid cultures of a panel of 145 whole-genome sequenced strains of Staphylococcus spp. and Enterococcus faecalis, the novel multiplex SIA detected and differentiated the eight SEs with assay accuracies of 86.9–100%. Notably, the multiplex SIA covered one to four sequence variants for each of the individual SEs. Validation confirmed high recovery rates and reliable performance in three representative complex food matrices. The implementation of the novel mAbs in a multiplex SIA enabled, for the first time, simultaneous detection, differentiation, and quantification of multiple SEs from minimal sample volumes using Luminex^® technology. As a result, the multiplex SIA will help strengthen food safety protocols and public health response capabilities. Full article

Background: Monoclonal antibodies against the sodium-dependent phosphate transporter NaPi2b (SLC34A2) represent a promising approach in the treatment of ovarian and lung cancer. Of particular interest is the potential cancer-specific MX35 epitope of NaPi2b, as it serves as a target for monoclonal antibodies studied at various stages of preclinical and clinical trials. However, variations in the NaPi2b protein structure may limit the efficacy of therapeutic antibodies by affecting the accessibility of the MX35 epitope. Methods: An in silico analysis was performed using data from 101,562 tumor samples. Genomic DNA sequencing was conducted on blood samples from patients with ovarian carcinoma, breast cancer, and renal carcinoma to access the frequency of germline mutations in the SLC34A2 gene region encoding the MX35 epitope. To assess the impact of the selected mutation, we generated a model cell line through site-directed mutagenesis carrying the mutant NaPi2b variant. Results: Using in silico analysis, we identified 17 unique variants in the SLC34A2 gene leading to amino acid substitutions within the MX35 epitope of the NaPi2b. Among these, the most prevalent mutation, c.989C>T, resulting in p.T330M substitution, was detected in 5 out of 64 patients through genomic DNA sequencing. Using site-directed mutagenesis, we created the OVCAR-8/NaPi2b^p.T330M model cell line. L3 (28/1) monoclonal antibodies specific to the MX35 epitope failed to recognize the mutant NaPi2b^p.T330M variant compared to the wild-type of the NaPi2b in both Western blot and confocal microscopy experiments. Conclusions: The obtained data may serve as a basis for predicting the efficacy of monoclonal antibody-based targeted therapy binding to the MX35 epitope of NaPi2b in the treatment of oncological diseases. Full article

Background: Monoclonal antibodies (mAbs) are potent treatment options for infectious diseases. The rapid isolation and in vivo validation of therapeutic mAb candidates, including mAb cocktails, are essential to combat novel or rapidly mutating pathogens. The rapid selection and production of mAb candidates in sufficient amount and quality for preclinical studies are a major limiting step in the mAb development pipeline. Methods: Here, we developed a method to facilitate the screening of therapeutic mAbs in mouse models. Four conventional mAbs were transformed into single-chain variable fragments fused to the fragment crystallizable (Fc) region of a human IgG1 (scFv-IgG). These scFv-IgG were expressed individually or as a cocktail in vitro and in mice following transfection or hydrodynamic delivery of the corresponding plasmids. Results: This method induced high expression of all scFv-IgG and provided protection in two murine infection models. Conclusions: This study highlights the benefits of this approach for the rapid, low-cost screening of therapeutic mAb candidates. Full article

The African swine fever virus (ASFV) is a large enveloped DNA virus that causes a highly pathogenic hemorrhagic disease in both domestic pigs and wild boars. The ASFV genome contains a double-stranded DNA encoding more than 150 proteins. The ASFV possesses only one protease, pS273R, which is important for virion assembly and host immune evasion. Therefore, the specific monoclonal antibody (mAb) against pS273R is useful for ASFV research. Here, we generated two specific anti-pS273R mAbs named 2F3 and 3C2, both of which were successfully applied for ELISA, Western blotting, and immunofluorescence assays. Further, we showed that both 2F3 and 3C2 mAbs recognize a new epitope of N terminal 1–25 amino acids of pS273R protein, which is highly conserved across different ASFV strains including all genotype I and II strains. Based on the recognized epitope, an indirect ELISA was established and was effective in detecting antibodies during ASFV infection. To conclude, the specific pS273R mAbs and corresponding epitope identified will strongly promote ASFV serological diagnosis and vaccine research. Full article

The African swine fever virus (ASFV) is an ancient, structurally complex, double-stranded DNA virus that causes African swine fever. Since its discovery in Kenya and Africa in 1921, no effective vaccine or antiviral strategy has been developed. Therefore, the selection of more suitable vaccines or antiviral targets is the top priority to solve the African swine fever virus problem. B125R, one of the virulence genes of ASFV, encodes a non-structural protein (pB125R), which is important in ASFV infection. However, the epitope of pB125R is not well characterized at present. We observed that pB125R is specifically recognized by inactivated ASFV-positive sera, suggesting that it has the potential to act as a protective antigen against ASFV infection. Elucidation of the antigenic epitope within pB125R could facilitate the development of an epitope-based vaccine targeting ASFV. In this study, two strains of monoclonal antibodies (mAbs) against pB125R were produced by using the B cell hybridoma technique, named 9G11 and 15A9. The antigenic epitope recognized by mAb 9G11 was precisely located by using a series of truncated ASFV pB125R. The ⁵²DPLASQRDIYY⁶² (epitope on ASFV pB125R) was the smallest epitope recognized by mAb 9G11 and this epitope was highly conserved among different strains. The key amino acid sites were identified as D52, Q57, R58, and Y62 by the single-point mutation of 11 amino acids of the epitope by alanine scanning. In addition, the immunological effects of the epitope (pB125R-DY) against 9G11 were evaluated in mice, and the results showed that both full-length pB125R and the epitope pB125R-DY could induce effective humoral and cellular immune responses in mice. The mAbs obtained in this study reacted with the eukaryotic-expressed antigen proteins and the PAM cell samples infected with ASFV, indicating that the mAb can be used as a good tool for the detection of ASFV antigen infection. The B cell epitopes identified in this study provide a fundamental basis for the research and development of epitope-based vaccines against ASFV. Full article

DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This study reports the quantitative evaluation and localization of the administered pDNA over time and its association with corresponding mRNA levels and systemic protein concentrations. pDNA encoding the murine anti-HER2 4D5 mAb was administered to BALB/c mice via intramuscular injection followed by electroporation. Muscle biopsies and blood samples were taken at different time points (up to 3 months). In muscle, pDNA levels decreased 90% between 24 h and one week post treatment (p < 0.0001). In contrast, mRNA levels remained stable over time. The 4D5 antibody plasma concentrations reached peak levels at week two followed by a slow decrease (50% after 12 weeks, p < 0.0001). Evaluation of pDNA localization revealed that extranuclear pDNA was cleared fast, whereas the nuclear fraction remained relatively stable. This is in line with the observed mRNA and protein levels over time and indicates that only a minor fraction of the administered pDNA is ultimately responsible for the observed systemic mAb levels. In conclusion, this study demonstrates that durable expression is dependent on the nuclear uptake of the pDNA. Therefore, efforts to increase the protein levels upon pDNA-based gene therapy should focus on strategies to increase both cellular entry and migration of the pDNA into the nucleus. The currently applied methodology can be used to guide the design and evaluation of novel plasmid-based vectors or alternative delivery methods in order to achieve a robust and prolonged protein expression. Full article

Monoclonal antibodies (mAbs), the new revolutionary class of medications, are fast becoming tools against various diseases thanks to a unique structure and function that allow them to bind highly specific targets or receptors. These specialized proteins can be produced in large quantities via the hybridoma technique introduced in 1975 or by means of modern technologies. Additional methods have been developed to generate mAbs with new biological properties such as humanized, chimeric, or murine. The inclusion of mAbs in therapeutic regimens is a major medical advance and will hopefully lead to significant improvements in infectious disease management. Since the first therapeutic mAb, muromonab-CD3, was approved by the U.S. Food and Drug Administration (FDA) in 1986, the list of approved mAbs and their clinical indications and applications have been proliferating. New technologies have been developed to modify the structure of mAbs, thereby increasing efficacy and improving delivery routes. Gene delivery technologies, such as non-viral synthetic plasmid DNA and messenger RNA vectors (DMabs or mRNA-encoded mAbs), built to express tailored mAb genes, might help overcome some of the challenges of mAb therapy, including production restrictions, cold-chain storage, transportation requirements, and expensive manufacturing and distribution processes. This paper reviews some of the recent developments in mAb discovery against viral infections and illustrates how mAbs can help to combat viral diseases and outbreaks. Full article

Coronavirus disease 2019 (COVID-19) caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the most severe health crisis, causing extraordinary economic disruption worldwide. SARS-CoV-2 is a single-stranded RNA-enveloped virus. The process of viral replication and particle packaging is finished in host cells. Viral proteins, including both structural and nonstructural proteins, play important roles in the viral life cycle, which also provides the targets of treatment. Therefore, a better understanding of the structural function of virus proteins is crucial to speed up the development of vaccines and therapeutic strategies. Currently, the structure and function of proteins encoded by the SARS-CoV-2 genome are reviewed by several studies. However, most of them are based on the analysis of SARS-CoV-1 particles, lacking a systematic review update for SARS-CoV-2. Here, we specifically focus on the structure and function of proteins encoded by SARS-CoV-2. Viral proteins that contribute to COVID-19 infection and disease pathogenesis are reviewed according to the most recent research findings. The structure-function correlation of viral proteins provides a fundamental rationale for vaccine development and targeted therapy. Then, current antiviral vaccines are updated, such as inactive viral vaccines and protein-based vaccines and DNA, mRNA, and circular RNA vaccines. A summary of other therapeutic options is also reviewed, including monoclonal antibodies such as a cross-neutralizer antibody, a constructed cobinding antibody, a dual functional monoclonal antibody, an antibody cocktail, and an engineered bispecific antibody, as well as peptide-based inhibitors, chemical compounds, and clustered regularly interspaced short palindromic repeats (CRISPR) exploration. Overall, viral proteins and their functions provide the basis for targeted therapy and vaccine development. Full article

Natural killer (NK)/T-cell lymphomas are aggressive malignancies. Epstein–Barr virus (EBV) infection in lymphoma cells is invariable. NK/T-cell lymphomas are divided into nasal, non-nasal, and disseminated subtypes. Nasal NK/T-cell lymphomas involve the nasal cavity and the upper aerodigestive tract. Non-nasal NK/T-cell lymphomas involve the skin, gastrointestinal tract, testis and other extranodal sites. Disseminated NK/T-cell lymphoma involves multiple organs, rarely presenting with a leukaemic phase. Lymphoma cells are positive for CD3ε (not surface CD3), CD56, cytotoxic molecules and EBV-encoded small RNA. There is a predilection for Asian and Central/South American populations. Genome-wide association studies have identified lymphoma susceptibility loci in Asian patients. Positron emission tomography computed tomography and plasma EBV DNA quantification are crucial evaluations at diagnosis and follow-up. Stage I/II patients typically receive non-athracycline regimens containing asparaginse, together with sequential/concurrent radiotherapy. Anthracycline-containing regimens are ineffective. Stage III/IV patients are treated with asparaginase-containing regimens, followed by allogeneic haematopoietic stem cell transplantation (HSCT) in suitable cases. Autologous HSCT does not improve outcome. In relapsed/refractory patients, novel approaches are needed, involving PD1/PD-L1 targeting, EBV-specific cytotoxic T-cells, and monoclonal antibodies. Small molecules including histone deacetylase inhibitors may be beneficial in selected patients. Future strategies may include targeting of signalling pathways and driver mutations. Full article

Since the first approval of monoclonal antibodies by the United States Food and Drug Administration (FDA) in 1986, therapeutic antibodies have become one of the predominant classes of drugs in oncology and immunology. Despite their natural function in contributing to antiviral immunity, antibodies as drugs have only more recently been thought of as tools for combating infectious diseases. Passive immunization, or the delivery of the products of an immune response, offers near-immediate protection, unlike the active immune processes triggered by traditional vaccines, which rely on the time it takes for the host’s immune system to develop an effective defense. This rapid onset of protection is particularly well suited to containing outbreaks of emerging viral diseases. Despite these positive attributes, the high cost associated with antibody manufacture and the need for a cold chain for storage and transport limit their deployment on a global scale, especially in areas with limited resources. The in vivo transfer of nucleic acid-based technologies encoding optimized therapeutic antibodies transform the body into a bioreactor for rapid and sustained production of biologics and hold great promise for circumventing the obstacles faced by the traditional delivery of antibodies. In this review, we provide an overview of the different antibody delivery strategies that are currently being developed, with particular emphasis on in vivo transfection of naked plasmid DNA facilitated by electroporation. Full article

H7 subtype avian influenza viruses have caused outbreaks in poultry, and even human infection, for decades in both Eurasia and North America. Although effective vaccines offer the best protection against avian influenza viruses, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of cross-protective vaccine candidates. In this study, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to develop four consensus H7 antigens (CH7-22, CH7-24, CH7-26, and CH7-28). In vitro experiments confirmed the binding of monoclonal antibodies to the head and stem domains of cell surface-expressed consensus HAs, indicating display of their antigenicity. Immunization with DNA vaccines encoding the four antigens was evaluated in a mouse model. Broadly reactive antibodies against H7 viruses from Eurasian and North American lineages were elicited and detected by binding, inhibition, and neutralizing analyses. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that CH7-22 and CH7-24 conferred the most effective protection against hetero-lethal challenge. Our data showed that the consensus H7 vaccines elicit a broadly reactive, protective response against Eurasian and North American lineage H7 viruses, which are suitable for development against other zoonotic influenza viruses. Full article