Search Results (44)

Background: The development of an effective HIV-1 vaccine remains a major challenge due to HIV-1’s extraordinary diversity, high mutation rate, and the rarity of broadly neutralizing antibody (bnAb) precursors. To address these challenges, we have previously immunized rabbits with mRNA-LNPs encoding for HIV-1 envelope glycoproteins (Envs), together with mRNA-LNPs encoding for HIV-1 Gag, which likely mediated the generation of virus-like particles presenting HIV-1 Envs to the immune system in vivo. Methods: Here, we investigated whether an escalating dose (ED) immunization using mRNA-LNP priming, followed by boosts with synthetic, protein-based, virus-like particles (synVLPs) displaying HIV-1 SOSIP trimers via SpyTag/SpyCatcher conjugation (group 1), could improve the quality and durability of the antibody responses compared to conventional bolus immunization (group 2). Previous studies have shown that, in contrast to single bolus immunization, the ED priming strategy could enhance B cell activation and prolong affinity maturation, resulting in higher-quality antibody responses. Results: Upon vaccination, rabbits from both groups developed strong homologous anti-Env antibody responses, with an increasing ability of sera from immunized rabbits to bind Envs following subsequent boosts. Antibodies in rabbit sera bound heterologous Envs, but there was no statistically significant difference in binding between the two groups. Overall, antibody responses were comparable across all animals and declined similarly over time in both groups, indicating that neither the adjuvants nor the ED priming led to any marked differences within this small sample size. Neutralization activity against homologous tier-2 HIV-1_AD8 (mRNA prime) and tier-2 HIV-1₁₀₅₉ (protein boost) was generally low across both groups; however, a higher neutralization titer was observed for the ED group against HIV-1_AD8 following the final boost. One of the rabbits from the bolus group exhibited exceptionally high neutralization titers that correlated with elevated Env-specific binding against HIV-1₁₀₅₉. Conclusions: These results highlight the challenges in eliciting broad and potent neutralizing antibody (nAb) responses. Our findings underscore the need for the continued development and refinement of immunogen design and delivery strategies to guide the elicitation of nAb. Full article

The emergence of multidrug-resistant Pseudomonas aeruginosa strains is increasingly becoming a critical threat to global health. Among the resistance mechanisms, the MexAB–OprM efflux pump confers P. aeruginosa with an efficient method to export a broad spectrum of antibiotics. The antimicrobial peptide Esc (1-21)-1c was shown to downregulate this efflux system, though its mechanism of action has not been unveiled thus far. Here, we employed a combination of molecular modeling and experimental methods to investigate the precise peptide inhibitory mechanism. Functional proteomic experiments revealed the P. aeruginosa protein Q9I5H3, homologous to E. coli QseB, as a putative key target of Esc(1-21)-1c. Molecular docking predicted stable peptide–protein interactions, which were experimentally validated through fluorescence spectroscopy. Furthermore, electrophoretic mobility shift assays demonstrated that Q9I5H3 specifically binds the MexAB–OprM promoter and that Esc(1-21)-1c competitively inhibits this interaction in a dose-dependent manner. These findings reveal a previously uncharacterized regulatory pathway for efflux pump control and highlight Q9I5H3 as a promising therapeutic target against multidrug-resistant P. aeruginosa. Full article

Congenital eye malformations like microphthalmia–anophthalmia–coloboma (MAC), anterior segment dysgenesis (ASD), primary congenital glaucoma (PCG) and congenital cataracts (CC) are significant causes of childhood visual impairment. Phenotypic heterogeneity often complicates diagnosis. The goal of this study was to optimize the diagnostic strategy for next-generation sequencing (NGS)-based procedures, thereby aiming to identify genetic causes of congenital eye malformations. Forty patients with congenital eye malformations were included. A primary diagnostic testing (PD) of a limited number of genes was followed by multigene panel (MGP) testing, including 186 eye-related genes, and exome sequencing. Causative variants were identified in 17 patients (43%) and clinically relevant variants of uncertain significance (VUS) in 6 patients (15%). PD had a diagnostic yield (DY) of 15%, MGP of 29% and exome sequencing of 4%, leading to a cumulative DY of 43%. Diagnostic rates were highest in CC (75%), bilateral cases (46%), complex ocular phenotypes (78%), patients with extraocular manifestations (55%) and positive family history (70%). Rare and possible new genotype–phenotype correlations and candidate genes (FAT1, POGZ) could be identified. In total, eight (likely) pathogenic variants in six genes (CYP1B1, ADAMTS18, MAB21L2, NHS, MFRP, CRYBB1) were not yet reported. A stepwise genetic testing approach starting with a broad multigene panel followed by exome sequencing provides higher diagnostic yield than limited phenotype-specific testing. Comprehensive genetic diagnosis is essential for prognosis, treatment and genetic counseling, underscoring the need for routine genetic testing and interdisciplinary collaboration in managing congenital eye malformations. Full article

Background: B7-H3, an immunoregulatory protein of the B7 family, has been associated with both anti-cancer immunity and tumor promotion, with its expression commonly correlated with poor prognosis. Although it is frequently expressed across cancers, its heterogeneity may limit the effectiveness of B7-H3-targeted therapies. Consequently, a sensitive and non-invasive method is needed to assess B7-H3 expression for patient selection and stratification. Single-domain antibody fragments (sdAbs) offer a promising platform for developing such a diagnostic tool. Methods: To generate B7-H3 sdAbs, two Ilamas were immunized with the recombinant human B7-H3 protein. Positive clones were selected through Phage biopanning and characterized for thermal stability, binding specificity, and affinity to human and murine B7-H3 proteins. Selected sdAbs were radiolabeled with Technetium-99m (^99mTc) and evaluated for B7-H3 detection in two xenograft tumor models using micro-SPECT/CT imaging and dissection studies. Results: Sixteen purified sdAbs bound specifically to recombinant B7-H3 proteins and cells expressing native B7-H3 antigens, with nanomolar affinities. The four best-performing sdAbs bound promiscuously to tested mouse and human B7-H3 isoforms. Lead sdAb C51 labeled with ^99mTc displayed specific accumulation across two human B7-H3⁺ tumor models, achieving high contrast with a tumor-to-blood ratio of up to 10 ± 3.16, and a tumor uptake of up to 4.96 ± 1.4%IA/g at 1.5 h post injection. Conclusions: The lead sdAb enabled rapid, specific, and non-invasive imaging of human B7-H3⁺ tumors. Its isoform promiscuity supports broad applicability across cancers expressing different human B7-H3 isoforms. These results support further development for clinical translation to enable patient selection and improved B7-H3-targeted therapies. Full article

Monoclonal antibodies (mAbs), as potent therapeutic agents, have been widely applied in the treatment of various major diseases, including infectious diseases, autoimmune disorders, cancers, and neurodegenerative diseases. However, early-generation mAbs were limited by high immunogenicity, short half-life, and insufficient affinity, which compromised their therapeutic efficacy. With technological advancements, novel approaches such as high-throughput screening and glycosyl modification have been introduced to improve the performance of mAbs. Furthermore, computer-aided design techniques—including molecular docking, molecular dynamics simulations, and artificial intelligence -based methods—are increasingly being employed to accelerate the optimization process. This review summarizes recent progress in the optimization of therapeutic mAbs, with a focus on technological breakthroughs and applications in affinity enhancement, development of broad-spectrum mAbs, specificity modulation, immunogenicity reduction, and stability improvement. Additionally, it discusses current challenges and future directions in antibody optimization. This review aims to provide insights and references for the development and optimization of next-generation antibody drugs, ultimately promoting the clinical application of safer and more effective mAb-based therapies. Full article

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics. 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

Background/Objectives: Since the World Health Organization (WHO) recommended HIV self-testing as an alternative to traditional facility-based testing in 2016, it has been increasingly adopted worldwide. This study aimed to evaluate the performance of the ConfiSign HIV Self-Test (GenBody Inc., Republic of Korea), a newly developed blood-based immunochromatographic assay for the qualitative detection of total antibodies (IgG and IgM) against HIV-1/HIV-2. Methods: The evaluation included four components: (1) retrospective analysis of 1400 archived serum samples (400 HIV-positive and 1000 HIV-negative samples), (2) prospective self-testing by 335 participants (112 HIV-positive participants and 223 individuals with an unknown HIV status, including healthy volunteers), (3) assessment using seroconversion panels and diverse HIV subtypes, and (4) analytical specificity testing for cross-reactivity and interference. The Elecsys HIV combi PT and Alinity I HIV Ag/Ab Combo assays were used as reference assays. Results: In retrospective testing, the ConfiSign HIV Self-Test achieved a positive percent agreement (PPA) of 100%, a negative percent agreement (NPA) of 99.2%, and a Cohen’s kappa value of 0.986, showing excellent agreement with the reference assays. In the prospective study, the test showed 100% sensitivity and specificity, with a low invalid result rate of 1.8%. All HIV-positive samples, including those with low signal-to-cutoff (S/Co) values in the Alinity I assay, were correctly identified. The test also reliably detected early seroconversion samples and accurately identified a broad range of HIV-1 subtypes (A, B, C, D, F, G, CRF01_AE, CRF02_AG, and group O) as well as HIV-2. No cross-reactivity or interference was observed with samples that were positive for hepatitis viruses, cytomegalovirus, Epstein–Barr virus, varicella zoster virus, influenza, HTLV-1, HTLV-2, or malaria. Conclusions: The ConfiSign HIV Self-Test demonstrated excellent sensitivity, specificity, and robustness across diverse clinical samples, supporting its reliability and practicality as a self-testing option for HIV-1/2 antibody detection. Full article

Merbecovirus, a subgenus of Betacoronavirus, includes MERS-CoV and multiple bat-derived viruses with zoonotic potential. Given the unpredictable emergence of these viruses and their genetic diversity, development of broad-spectrum diagnostic tools is expected. In this study, we established a sandwich ELISA targeting the nucleocapsid (N) protein of merbecoviruses. We generated monoclonal antibodies (mAbs) using recombinant N protein of a bat merbecovirus, VsCoV-1, and selected cross-reactive clones for other merbecoviruses. Three mAbs showed strong reactivities with multiple merbecoviruses but not with SARS-CoV-2 or endemic human coronaviruses. Pairwise ELISA screening identified 1A8/10H6 mAbs as the optimal combination for detection of N protein from six merbecoviruses—VsCoV-1, EjCoV-3, MERS-CoV, NeoCoV, HKU4, and HKU5—with limits of detection (LODs) below 7.81 ng/mL, including 1.25 ng/mL for VsCoV-1. Infectious bat merbecovirus EjCoV-3 was detected at 1.3 × 10³ PFU/mL. No cross-reactivity was observed with non-merbecoviruses, indicating its high specificity. This sandwich ELISA offers a rapid, reproducible, and cost-effective diagnostic platform with potential for high-throughput screening and automation. Moreover, its design is amenable to adaptation into point-of-care formats such as lateral flow assays, highlighting its value for field-based surveillance and pandemic preparedness. Full article

Background/Objectives: Monkeypox, twice declared a public health emergency of international concern by the WHO, currently lacks approved targeted therapeutics. This study focused on the development of monkeypox virus (MPXV) E8-specific human monoclonal antibodies (mAbs) derived from recipients of the recombinant vaccinia vaccine (rTV), with subsequent evaluation of their cross-neutralizing activity against orthopoxviruses, including the vaccinia virus (VACV) and MPXV. Methods: Three mAbs (C5, C9, and F8) were isolated from rTV vaccinees. Structural mapping characterized their binding domains on the MPXV E8 and VACV D8 proteins. Neutralization potency was assessed against the VACV TianTan strain and MPXV clade IIb. A combo was further evaluated in a VACV-infected mice model for clinical recovery and viral load reduction. Complement-dependent enhancement mechanisms were also investigated in vitro. Results: C9 targets the virion surface region of E8 and both the virion surface region and intravirion region of D8, showing cross-neutralization activity against the MPXV (IC₅₀ = 3.0 μg/mL) and VACV (IC₅₀ = 51.1 ng/mL) in vitro. All three antibodies demonstrated potent neutralization against the VACV in vitro: C5 (IC₅₀ = 3.9 ng/mL), C9 (IC₅₀ = 51.1 ng/mL), and F8 (IC₅₀ = 101.1 ng/mL). Notably, complement enhanced neutralization against the VACV by >50-fold, although no enhancement was observed for the MPXV. In vivo administration accelerated clinical recovery by 24 h and achieved significant viral clearance (0.9-log reduction). Conclusions: E8-targeting mAbs exhibited broad-spectrum neutralization against orthopoxviruses, demonstrating therapeutic potential against both historical (VACV) and emerging (MPXV) pathogens. However, MPXV’s resistance to complement-dependent enhancement highlights the necessity for pathogen-adapted optimization. These findings establish E8 as a critical conserved target for pan-poxvirus VACV and MPXV countermeasure development. Full article

Avian infectious bronchitis is caused by the avian infectious bronchitis virus (IBV), which poses a significant threat to the poultry industry and public health. The S1 protein of IBV plays a crucial role in the process of the virus invading host cells. To investigate the significant antigenic targets within the S1 protein, in this study, the truncated S1 sequence of the IBV M41 strain was cloned with approximately 660 bp and expressed. After purification and renaturation, the recombinant S1 protein was immunized into BALB/c mice. Then, following fusion with lymphocytes and SP2/0 cells, the indirect ELISA and Western blotting techniques were employed to screen hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the S1 protein. Antigenic epitopes of the mAbs were identified using truncated S1 fragments and peptide scanning. The results indicated that three hybridoma cell lines stably secreting S1 protein-specific mAbs (2A10, 4E9, and 5E12) were screened. The heavy chains of the three mAbs were IgG1, and all three mAbs contained kappa light chains. The identified minimal B-cell epitopes were ¹³²RVSAMK¹³⁷ and ¹⁴²FYNLTV¹⁴⁷. Homology analysis showed these both epitopes were conserved across IBV subtypes and located on the S1 protein surface. The conserved β-sheet epitope ¹³²RVSAMK¹³⁷ and the surface-exposed, flexible loop epitope ¹⁴²FYNLTV¹⁴⁷ serve as ideal targets for broad-spectrum diagnostics and early infection detection, respectively. These epitopes provide unique structural advantages for antibody binding, enabling the design of multivalent epitope vaccines or the development of immunomodulatory drugs. They offer novel biomaterials and targets for antibody-based drug development and rapid detection methods for avian infectious bronchitis virus (IBV), holding significant potential for the prevention and control of IBV. Full article

Background: In this study, the mechanisms implicated in delafloxacin resistance in Klebsiella pneumoniae strains were investigated. Delafloxacin is a novel, broad-spectrum fluoroquinolone that has been approved for clinical application. Methods: In our study, 43 K. pneumoniae strains were assessed, antimicrobial susceptibility testing was performed via the broth microdilution method, and the minimum inhibitory concentration (MIC) values for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were determined. Four delafloxacin-resistant K. pneumoniae strains were selected for whole-genome sequencing (WGS). Results: The MIC50 values for the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L. All four selected delafloxacin-resistant K. pneumoniae strains showed extended-spectrum beta-lactamase production, and one strain exhibited carbapenem resistance. WGS enabled us to determine the sequence types (STs) of these strains, namely, ST307 (two strains), ST377, and ST147. Multiple mutations in quinolone-resistance-determining regions (QRDRs) were detected in all the delafloxacin-resistant K. pneumoniae strains; specifically, gyrA Ser83Ile and parC Ser80Ile were uniformly present in the strains of ST307 and ST147. However, in the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile, amino acid substitutions were detected. We also identified OqxAB and AcrAB efflux pumps in all delafloxacin-resistant K. pneumoniae strains. The association between beta-lactamase production and delafloxacin resistance was determined; specifically, CTX-M-15 production was detected in the ST147, ST307, and ST377 strains. Moreover, NDM-1 was detected in ST147. Conclusions: We conclude that multiple mutations in QRDRs, in combination with OqxAB and AcrAB efflux pumps, achieved delafloxacin resistance in K. pneumoniae. In our study, we report on NDM-1-producing K. pneumoniae ST147 in Hungary. Full article

Monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy, offering unprecedented specificity and diverse mechanisms to combat malignant cells. These biologic agents have emerged as a cornerstone in targeted cancer treatment, binding to specific antigens on cancer cells and exerting their therapeutic effects through various mechanisms, including inhibition of signaling pathways, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). The unique ability of mAbs to engage the immune system and directly interfere with cancer cell function has significantly enhanced the therapeutic armamentarium against a broad spectrum of malignancies. mAbs were initially studied in oncology; however, today, treatments have been developed for eye diseases. This review discusses the current applications of mAbs for the treatment of ocular diseases, discussing the specificity and the variety of mechanisms by which these molecules exhibit their therapeutic effects. The benefits, drawbacks, effectiveness, and risks associated with using mAbs in ophthalmology are highlighted, focusing on the most relevant ocular diseases and mAbs currently in use. Technological advances have led to in vitro production methods and recombinant engineering techniques, allowing the development of chimeric, humanized, and fully human mAbs. Nowadays, many humanized mAbs have several applications, e.g., for the treatment of age-related macular disease, diabetic retinopathy, and uveitis, while studies about new applications of mAbs, such as for SARS-CoV-2 infection, are also currently ongoing to seek more efficient and safe approaches to treat this new ocular disease. Full article

Biological barriers remain a major obstacle for the development of innovative therapeutics. Depending on a disease’s pathophysiology, the involved tissues, cell populations, and cellular components, drugs often have to overcome several biological barriers to reach their target cells and become effective in a specific cellular compartment. Human biological barriers are incredibly diverse and include multiple layers of protection and obstruction. Importantly, biological barriers are not only found at the organ/tissue level, but also include cellular structures such as the outer plasma membrane, the endolysosomal machinery, and the nuclear envelope. Nowadays, clinicians have access to a broad arsenal of therapeutics ranging from chemically synthesized small molecules, biologicals including recombinant proteins (such as monoclonal antibodies and hormones), nucleic-acid-based therapeutics, and antibody-drug conjugates (ADCs), to modern viral-vector-mediated gene therapy. In the past decade, the therapeutic landscape has been changing rapidly, giving rise to a multitude of innovative therapy approaches. In 2018, the FDA approval of patisiran paved the way for small interfering RNAs (siRNAs) to become a novel class of nucleic-acid-based therapeutics, which—upon effective drug delivery to their target cells—allow to elegantly regulate the post-transcriptional gene expression. The recent approvals of valoctocogene roxaparvovec and etranacogene dezaparvovec for the treatment of hemophilia A and B, respectively, mark the breakthrough of viral-vector-based gene therapy as a new tool to cure disease. A multitude of highly innovative medicines and drug delivery methods including mRNA-based cancer vaccines and exosome-targeted therapy is on the verge of entering the market and changing the treatment landscape for a broad range of conditions. In this review, we provide insights into three different disease entities, which are clinically, scientifically, and socioeconomically impactful and have given rise to many technological advancements: acquired immunodeficiency syndrome (AIDS) as a predominant infectious disease, pancreatic carcinoma as one of the most lethal solid cancers, and hemophilia A/B as a hereditary genetic disorder. Our primary objective is to highlight the overarching principles of biological barriers that can be identified across different disease areas. Our second goal is to showcase which therapeutic approaches designed to cross disease-specific biological barriers have been promising in effectively treating disease. In this context, we will exemplify how the right selection of the drug category and delivery vehicle, mode of administration, and therapeutic target(s) can help overcome various biological barriers to prevent, treat, and cure disease. Full article

Disease-modifying therapies (DMTs) impact the cellular immune response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccines in patients with multiple sclerosis (pwMS). In this study, we aim to elucidate the characteristics of the involved antigen-specific T cells via the measurement of broad cytokine profiles in pwMS on various DMTs. We examined SARS-CoV-2-specific T cell responses in whole blood cultures characterized by the release of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α), as well as antibodies (AB) targeting the SARS-CoV-2 spike protein in pwMS following either two or three doses of mRNA or viral vector vaccines (VVV). For mRNA vaccination non-responders, the NVX-CoV2373 protein-based vaccine was administered, and immune responses were evaluated. Our findings indicate that immune responses to SARS-CoV-2 vaccines in pwMS are skewed towards a Th1 phenotype, characterized by IL-2 and IFN-γ. Additionally, a Th2 response characterized by IL-5, and to a lesser extent IL-4, IL-10, and IL-13, is observed. Therefore, the measurement of IL-2 and IL-5 levels could complement traditional IFN-γ assays to more comprehensively characterize the cellular responses to SARS-CoV-2 vaccines. Our results provide a comprehensive cytokine profile for pwMS receiving different DMTs and offer valuable insights for designing vaccination strategies in this patient population. Full article