Search Results (428)

This study characterized collagen remodeling in an electron-beam-sterilized porcine acellular dermal matrix (E-PADM) by evaluating host response kinetics during wound healing. E-PADM (n = 6 lots/time point) was implanted in an abdominal wall bridging defect in nonhuman primates (N = 24). Histological, immunohistochemical, and biochemical assessments were conducted. Pro-inflammatory tissue cytokines peaked 1 month post-implantation and subsided to baseline by 6 months. E-PADM-specific serum immunoglobulin G antibodies increased by 213-fold from baseline at 1 month, then decreased to <10-fold by 6–9 months. The mean percentage tissue area staining positively for matrix metalloproteinase-1 plateaued at 3 months (40.3 ± 16.9%), then subsided by 6 months (16.3 ± 11.1%); tissue inhibitor matrix metalloproteinase-1 content plateaued at 1 month (39.0 ± 14.3%), then subsided by 9 months (13.0 ± 8.8%). Mean E-PADM thickness (1.7 ± 0.2 mm pre-implant) increased at 3 months (2.9 ± 1.5 mm), then decreased by 9 months (1.9 ± 1.1; equivalent to pre-implant). Histology demonstrated mild inflammation between 1–3 months, then a peak in host tissue deposition, with ≈75%–100% E-PADM collagen turnover, and fibroblast infiltration and neovascularization between 3–6 months. Picrosirius red staining revealed that mature E-PADM collagen was replaced by host-associated neo-collagen by 6 months. E-PADM implantation induced wound healing, which drove dermal E-PADM collagen remodeling to native, functional fascia-like tissue at the implant site. Full article

Oxidized phospholipids (OxPLs) are increasingly recognized as biologically active lipids involved in various pathologies. Both exposure to pathogenic factors and the efficacy of protective mechanisms are critical to disease development. In this study, we characterized an immunoassay that quantified the total capacity of the plasma to degrade or mask OxPLs, thereby preventing their interaction with cells and soluble proteins. OxLDL-coated plates were first incubated with human blood plasma or a control vehicle, followed by an ELISA using a monoclonal antibody specific to oxidized phosphatidylethanolamine. Pretreatment with the diluted blood plasma markedly inhibited mAb binding. The masking assay was optimized by evaluating the buffer composition, the compatibility with various anticoagulants, potential interfering compounds, the kinetic parameters, pre-analytical stability, statistical robustness, and intra- and inter-individual variability. We propose that this masking assay provides a simple immunological approach to assessing protective mechanisms against lipid peroxidation products. Establishing this robust and reproducible method is essential for conducting clinical association studies that explore masking activity as a potential biomarker of the predisposition to a broad range of lipid-peroxidation-related diseases. Full article

PRRSV continues to evolve, complicating its epidemiological landscape in China. In this study, we isolated a novel PRRSV strain, GZ2022, from a swine farm in Guizhou Province. Subsequent analyses performed on this isolate included complete genome sequencing, phylogenetic analysis, recombination assessment, and characterization of its biological properties. Phylogenetic analysis revealed that GZ2022 clusters within Lineage 1 (NADC30-like) and features a 131-amino-acid deletion in NSP2, consistent with NADC30-derived strains. Recombination analysis identified NADC30 as the major parental strain (75% genomic contribution), with a minor recombinant region (25%) derived from the highly pathogenic HuN4 strain. In vitro growth kinetics revealed peak viral titers in Marc-145 cells at 72 h post infection (hpi). Pathogenicity was evaluated in 21-day-old piglets infected with GZ2022, the highly pathogenic PRRSV strain WUH3, or negative controls. Both infected groups exhibited typical PRRS clinical signs (fever, respiratory distress) and histopathological lesions (interstitial pneumonia, pulmonary consolidation). However, GZ2022-infected piglets exhibited attenuated virulence compared to WUH3, with reduced pulmonary hemorrhage and 0% mortality compared to 80% in the WUH3 group. Seroconversion (N-protein antibodies) was observed at 14 dpi (days post inoculation) in GZ2022-infected animals, persisting throughout the 28-day trial. Viral shedding dynamics aligned with moderate pathogenicity. These findings classify GZ2022 as a moderately virulent NADC30-like recombinant strain with partial HuN4-derived genomic regions. The emergence of such strains underscores the need for sustained surveillance of PRRSV genetic diversity and systematic evaluation of the biological properties of novel variants to refine control measures and inform vaccine development. Full article

Background: Since the massive outbreak of foot-and-mouth disease (FMD) in South Korea in 2010–2011, cloven-hoofed livestock have been immunized with serotype O and A vaccines across the country. Other serotypes of FMD vaccines were stockpiled in overseas FMD vaccine factories as antigen banks. Once a manufacturing facility has been established in South Korea, the overseas antigen banks will be replaced by domestic one. Therefore, this study aimed to evaluate the commercial potential of the previously developed SAT2 vaccine candidate (SAT2 ZIM-R). Methods: The optimal condition was determined at various virus concentrations, infection times, and pH levels, resulting in 0.01 MOI for SAT2 ZIM-R for 24 h infection at a pH of 7.5. Results: When the SAT2 ZIM-R virus was produced in flasks from 40 to 1000 mL in fivefold increments, all scales of production yielded > 7.0 µg/mL of antigens. Using a bioreactor, 5.6 µg/mL of antigens was recovered from a 1 L viral culture. The optimal conditions of viral inactivation kinetics were determined to be 1 mM of binary ethyleneimine (BEI) treatment at 26 °C for 24 h, with approximately 91% of the antigen being retained after virus inactivation. When the SAT2 ZIM-R experimental vaccine was administered twice to pigs, the neutralizing antibody titer increased approximately 500-fold after booster immunization. Conclusions: To the best of our knowledge, this is the first study to evaluate the antigen productivity, viral inactivation kinetics, and immunogenicity of the SAT vaccine strain in pigs. In the future, the SAT2 ZIM-R vaccine may be a useful candidate vaccine for a domestic antigen bank. Full article

Background/Objectives: The administration of human monoclonal antibodies (mAb) in preclinical pharmacokinetics and toxicology studies often triggers an immune response, leading to the formation of anti-drug antibodies (ADA). To mitigate this effect, we have recently performed and reported on studies using short-term immunosuppressive regimens to induce prolonged immune tolerance towards a human mAb, erenumab, in rats. Here, we report on the development of a semi-mechanistic modeling approach that quantitatively integrates pharmacokinetic and immunogenicity assessments from immune tolerance induction studies to provide a framework for the simulation-based evaluation of different immune induction scenarios for the maintenance of prolonged immune tolerance towards human mAbs. Methods: The integrated pharmacokinetic/pharmacodynamic (PK/PD) modeling approach combined a semi-mechanistic model of the adaptive immune system to predict ADA formation kinetics with a population pharmacokinetic model to assess the impact of the time course of the ADA magnitude on the PK of erenumab in rats. Model-derived erenumab concentration–time profiles served as input for a quantitative system pharmacology-style semi-mechanistic model of the adaptive immune system to conceptualize the ADA response as a function of the kinetics of CD4⁺ T helper cells and T regulatory cells. Results: The model adequately described the observed ADA magnitude–time profiles in all treatment groups and reasonably simulated the kinetics of selected immune cells responsible for ADA formation. It also successfully captured the impact of tacrolimus/sirolimus immunomodulation on ADA formation, demonstrating that the regimen effectively suppressed ADA formations and induced immune tolerance. Conclusions: This work demonstrates the utility of modeling approaches to integrate pharmacokinetic and immunogenicity assessment data for the prospective planning of long-term toxicology studies to support the preclinical development of mAbs. Full article

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case by targeting P2X4. After nerve injury, ATP release activates/upregulates P2X4 receptors (P2X4R) sequestered in late endosomes, triggering a cascade of chronic pain-related events. Nine humanized scFv (hscFv) variants targeting a specific extracellular 13-amino-acid peptide fragment of human P2X4R were generated via CDR grafting. ELISA analysis revealed nanomolar binding affinities, with most humanized molecules exhibiting comparable or superior affinity compared to the original murine antibody. Octet measurements confirmed that the lead, HC3-LC3, exhibited nanomolar binding kinetics (KD = 2.5 × 10⁻⁹ M). In vivo functional validation with P2X4R hscFv reversed nerve injury-induced chronic pain-related behaviors with a single dose (0.4 mg/kg, intraperitoneal) within two weeks. The return to naïve baseline remained durably reduced > 100 days. In independent confirmation, the spared nerve injury (SNI) model was similarly reduced. This constitutes an original method whereby durable reversals of chronic nerve injury pain, anxiety and depression measures are accomplished. Full article

Accurate detection of biomolecular interactions is essential in many areas, from the detection of the presence of biomarkers in the clinic to the development of therapeutic drugs and biologics in biopharma to the understanding of various biological processes in basic research. Traditional endpoint approaches can suffer from false-negative results for biomolecular interactions with fast kinetics. By contrast, real-time detection techniques like surface plasmon resonance (SPR) monitor interactions as they form and disassemble, reducing the risk of false-negative results. By leveraging cell-free expressed proteins captured on either glass or SPR biosensors and using two different commercial antibodies with variable off-rates that both target HaloTag antigens as a model, we compare and contrast results from a fluorescence endpoint assay versus real-time sensor-integrated proteome on chip (SPOC^®) SPR-based detection. In this study, we illustrate the limitations of the representative immunofluorescent endpoint assay when investigating transient interactions characterized by fast dissociation rates. We highlight the importance of choosing reagents well suited to the selected assay, as well as the importance of considering binding kinetics and protein ligand conformational states when interpreting results from binding assays, especially for applications as critical as the off-target screening of therapeutics. Full article

Background/Objectives: This study examines the longitudinal dynamics of anti-nucleocapsid (anti-N) and anti-spike (anti-S) antibody responses to SARS-CoV-2 infection and mRNA vaccination based on 81,878 serum samples from 23,616 healthcare workers (HCWs) across five European countries. It includes data across four scheduled vaccine doses—predominantly BNT162b2—with 25% of samples originating from individuals with confirmed prior infection, as evidenced by elevated anti-S levels, positive Anti-N antibodies, or PCR results. Methods: The study employed a shifted transformation method for data normalization and utilized the Bass diffusion model to predict antibody titer dynamics influenced by both internal factors—such as immune activation contextualized through sociodemographic issues—and external factors, including infection and vaccination. Despite the absence of direct measurements for some internal variables, the model effectively inferred their impact, enabling a rigorous and nuanced delineation of immune response profiles. Results: The Bass diffusion model rigorously captured variations in antibody titers, analyzed through demographic factors such as gender, age, and job role, while thoroughly accounting for pre-infection status. The results indicate that Anti-N antibodies, exclusively produced post-infection, exhibited a rapid decline, while anti-S antibodies, generated from both infection and vaccination, demonstrated prolonged persistence. A significant decline in anti-S levels was observed 3–5 months post-vaccination, with adaptive immunity—characterized by the dominance of internal factors effects relative to external ones—achieved in most groups after the fourth dose. However, adaptive immunity post second dose was limited to specific demographics. Conclusions: These findings emphasize the significance of the Bass Method in predicting vaccine-induced, hybrid immune responses and detecting adaptive immunity by overcoming limitations in internal factor data, thereby advancing effective vaccination and infection control strategies during public health crises. These findings highlight the Bass Method’s value in predicting vaccine-induced and hybrid immunity, effectively addressing internal factor data gaps to enhance vaccination and infection control strategies. Full article

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity. Full article

SPR biosensors operate on the principle of evanescent wave propagation at metal–dielectric interfaces in total internal reflection conditions, with consequent photonic energy attenuation. This plasmonic excitation occurs in specific conditions of incident light wavelength, angle, and the dielectric refractive index. This principle has been the basis for SPR-based biosensor setups wherein mass/concentration-induced changes in the refractive indices of dielectric media reflect as plasmonic resonance condition changes quantitatively reported as arbitrary response units. SPR biosensors operating on this conceptual framework have been designed to study biomolecular interactions with real-time readout and in label-free setups, providing key kinetic characterization that has been valuable in various applications. SPR biosensors often feature antibodies as target affinity probes. Notably, the operational challenges encountered with antibodies have led to the development of aptamers—oligonucleotide biomolecules rationally designed to adopt tertiary structures, enabling high affinity and specific binding to a wide range of targets. Aptamers have been extensively adopted in SPR biosensor setups with promising clinical and industrial prospects. In this paper, we explore the growing literature on SPR setups featuring aptamers, specifically providing expert commentary on the current state and future implications of these SPR aptasensors for drug discovery as well as disease diagnosis and monitoring. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Botulinum neurotoxin serotype A (BoNT/A), the most toxic of the seven serotypes produced by Clostridium botulinum, poses significant public health risks because of its involvement in foodborne outbreaks and potential use in bioterrorism. In this study, we developed high-affinity monoclonal antibodies for BoNT/A detection using single-cell fluorescence-activated cell sorting and nested PCR. The optimized antibody pair demonstrated exceptional sensitivity, detecting recombinant BoNT/A at concentrations as low as 0.02 ng/mL with a linear range of 0.02–10 ng/mL, while maintaining high specificity against BoNT/B, E, and F. Biolayer interferometry confirmed superior binding kinetics, and a time-resolved fluoroimmunoassay (TRFIA) demonstrated consistent performance in complex food matrices, including ham sausage and soybean paste. These rabbit-derived monoclonal antibodies enable ultrasensitive detection of BoNT/A across diverse food matrices, offering a powerful tool for food safety monitoring and biosecurity. Full article

Porcine deltacoronavirus (PDCoV) is a newly discovered enteropathogenic coronavirus primarily responsible for diarrhea and mortality in piglets, with the potential to infect humans, thereby posing a significant threat to both human health and the global pig industry. Currently, there is no commercially available live-attenuated vaccine for PDCoV. In this study, an isolated virulent PDCoV strain, DHeB1, was continuously passaged in LLC-PK1 cells for up to 110 passages. The virus growth kinetics in cell culture and complete genome sequences of various passages (F11, F40, F70, F90, and F110) were determined. The results indicated significant increases in virus titers at passages F40 and F90. Sequence analysis revealed that only a few single-nucleotide mutations (some of which resulted in amino acid changes) and one nucleotide insertion were observed throughout successive passages. Notably, the eight and seven amino acid mutations that emerged in F40 and F70, respectively, remained stable in subsequent passages and were predominantly located in the S glycoprotein. The pathogenicity of F11, F40, F70, and F90 was assessed in 5-day-old piglets, revealing markedly reduced clinical symptoms, histopathological lesions, and intestinal PDCoV antigen distributions in piglets inoculated with F70 or F90. Importantly, F90 exhibited little to no virulence in piglets. The immunogenicity of F70, F90, and F110 was further evaluated in weaned piglets, with results indicating that the neutralizing antibody titers induced by F70 and F90 were comparable and significantly higher than those induced by F110. Collectively, these findings suggest that the PDCoV strain DHeB1 has been attenuated and can be used to develop a live-attenuated vaccine against PDCoV. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered a promising viral vector for the expression and delivery of foreign genes for the development of a new generation of multi-valent vaccines against PRRSV and other porcine viruses, as well as for analyses of the immune response against PRRSV and anti-PRRSV component screening. In the present study, the junction site between nsp1β and nsp2 in the PRRSV genome was tested for the insertion and expression of foreign genes. Three foreign genes, including eGFP, iLOV3, and TEVp, were inserted into the intergenic junction between nsp1β and nsp2 and expressed by the respective recombinant PRRSVs (rPRRSV-SH01-eGFP, rPRRSV-SH01-iLOV3, and rPRRSV-SH01-TEVp) in vitro in mammalian cells. Analysis of the growth kinetics of the rescued recombinant PRRSVs showed no significant differences between the recombinant PRRSVs and their parental viruses. The inserted genes were consistently present in the viral genome during serial passage in vitro (for at least 20 passages). In addition, rPRRSV-SH01-eGFP can be used as a reporter virus for rapid detection of neutralizing antibodies against PRRSV through a fluorescent focus unit reduction-based assay. These data demonstrate that the junction between nsp1β and nsp2 is a new site that is suitable for the insertion and expression of foreign genes, providing a new option to express and deliver foreign genes using PRRSV-based vectors for different purposes, such as the development of multi-valent vaccines against PRRSV and other porcine viruses. Full article

The use of adalimumab biosimilars has become increasingly common in clinical practice, reflecting their growing acceptance and efficacy as therapeutic alternatives to reference biologics. However, studies investigating the molecular interactions between anti-adalimumab antibodies (AAA) elicited in patients and different adalimumab biosimilars remain limited. This study aims to characterize the kinetic interactions between purified AAA from pediatric patients with Juvenile Idiopathic Arthritis and three adalimumab formulations: the originator Humira^®, and the biosimilars GP2017 (Hyrimoz^®) and SB5 (Imraldi^®). For this purpose, adalimumab formulations were immobilized on a gold chip, and purified AAA were flowed to perform further kinetic analysis using the surface plasmon resonance (SPR) technology. Results showed that the K_D values for purified AAA from patients treated with biosimilars GP2017 (Hyrimoz^®) or SB5 (Imraldi^®) were comparable across all formulations tested, including the originator Humira^®. AAA interacted with Humira^®, Hyrimoz^®, and Imraldi^® with similar apparent affinity (10⁻⁹ M > K_D > 10⁻¹⁰ M); slight variations have been observed among patients, less among biosimilars. The similarity in K_D values across biosimilars and the originator supports the notion that, at the level of immunogenicity, biosimilars can be considered clinically comparable to the originator. Full article