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Science and Technology of Advanced... 2024Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly...
Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.
PubMed: 38817250
DOI: 10.1080/14686996.2024.2351791 -
Frontiers in Immunology 2024Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first...
INTRODUCTION
Current SARS-CoV-2 strains continue to mutate and attempt to evade the antibody response elicited by previous exposures and vaccinations. In September of 2022, the first updated SARS-CoV-2 vaccines, designed to create immune responses specific for the variants circulating in 2022, were approved. These new vaccines, known commonly as the bivalent boost(er), include mRNA that encodes both the original Wuhan-Hu-1 spike protein as well as the spike protein specific to the Omicron BA.4 and BA.5 variants.
METHODS
We recruited volunteers from University of Massachusetts student, faculty and staff members to provide samples of blood and saliva at four different time points, including pre-boost and three times post boost and analyzed samples for antibody production as well as neutralization of virus.
RESULTS
Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response.
CONCLUSION
We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.
Topics: Humans; SARS-CoV-2; Antibodies, Viral; COVID-19; Saliva; COVID-19 Vaccines; Immunization, Secondary; Spike Glycoprotein, Coronavirus; Male; Female; Adult; Antibodies, Neutralizing; Middle Aged; Antibody Formation; Young Adult
PubMed: 38812500
DOI: 10.3389/fimmu.2024.1401209 -
Antibodies (Basel, Switzerland) May 2024The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the...
The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.
PubMed: 38804304
DOI: 10.3390/antib13020036 -
Frontiers in Immunology 2024() is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by in equid...
Expression of IL-10 and TGF-β1 in horses experimentally infected with merozoites is associated with antibody production but not modulation of pro-inflammatory responses.
() is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by in equid hosts impeding understanding of the host parasite relationship and development of potent vaccines for control of infections. The objective of this study was to evaluate the host-parasite dynamics between merozoites and infected horses by assessing cytokine expression during primary and secondary parasite exposure, and to determine whether the pattern of expression correlated with clinical indicators of disease. Our findings showed that the expression of pro-inflammatory cytokines was very low and inconsistent during both primary and secondary infection. There was also no correlation between the symptoms observed during primary infection and expression of the cytokines. This suggests that the symptoms might have occurred primarily due to hemolysis and likely not the undesirable effects of pro-inflammatory responses. However, IL-10 and TGF-β1 were highly expressed in both phases of infection, and their expression was linked to antibody production but not moderation of pro-inflammatory cytokine responses.
Topics: Animals; Horses; Theileriasis; Interleukin-10; Theileria; Transforming Growth Factor beta1; Horse Diseases; Merozoites; Antibodies, Protozoan; Antibody Formation; Cytokines; Host-Parasite Interactions
PubMed: 38803499
DOI: 10.3389/fimmu.2024.1370255 -
Journal of Pharmaceutical Sciences May 2024Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the...
Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.
PubMed: 38801973
DOI: 10.1016/j.xphs.2024.05.024 -
BioRxiv : the Preprint Server For... May 2024B cell activation is accompanied by dynamic metabolic reprogramming, supported by a multitude of nutrients that include glucose, amino acids and fatty acids. While...
B cell activation is accompanied by dynamic metabolic reprogramming, supported by a multitude of nutrients that include glucose, amino acids and fatty acids. While several studies have indicated that fatty acid mitochondrial oxidation is critical for immune cell functions, contradictory findings have been reported. Carnitine palmitoyltransferase II (CPT2) is a critical enzyme for long-chain fatty acid oxidation in mitochondria. Here, we test the requirement of CPT2 for humoral immunity using a mouse model with a lymphocyte specific deletion of CPT2. Stable C isotope tracing reveals highly reduced fatty acid-derived citrate production in CPT2 deficient B cells. Yet, CPT2 deficiency has no significant impact on B cell development, B cell activation, germinal center formation, and antibody production upon either thymus-dependent or -independent antigen challenges. Together, our findings indicate that CPT2 mediated fatty acid oxidation is dispensable for humoral immunity, highlighting the metabolic flexibility of lymphocytes.
PubMed: 38798358
DOI: 10.1101/2024.05.15.594133 -
BioRxiv : the Preprint Server For... May 2024Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet...
Platelets are highly reactive fragments of megakaryocytes that play a fundamental role in thrombosis and hemostasis. Predictably, all conventional anti-platelet therapies elicit bleeding, raising the question whether the thrombotic activity of platelets can be targeted separately. In this study, we describe a novel approach of inhibiting platelet activation through the use of bispecific single-chain variable fragments (bi-scFvs), termed cis-acting platelet receptor inhibitors (CAPRIs) that harness the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing co-inhibitory receptor G6b-B (G6B) to suppress immunoreceptor tyrosine-based (ITAM)-containing receptor-mediated platelet activation. CAPRI-mediated hetero-clustering of G6B with either the ITAM-containing GPVI-FcR γ-chain complex or FcγRIIA (CD32A) inhibited collagen- or immune complex-induced platelet aggregation. G6B-GPVI CAPRIs strongly and specifically inhibited thrombus formation on collagen under arterial shear, whereas G6B-CD32A CAPRI strongly and specifically inhibited thrombus formation to heparin-induced thrombocytopenia, vaccine-induced thrombotic thrombocytopenia and antiphospholipid syndrome complexes on Von Willebrand Factor-coated surfaces and photochemical-injured endothelial cells under arterial shear. Our findings provide proof-of-concept that CAPRIs are highly effective at inhibiting ITAM receptor-mediated platelet activation, laying the foundation for a novel family of anti-thrombotic therapeutics with potentially improved efficacy and fewer bleeding outcomes compared with current anti-platelet therapies. .
PubMed: 38798354
DOI: 10.1101/2024.05.10.593500 -
International Dental Journal May 2024The purpose of this work was to develop an anti-CAT-SYI immunoglobulin Y (IgY) antibody that targeted both GtfB (glucosyltransferase B) and GbpB (glucan-binding protein...
OBJECTIVE
The purpose of this work was to develop an anti-CAT-SYI immunoglobulin Y (IgY) antibody that targeted both GtfB (glucosyltransferase B) and GbpB (glucan-binding protein B) and test its anticaries properties in rats.
METHODS
A new CAT-SYI fusion gene was created utilising functional DNA fragments from the GtfB and GbpB genes. The recombinant antigens, comprising the fused CAT-SYI antigen, GtfB, and GbpB, were expressed and purified using a prokaryotic expression and purification system. The purified recombinant antigens were utilised to immunise laying hens against particular IgY production. The biological activities of these particular IgY antibodies were then assessed both in vitro and in vivo, including their capacity to suppress biofilm formation and tooth caries.
RESULTS
Results indicated that these produced IgY antibodies demonstrated a high antibody titer (>0.1 μg/mL) and could precisely recognise and bind to their respective antigens. Furthermore, it was discovered that these specific IgY antibodies successfully bind to Streptococcus mutans and significantly reduce biofilm development. After 8 weeks of ingesting antigen-specific IgY meals, comprising anti-GtfB IgY and anti-GbpB IgY, the severity of dental caries was dramatically reduced in S mutans-infected Sprague-Dawley rats (P < .01). Anti-CAT-SYI IgY therapy significantly reduced tooth cavities by 89.0% in vivo (P < .05) compared to other treatment groups.
CONCLUSIONS
The anti-CAT-SYI IgY, a multitarget antibody that targets both GtfB and GbpB, displayed excellent inhibitory effects against S mutans, making it a promising targeted method with improved anticaries efficacy and significant application opportunities.
PubMed: 38797634
DOI: 10.1016/j.identj.2024.05.006 -
International Journal of Biological... Jun 2024Monoclonal antibodies (mAbs) have garnered substantial attention within the field of ophthalmology and can be used to suppress scar formation after minimally invasive...
Monoclonal antibodies (mAbs) have garnered substantial attention within the field of ophthalmology and can be used to suppress scar formation after minimally invasive glaucoma surgeries. Here, by controlling mAb passive diffusion, we developed a polymeric, rate-controlling membrane reservoir loaded with poly(lactic-co-glycolic acid) microspheres to deliver mAb for several weeks. Different parameters were tested to ensure that the microspheres achieved a good quality characteristic, and our results showed that 1 %W/V emulsifier with 5 %W/V NaCl achieved mAb-loaded microspheres with the highest stability, encapsulation efficiency and minimal burst release. Then, we fabricated and compared 10 types of microporous films based on polylactic acid (PLA), polycaprolactone (PCL), and polyethylene glycol (PEG). Our results revealed distinct pore characteristics and degradation patterns in different films due to varying polymer properties, and all the polymeric film formulations showed good biocompatibility in both human trabecular meshwork cells and human conjunctival fibroblasts. Finally, the optimized microspheres were loaded into the reservoir-type polymeric implant assembled by microporous membranes with different surface coating modifications. The implant formulation, which was fabricated by 60 PCL: 40 PEG (3 %W/V) polymer with 0.1 %W/V poly(lactic-co-glycolic acid) barrier, exerted the best drug release profile that can sustained release mAb (83.6 %) for 4 weeks.
Topics: Humans; Glaucoma; Antibodies, Monoclonal; Microspheres; Minimally Invasive Surgical Procedures; Polylactic Acid-Polyglycolic Acid Copolymer; Polyesters; Drug Delivery Systems; Drug Liberation; Polymers; Polyethylene Glycols; Porosity; Drug Carriers
PubMed: 38797299
DOI: 10.1016/j.ijbiomac.2024.132655 -
NPJ Vaccines May 2024Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was...
Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was previously shown to expand germinal center formation and improve humoral responses in adult mice. We report that sustained antigen delivery through sequential dosing overcomes neonatal limitations to form germinal center reactions and improves humoral immunity. Thus, vaccine strategies that extend the release of vaccine antigens may reduce the number of doses, and time needed, to achieve protective immunity in neonates and young infants.
PubMed: 38796539
DOI: 10.1038/s41541-024-00875-3