Search Results (114)

Background/Objectives: Polyethylene glycol (PEG) is widely used to enhance the stability and pharmacokinetics of nanomedicines, including lipid nanoparticle (LNP)-based mRNA vaccines. However, both pre-existing and vaccine-induced anti-PEG antibodies may compromise the efficacy and safety of PEGylated therapeutics. Methods: In this study, we analyzed the specificity and avidity of anti-PEG antibodies in human blood donor samples from Unvaccinated individuals and recipients of PEGylated mRNA-LNP vaccines (Comirnaty and Spikevax), polysorbate-containing vaccines, or vaccines lacking both PEG and polysorbates. Quantitative ELISA was used to characterize anti-PEG and anti-polysorbate IgM and IgG responses in 325 plasma samples, while an equilibrium titration method was applied to assess IgG binding to PEG molecules, micelles, and PEGylated liposomes with defined structural features in 36 plasma samples. Results: Vaccination with PEGylated mRNA-LNPs was associated with increased anti-PEG antibody levels and qualitative changes in antibody binding behavior. Anti-PEG IgG antibodies displayed progressively higher avidity toward larger and structurally more complex PEG-containing antigens, with the strongest binding observed for PEGylated liposomes. Notably, vaccinated individuals, particularly those who received Spikevax, showed increased end-group and backbone-specific avidity, as well as an enhanced ability of antibody paratopes to engage shorter PEG chains. In contrast, polysorbate-containing or PEG-free vaccines did not elicit comparable effects. Conclusions: These findings suggest that vaccination with PEGylated mRNA-LNPs is associated with the emergence of altered antibody populations with increased end-group reactivity and higher avidity toward PEG-directed immune responses, despite PEG being a synthetic, nonprotein polymer. Antibody binding to LNPs, accompanied by the emergence of high-avidity anti-PEG IgG seems to be consistent with an increased risk of adverse events, particularly following repeated vaccinations, including complement activation-related pseudoallergy (CARPA) and anaphylaxis. It may also contribute to altered immune protection against the vaccine target, underscoring the need for avidity-aware risk-benefit assessment of PEGylated therapeutics. Full article

Objectives: Membrane-enveloped virus-like particles (VLPs) constitute a versatile vaccine platform allowing for the display of heterologous viral surface antigens. The density of displayed antigens is paramount for the efficient elicitation of a strong cellular and humoral immune response. SARS-CoV-2 spike protein variants with engineered cytoplasmic tails (CTs) were generated to enhance decoration efficiency on the surface of VLPs formed by the HIV core protein Gag. These HIV (SARS-CoV-2) chimeric particles serve as a vaccine component prototype. Methods: Spike variants were first analyzed for cellular and surface expression as well as incorporation into extracellular vesicles (EVs) and VLPs using flow cytometric analysis and Western blot analysis. Receptor binding, fusogenicity, i.e., mediating the fusion of spike-positive with receptor-containing membranes, and the proteins’ potential to mediate lentiviral vector gene transduction into susceptible target cells was examined by employing syncytia-formation assays and vector titration experiments. The display of a neutralization-sensitive epitope was examined utilizing immuno-precipitation using a neutralizing antibody. Results: All four variants were shown to be cell-surface expressed, to recruit the cognate receptor, to mediate membrane fusion and cell entry of lentiviral pseudotype vector particles and to decorate VLPs and EVs. However, the spike variant encompassing a truncated CT derived from the gibbon ape leukemia virus (GaLV) transmembrane (TM) envelope protein was most efficiently incorporated into HIV Gag-formed VLPs. All variants exposed a neutralization-sensitive epitope in the receptor binding domain. Conclusions: Engineering of the CTs of viral surface antigens can enhance VLP decoration, while required functionality of the ecto-domain such as receptor recognition, fusogenicity and neutralization-sensitive epitope presentation are not abrogated. This indicates the preservation of the structural integrity of the antigen required to elicit a neutralizing humoral immunity upon vaccination. The identified truncated CT of GaLV TM may be of utility to improve the incorporation of other viral surface antigens into a variety of membrane-enveloped VLPs derived from a range of different parental viruses. Full article

Imprinted proteins (IPs) are promising materials for producing artificial alternatives to natural recognition systems (antibodies, aptamers, etc.) due to their high sorption properties and specificity. However, contemporary understanding of the imprinting process at the atomic level is rather limited, which hinders the rational design of more efficient IPs. In this paper, we use computational modeling to provide a description of fundamental principles of protein imprinting at the atomic level. We have modeled several potential associates between the protein matrix and template molecules that form during the imprinting process up to the addition of the cross-linking agent. We used bovine serum albumin (BSA) as the protein matrix and 4-hydroxycoumarin (4–HC) as a molecular template. In combination with computational modeling, extensive experimental analyses including isothermal titration calorimetry (ITC) and NMR spectroscopic methods (¹H NMR and diffusion-ordered NMR spectroscopy (DOSY)) were used to evaluate the potential efficiency of imprinted BSA. This study represents a step toward the future rational in silico design of IPs. Full article

This study aimed to express the canine parvovirus (CPV) VP2 protein prokaryotically and develop an indirect ELISA for detecting CPV-specific antibodies in canine serum. The VP2 gene from a laboratory-isolated CPV strain was amplified and cloned into the pET-28a vector. Following prokaryotic expression optimization, the recombinant protein was purified via Ni-NTA affinity chromatography and validated using Western blotting. An indirect ELISA was established utilizing the purified VP2 as the coating antigen, with optimal parameters determined by checkerboard titration. A 1773 bp VP2 fragment was amplified. Optimal expression of the 64.8 kDa recombinant VP2 was achieved with 2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 32 °C for 8 h. For the indirect ELISA, the optimal antigen coating concentration was 2 μg/mL, alongside primary (canine serum) and secondary antibody dilutions of 1:320 and 1:4000, respectively. The diagnostic cut-off optical density at 450 nm (OD₄₅₀) threshold was established at ≥0.2066, and the analytical sensitivity reached a serum dilution of 1:5120. Compared with the hemagglutination inhibition (HI) assay using 192 clinical serum samples, the ELISA showed a diagnostic sensitivity of 85.94%, a diagnostic specificity of 88.28%, and an overall agreement rate of 87.50%. The mean intra-assay and inter-assay coefficients of variation were 4.39% and 3.02%, respectively. These findings indicate that the recombinant VP2-based indirect ELISA showed good analytical sensitivity, reproducibility, and diagnostic agreement with the HI assay for detecting CPV-specific antibodies in canine serum under the tested conditions, although broader cross-reactivity validation is still required. Full article

The global tilapia industry is threatened due to the mass die-off of farmed tilapia caused by an emerging virus, Tilapia Lake Virus (TiLV). The threat of TiLV disease demands for cost-effective diagnostics and preventive measures such as protein and antibody-based detection tools and even vaccines like protein subunit vaccines. In this study, we aimed to recombinantly express the entire S4 protein of TiLV in vitro and in vivo using an extended host-range baculovirus expression vector system (EHR-BEVS) and used the purified protein to generate polyclonal anti-S4 antibodies. The parental extended host-range ABM-eIF4E baculovirus previously developed in our laboratory was used to generate the recombinant ABM-eIF4E-S4-His baculovirus expression vector using homologous recombination and was purified through multiple rounds of plaque selection and end-point titration, eliminating the need for the use of antibiotic resistance gene selection marker. Different insect cell lines including Trichoplusia ni Hübner, 1802 larvae were infected with ABM-eIF4-S4-His to produce recombinant S4 protein. Western blotting was used to detect the recombinantly expressed S4 protein. We generated an antibiotic resistance gene-free EHR-BEV and observed the permissibility of all the insect cell lines tested including T. ni larvae to infection with the recombinant ABM-eIF4E-S4-His. Secondly, immunogenic S4 protein capable of eliciting rabbit polyclonal anti-S4 antibodies was recombinantly expressed in the infected insect cells and larvae. TiLV S4 protein was recombinantly expressed in vitro and in vivo using extended host-range ABM-eIF4E-S4-His baculovirus expression vector and was shown to induce rabbit antibody production that can recognize S4 protein supporting the potential of insect larvae as alternative biofactories in the production of recombinant TiLV protein subunit for the development of diagnostics and preventive vaccines against TiLV disease. Full article

Background/Objectives: International guidelines advocate for personalized vaccination protocols using point-of-care (POC) antibody titration to identify dogs requiring boosters for CPV-2, CDV, and CAdV-1. As traditional venipuncture can be challenging in specific patients, this study evaluate the clinical agreement of a novel minimally invasive capillary blood sampling technique (ear-prick) for core vaccine antibody titration. Methods: Paired blood samples were collected from 55 healthy dogs using venipuncture and an ear-prick technique with a portable lancet. Antibody titers were determined using a semi-quantitative POC kit (VacciCheck^® Canine). The procedure was optimized comparing 28G and 21G lancets, with the latter used in 43 dogs to ensure adequate blood flow. Comprehensive statistical methods evaluated the correlation and agreement between the two sampling techniques. Results: Statistical analysis showed no significant differences between sampling methods (p > 0.05). In the optimized group (21G lancet), full match rates reached 81.4% for CPV-2, 76.8% for CDV, and 74.4% for CAdV-1. Clinical concordance was exceptionally high: 95.3% for CPV-2, 90.7% for CDV, and 100% for CAdV-1. Statistical analysis confirmed perfect agreement (1.00) for CPV-2 and CAdV-1, and moderate agreement (0.48) for CDV. Conclusions: The ear-prick technique using a 21G lancet is a reliable, minimally invasive alternative to venipuncture for antibody titration. This method simplifies clinical procedures and facilitates personalized immunization monitoring. Given the minimal blood volume required, it represents a versatile approach for evaluating immune status and protection levels to core vaccines in diverse settings, including pediatric and shelter medicine. Full article

This study developed a dual-antigen enzyme-linked immunosorbent assay (ELISA) based on σB protein and genotype 5-specific σC protein of avian reovirus (ARV). First, σB and σC proteins were expressed and purified using recombinant technology. Through optimization of coating conditions, the optimal antigen combination was determined to be a mixture of the two proteins at a 1:3 molecular ratio (total concentration: 0.8 μg/mL). Key parameters of the indirect ELISA were optimized via checkerboard titration. Validation confirmed that the dual-antigen ELISA exhibited a sensitivity of 1:3200 against genotype 5 ARV-positive sera, with no cross-reactivity and a coefficient of variation of 2.9–8.6%, demonstrating excellent reproducibility. In application testing, the method specifically detected serum antibodies against genotype 5 ARV variant strains, achieving a 100% positive detection rate in experimental chickens within the first week post-challenge and effectively monitoring dynamic antibody changes in infected flocks. Furthermore, the detection rate for genotype 5-positive serum samples (100%) was significantly higher than that of a commercial kit (75%). This dual-antigen indirect ELISA overcomes the sensitivity limitations associated with conventional genotype 5 ARV detection methods and provides a reliable tool for epidemiological surveillance and infection monitoring. Full article

Background: Hypothyroidism is one of the most common endocrine conditions during pregnancy and has been associated with poor obstetric and perinatal outcomes. There is still a lack of data from Middle Eastern populations, despite its clinical significance. This study aimed to evaluate thyroid management patterns during pregnancy and examine the association between thyroid function control and maternal and perinatal outcomes in women with hypothyroidism at a tertiary care center in Qatar. Methods: A retrospective cohort study including 379 pregnant women with hypothyroidism diagnosed between January 2019 and November 2022 was conducted at Sidra Medicine in Doha, Qatar. Based on trimester-specific Thyroid-stimulating hormone (TSH )reference values, participants were categorized as having adequately or inadequately controlled thyroid function. Data on obstetrics, biochemistry, and demographics were taken from electronic medical records (EMR). Statistical analyses were performed using chi-square tests for categorical variables and t-tests for continuous variables, with a significance threshold of p < 0.05. Results: Participants had a mean Body Mass Index (BMI) of 30.33 ± 6.14 kg/m² and an average age of 32.65 ± 4.99 years; 54% of them were Qataris. Of the patients, 58.5% had positive thyroid antibodies and 55.7% had pre-gestational hypothyroidism. Women with pre-gestational hypothyroidism required significantly higher levothyroxine doses compared with those with gestational hypothyroidism (93.2 ± 47.5 mcg/day vs. 67.6 ± 30.1 mcg/day; p < 0.001). Treatment adjustment was demonstrated by the improvement in TSH normalization from 51.3% in the first trimester to 64.2% in the third trimester (p = 0.041). No significant associations were observed with pre-eclampsia, preterm delivery, hypertension, or placental abruption. However, women with normal third-trimester TSH had a higher prevalence of gestational diabetes mellitus (GDM) compared with those with elevated TSH (51.6% vs. 36.8%; p = 0.013). Conclusions: Appropriate trimester-specific monitoring and timely levothyroxine titration was associated with improved biochemical control without adverse maternal outcomes. Greater levothyroxine requirements in women with pre-gestational hypothyroidism emphasize the importance of early intervention. These findings highlight the potential benefit of structured thyroid monitoring and multidisciplinary care approaches in pregnancy and may help inform future regional clinical practice guidelines. Full article

Treponema pallidum is the etiological cause of syphilis, and in recent years, reemergence has been reported, especially among men who have sex with men and people living with HIV (PLWH). Certain cytokines may act as hallmark biomarkers in the progression of syphilis in PLWH, and studying how the immune system works against T. pallidum is important, especially in PLWH, whose immune system is compromised. We evaluated the serum expressions of IFN, TNF, IL-10, TGF-β1 and IL-17 in men living with HIV (MLWH) and their association with distinct stages of syphilis. We recruited MLWH from March to October 2022. A blood sample was requested, syphilis was detected using the reverse algorithm, and antibodies were titrated to determine the stage. Each of the cytokines studied was quantified using commercial ELISA kits. The following groups were formed: active syphilis (n = 217), cured syphilis (n = 134), and without syphilis (n = 159). The prevalence of elevated TGF-β1 differed between groups, being highest in individuals with active syphilis (51.6%; median 319 pg/mL), followed by those with cured syphilis (41.0%; median 137.0 pg/mL). Younger participants and persons without a history of sexually transmitted infections were more likely to present with high TGF-β1 levels. TGF-β1 may act as a biomarker in active syphilis and could suppress the inflammatory response against spirochetes. Full article

Background: Rift Valley fever virus (RVFV) is a significant mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Reliable diagnostic assays for detecting antibodies and assessing their functional neutralizing capacity are essential for surveillance programs, vaccine monitoring, and outbreak preparedness. Objective: This study evaluates and compares the analytical performance of a competitive enzyme-linked immunosorbent assay (cELISA) and a virus neutralization test (VNT) for detecting RVFV antibodies in vaccinated sheep sera, establishing an integrated laboratory workflow for virus titration, serological detection, and functional neutralization. Methods: Twenty serum samples were collected from sheep pre-vaccination and one month post-vaccination with Smithburn live attenuated RVFV vaccine. Sera were tested using a commercial multispecies RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and 50% tissue culture infective dose (TCID₅₀/0.1 mL) was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 h after infection with different viral doses (10² to 10⁵ TCID₅₀/0.1 mL), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by cytopathic effect (CPE) inhibition. Results: ELISA revealed robust antibody signals up to a 1:32 dilution, with signal-to-noise (S/N) < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 10^6.5 TCID₅₀/0.1 mL. The VNT exhibited time- and dose-dependent kinetics; high protection rates (≥97) were observed at 1:2–1:8 dilutions against 10²–10³ TCID₅₀/0.1 mL challenge doses; however, neutralizing efficacy decreased significantly at higher viral loads and higher serum dilutions. While cELISA and VNT results correlated strongly at low serum dilutions, the cELISA showed decreased sensitivity at dilutions ≥ 1:64, where the VNT remained capable of detecting functional neutralizing activity. Conclusions/Discussion: The results demonstrate that while both assays correlate well at high antibody concentrations, they diverge at lower concentrations. This discrepancy highlights the functional difference between binding antibodies (N-protein) and neutralizing antibodies (Gn/Gc glycoproteins). Consequently, the cELISA is ideal for rapid screening, whereas the VNT is indispensable for confirming functional immunity. Integrating both assays provides a more accurate immunological profile for RVFV surveillance and vaccine evaluation. Full article

Nanobodies (VHHs or single-domain antibodies) are powerful affinity reagents, but their routine use is often limited by production constraints and by the lack of a conserved Fc region for secondary detection. We describe a simplified strategy in which functional GST–nanobody fusion proteins are expressed directly in the cytoplasm of Escherichia coli Origami^TM 2 (DE3), a strain that supports disulfide bond formation through trxB/gor mutations. Using well-characterized nanobodies against GFP (Lag2) and mCherry (C11), we designed N-terminal GST fusions and confirmed by AlphaFold3-based modeling that both constructs preserve the GST fold and the VHH (Variable domain of the Heavy-chain antibody of Heavy-chain-only antibodies) β-sandwich with defined CDR loops and a predicted intradomain disulfide bond. Following IPTG induction and purification by glutathione affinity and size-exclusion chromatography, we obtained soluble GST-nb-GFP and GST-nb-mCherry at ~8–12 mg/L. Isothermal titration calorimetry showed nanomolar binding to their antigens (K_d ~123 nM for GFP and ~199 nM for mCherry). Consistent with conformational epitope recognition, GST-nanobodies were reactive in native-state dot blots but not in denaturing Western blots under the conditions tested. The GST moiety enabled indirect immunofluorescence via anti-GST antibodies, yielding specific labeling of GFP- or mCherry-tagged TGN38 in HeLa and H4 cells. Finally, we demonstrate “GST-nanobody pulldown” as a robust method for affinity capture from cell lysates. Together, this platform provides a low-cost, versatile route to functional nanobody reagents without requiring tag removal, and complements other nanobody designs (e.g., VHH-Fc fusions) in an application-dependent manner. Full article

The IMMY cryptococcal antigen (CrAg) lateral flow assay (LFA) is a reliable diagnostic tool for Cryptococcus detection, but false-positive results may arise from procedural or reagent-related errors, underscoring careful operation of the assay to ensure diagnostic accuracy and prevent unnecessary treatment. Two patients who were initially reported as CrAg-positive by a peripheral laboratory were referred to Kiruddu Hospital in Kampala, Uganda, for clinical assessment and confirmatory testing. Repeat tests were conducted using specimen diluent following the manufacturer’s protocol, resulting in negative results. Semi-quantitative CrAg LFA testing and a series of control assays were performed to identify the source of error. We were able to consistently reproduce positive results when the titration diluent was inappropriately used instead of the specimen diluent. Serial dilutions confirmed persistent false positivity up to 1:80 when inappropriately using the titration diluent, while all dilutions that appropriately started with the specimen diluent remained negative. We hypothesize that the incorrect use of titration diluent instead of specimen diluent led to false-positive CrAg LFA results due to the absence of the blocking agent that neutralizes heterophilic antibodies. Procedural errors can lead to diagnostic misinterpretation and serious consequences in clinical management, emphasizing the importance of adherence to manufacturer’s instructions. Full article

Lumpy Skin Disease (LSD) is a transboundary bovine viral disease. It has a significant economic impact and is caused by the Lumpy Skin Disease Virus (LSDV). Effective surveillance tools are essential for the early detection of infection, outbreak control, and assessment of vaccination coverage in endemic regions such as India. In this study, an in-house ELISA based on inactivated whole-virus antigen (WVA) was developed, optimized, and validated for the detection of LSDV antibodies in cattle. Its field applicability was assessed through a cross-sectional seroprevalence survey conducted across five Indian states. A local field isolate of LSDV (strain 5-Chitra) was cultured in MDBK cells, inactivated using binary ethylenimine (BEI), and used as the antigen source. The assay was optimized by checkerboard titration and evaluated against the Serum Neutralization Test (SNT). Diagnostic sensitivity and specificity were evaluated using the receiver operating characteristic (ROC) curve and area under the curve (AUC) analyses, while cross-reactivity was assessed using sera positive for HS, IBR, BQ, MCF, GTP, SPP, CE, FMD, and Brucellosis. Assay reproducibility was confirmed through inter- and intra-laboratory validation. For the seroprevalence study, 3230 cattle serum samples were collected using a stratified random sampling design across five Indian states, and logistic regression analysis of a subset of 1302 samples was performed to assess the influence of age and sex on LSDV seropositivity. Checkerboard titration identified optimal ELISA conditions at 50 ng of antigen per well, a 1:150 serum dilution, and a 1:10,000 dilution of anti-bovine HRP-conjugated secondary antibody. The WVA-ELISA demonstrated excellent diagnostic performance, with 100% sensitivity, 95% specificity, and no cross-reactivity with other ruminant bacterial or viral pathogens, and showed high laboratory reproducibility (κ > 0.96). Seroprevalence ranged from 50.6% to 71.1% across the five states, indicating widespread exposure to LSDV. Risk factor analysis revealed significantly higher odds of seropositivity among calves (≤1 year old) and female cattle, suggesting age- and sex-dependent susceptibility. Full article

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and fatal disease. Accurate detection in the early stages of an outbreak relies on molecular methods, but serological monitoring at the population level is also crucial for assessing the extent of exposure and past infections. This experiment developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against ASFV, using three ASFV RNA polymerase subunits (H359L, C147L, and D339L) as coating antigens. The recombinant proteins were successfully expressed in Escherichia coli and purified. Using a checkerboard titration method, we systematically optimized key assay parameters, determining the optimal coating conditions to be a mixture of H359L, C147L, and D339L at a volume ratio of 1:2:2, with individual concentrations of 1 μg/mL, 0.4 μg/mL, and 0.5 μg/mL, respectively. Other optimized parameters included a serum dilution of 1:200, a blocking buffer containing 5% skim milk, and specific incubation conditions for the secondary antibody and substrate. The cut-off value was established at 0.430 ($\overline{x}$ + 4SD) based on 30 negative sera. The established triple-antigen indirect ELISA exhibited high sensitivity (detecting positives at dilutions up to 1:3200) and excellent specificity (no cross-reactivity with antisera against CSFV, PRRSV, PRV, PCV2, and PEDV. Both intra and inter assay repeatability were confirmed, with coefficients of variation ranging from 1.020% to 7.600%. Validation with 123 clinical serum samples demonstrated a 96.75% concordance rate with a commercial kit. In conclusion, the three-antigen indirect ELISA established in this study exhibits high specificity and sensitivity, making it suitable for serological surveillance and exposure assessment of ASFV antibodies. It can be combined with molecular detection for epidemiological investigations and integrated prevention and control measures. Full article

Human Epidermal Growth Factor Receptor 2 (HER2) is a key therapeutic target in breast cancer. However, the application of existing anti-HER2 antibody drugs is limited by such issues as large molecular weight and poor stability. In this study, a series of small protein minibinders targeting HER2 domain IV were de novo designed using the RFdiffusion method. Candidate molecules were selected through a combination of ProteinMPNN and AlphaFold2 screening, and their binding capabilities were further evaluated using Escherichia coli surface display coupled with flow cytometry analysis. By integrating molecular dynamics simulations, confocal fluorescence imaging, and isothermal titration calorimetry (ITC) experiments, a highly efficient minibinder (0_703_6) with nanomolar affinity and a smaller molecular size was finally identified. Compared with the existing drug molecules, the identified minibinder exhibited approximately threefold higher affinity and a threefold reduction in molecular size. This study provides strong support for the development of novel, stable, and easily expressible HER2-targeted therapeutic molecules and also offers new insights into the rapid development of robust breast cancer drugs that may serve as ideal alternatives to monoclonal antibodies. Full article