Search Results (273)

Nanobodies (single-domain antibodies, VHHs) have emerged as versatile tools for evaluating and treating Alzheimer’s disease (AD). They offer distinct engineering benefits compared with traditional antibodies and small molecules, including small size, stability, and specificity. In AD, nanobodies have been shown in preclinical models to neutralize toxic amyloid-β oligomers, inhibit tau generation and aggregation, and modulate neuroinflammation, thereby demonstrating significant therapeutic potential. However, all nanobody applications in AD are discussed strictly as preclinical therapeutic potential rather than established clinical therapies, and direct clinical evidence in patients with AD is still lacking. Advanced engineering strategies, including intranasal and intrathecal routes, receptor-mediated transport, plasma protein binding with albumin, and focused ultrasound to facilitate brain penetration. Additionally, to improve nanobody delivery precision, half-life, and efficacy, strategies such as integrating nanobodies with nanoparticles, dendrimers, liposomes, and viral vectors are being employed. In fact, nanobodies are applied beyond monotherapy across multiple technological platforms to optimize brain delivery and target multiple targets. Nanobodies have been used on bispecific and trispecific antibody platforms, as well as in CRISPR/Cas9 editing and AI-driven technologies, to expand their applications. Recently, preclinical evidence has been mounting on the efficacy of nanobodies in clearing Aβ and tau, preserving synapses, and normalizing biomarkers. Comparison with FDA-approved anti-Aβ monoclonal antibodies (aducanumab, lecanemab, and donanemab) highlights opportunities and current translational gaps, including safety testing, half-life extension, and delivery optimization. This review critically delineates the current molecular mechanisms, emerging strategies, and delivery platforms, and emphasizes the potential of nanobodies as promising therapeutic and diagnostic molecules in AD therapeutics. Full article

Multiple myeloma (MM) is an incurable hematologic malignancy arising from clonal plasma cells, with poor long-term outcomes due to inevitable relapse after conventional therapies. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed patients. Conventional CARs employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or VHHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. We designed and evaluated a novel anti-BCMA nanoCAR-T based on the VHH Nb17, compared with the conventional scFv-based CAR-T CT103a. Nb17 demonstrated strong BCMA binding and was incorporated into a CAR construct. Both nanoCAR-T and CT103a were generated via lentiviral transduction of primary T cells. Their cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression were assessed in vitro, along with antitumor activity in vivo. Nb17-nanoCAR-T demonstrated specific cytotoxicity, cytokine release (IL-2, TFNa, IFNg), and CD107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between both CARs. Upon rechallenge, both CARs showed enhanced proliferation compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated tumors in NSG mice. These findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative. Full article

Background: Healthcare workers (HCWs) are at high risk of breakthrough SARS-CoV-2 infections despite complete vaccination schedules. There are gaps in our understanding of the specific antibody isotypes and cytokine profiles produced during an infection following vaccination. In this study, we evaluated SARS-CoV-2⁻specific antibody isotypes and their association with cytokine production in HCWs with breakthrough infections. Methods: Serum samples from 114 HCWs were analyzed for antibody isotypes against the nucleoprotein (NCP) and the receptor binding domain (RBD) of the spike protein, as well as for a panel of 13 cytokines. Results: Vaccinated SARS-CoV-2⁺ HCWs showed a higher prevalence of anti-SARS-CoV-2 antibodies against NCP (IgM = 93.8%, IgG = 93.8%, IgA = 28.1%) and RBD (IgM = 46.9%, IgG = 100%, IgA = 90.6%). A specific IgM response to NCP was more frequent in vaccinated SARS-CoV-2⁺ individuals, whereas IgA responses were predominantly specific for RBD. Both pro- and anti-inflammatory cytokines were elevated in vaccinated HCWs with breakthrough infections compared with unvaccinated and uninfected individuals. Interestingly, infected IgG+ HCWs with IgM specific for both NCP and RBD exhibited significantly higher IL-8, IL-6, TNF-α, IFN-γ, IL-2, IL-10, and TGF-β concentrations. Conclusion. Our data show that breakthrough infections in vaccinated HCWs induce a robust pro-and anti-inflammatory cytokine profile, which is associated with a broader IgM response directed against both NCP and RBD. 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

Objectives: Bovine coronaviruses (BCoV) are endemic worldwide, causing diarrhea, winter dysentery, and bovine respiratory disease in newborn calves. These lead to higher calf mortality, reduced growth of fattening cows, and lower milk production in adult cows, resulting in significant losses to the cattle industry. Since commercial preventive drugs are not available in China, and existing treatments can only reduce the mortality of sick calves without fundamental control, the development of safe and effective vaccines is crucial. Methods: Two mRNA vaccines targeting the BCoV spiny receptor-binding domain (S-RBD) were prepared: XBS01 and XBS02. These two mRNAs, optimized for coding by AI and encapsulated in lipid nanoparticles (LNPs), were injected intramuscularly into mice (10 μg per mouse, twice, 2 weeks apart); a blank control group was not immunized. Serum antibodies, memory B/T cell activation and cytokine secretion were assessed by ELISA, flow cytometry and ELISpot. Results: Both vaccines induced humoral and cellular immunity:anti-S-RBD IgG titers were higher than those of the control group, and there was memory B-cell production and T-cell activation. XBS02 was superior to XBS01 in terms of peak antibody, memory B-cell frequency, T-cell activation rate, and IFN-γ/IL-2 secretion, and showed a stronger Th 1 response. Conclusions: Both BCoV S-RBD mRNA vaccines had good immunogenicity, with XBS02 providing better protection. This study supports the optimization and application of BCoV mRNA vaccines and accumulates data for mRNA technology in veterinary practice. Full article

Standardized, one-size-fits-all COVID-19 booster schedules may be suboptimal due to individual variation in immune backgrounds, particularly prior infection, which induces robust hybrid immunity. This study estimated optimal booster timing by modeling antibody decay in relation to surrogate correlates of protection (CoP). In a prospective cohort of 177 Japanese healthcare workers, we longitudinally monitored anti-spike receptor-binding domain (S-RBD) antibody titers following BNT162b2 vaccination. Participants were stratified into SARS-CoV-2-naïve and previously infected groups. Mixed-effects models were developed to predict when antibody titers would decline below predefined CoP thresholds. The model estimated optimal booster timing after a two-dose primary series to be 3–5 months for naïve individuals and approximately one year for those with prior infection. Following a third dose, the estimated interval extended to 8–12 months for the naïve group and 1.5–2 years for the previously infected group. These substantial differences underscore the limitations of uniform booster schedules. Our findings provide a quantitative framework for personalized vaccination strategies based on individual antibody profiles and immune status, thereby optimizing protection. Full article

Testing medical countermeasures for SARS-CoV-2 transmission using vertebrates can be hindered by legislation regulating animal experimentation, high costs, and ethical concerns. To overcome these challenges, we propose a new Caenorhabditis elegans strain that constitutively expresses the human angiotensin-converting enzyme 2 receptor (ACE2). This resulted in significant impairment of reproduction and a defect in pharyngeal function compared to wild-type (WT) worms. SARS-CoV-2 infection was simulated by treating worms with the receptor-binding domain (RBD) of the spike protein, which caused dose-dependent and time-dependent pharyngeal impairment in ACE2 worms but not in WT worms. The toxicity of RBD was prevented by administering an anti-human ACE2 antibody, demonstrating that interactions with the ACE2 receptor are essential. The ACE2-expressing worm strain was further used for pharmacological research with Raloxifene. In vitro, 1–3 μM of Raloxifene reduced the entry of lentiviral particles carrying the Wuhan variant and B.1.1.7 UK and B.1.1.529 Omicron strains into HEK293-ACE2, in addition to particles expressing N501Y-mutated or P681H-mutated spike proteins. Raloxifene (0.1–1 μM) completely counteracted RBD toxicity in ACE2 worms, indicating that this strain offers a cost-effective in vivo screening platform for molecules with effects involving interactions with the ACE2 receptor. Full article

The COVID-19 pandemic, driven by SARS-CoV-2, continues to challenge global health due to emerging variants and the potential risk posed by related sarbecoviruses. Neutralizing antibodies targeting the spike (S) glycoprotein, particularly the receptor-binding domain (RBD), play a crucial role in viral neutralization and vaccine design. Although broadly neutralizing anti-RBD antibodies have been identified, the nature of cross-reactive humoral responses induced by natural infection with ancestral SARS-CoV-2 strains remains incompletely understood. Here, we isolated 105 S-specific monoclonal antibodies (mAbs) from individuals recovered from prototype SARS-CoV-2 infection. Of these, 30 mAbs cross-recognized SARS-CoV-1, including 25 RBD-directed mAbs, of which 12 displayed cross-neutralizing activity against both viruses. Among them, mAb 12C2 potently neutralized SARS-CoV-1 and multiple SARS-CoV-2 variants, likely through mechanisms that include inhibition of membrane fusion and potential destabilization of the S trimer. Cryo-electron microscopy revealed that 12C2 engages the outer face of the RBD, overlapping with the epitope recognized by the broadly neutralizing antibody S309 derived from SARS-CoV-1 convalescent. Collectively, these findings demonstrate that ancestral SARS-CoV-2 infection can elicit robust cross-neutralizing antibody responses and provide valuable insights for the design of broadly protective antibodies and vaccines. Full article

Background: Gold nanoparticles (AuNPs) are a promising platform for vaccine antigen delivery due to their ability to stimulate both innate and adaptive immune responses. These effects depend strongly on physicochemical properties such as size, polydispersity, morphology, and surface charge, which are in turn determined by the synthesis method. While amino acids are often used as capping agents for AuNPs, their direct use as both reducing and stabilizing agents has been rarely investigated. Objectives: This study aimed to establish an ultrasound-assisted method for synthesizing AuNPs using amino compounds as both reducing and stabilizing agents, and assess their physicochemical characteristics, antigen-binding capacity, and immunogenicity. Methods: AuNPs were synthesized using L-cysteine, L-arginine, and cysteamine as dual reducing/stabilizing agents under ultrasonic conditions. The nanoparticles were combined with a recombinant receptor-binding domain (RBD) of SARS-CoV-2 and evaluated in mice for their ability to induce antibody responses. Results: The synthesized AuNPs exhibited hydrodynamic diameters ranging from 6.3 to 12.4 nm and zeta potentials from −40.5 to +36.5 mV, depending on the amino compound used. All formulations elicited robust anti-RBD IgG responses, but virus neutralization activity varied significantly. Notably, AuNP–arginine induced the strongest neutralizing response despite lower adsorption capacity and stability, suggesting that epitope preservation and antigen presentation quality were more decisive than antigen density. Conclusions: These findings underscore the importance of nanoparticle design in optimizing antigen presentation and highlight the potential of amino compound-synthesized AuNPs as effective antigen delivery vehicles for future vaccine development. Full article

Objectives: The aim of the present study was to better understand the antibody concentrations in healthcare workers (HCWs) from a hospital in Mexico City with a high density of COVID-19 patients. Methods: Up to 243 HCWs were recruited in 2020 and 2022 and were sorted into three groups: hybrid immunity (HI, natural infection plus vaccination), vaccine-induced immunity (VI), and unvaccinated but RT-qPCR negative at the beginning of the pandemic (UV). Peripheral blood and nasopharyngeal swab samples were obtained; additionally, saliva samples were obtained from the UV group. The titers of IgG, IgM, and IgA against the SARS-CoV-2 receptor-binding domain (RBD) and nucleocapsid (NCP) proteins were assessed using an in-house ELISA, and positivity to the virus was determined via RT-qPCR. Results: Most HI and VI participants were positive for serum anti-RBD IgG (92.8% and 100%, respectively), while 26.6% (for HI) and 19% (for VI) were positive for anti-NCP IgG. Regarding serum anti-RBD IgA, the VI and HI groups had positive rates of 87.3% and 66%, respectively. In contrast, the UV group showed a rate of 5.7% but the positivity for IgA in saliva was higher (52% for RBD and 35% for NCP). In addition, the highest antibody titers were obtained for anti-RBD IgG and IgA in the HI and VI groups, respectively. In saliva, the IgA antibody titer was higher for the RBD antigen (1:1280). Conclusions: These results strengthen our understanding of antibody concentrations in HCWs during two critical years of the pandemic in a general hospital with many COVID-19 patients. Full article

Background: The concentration of antigen-specific antibodies in serum is usually measured in international units/mL. Therefore, the actual concentration of virus-specific antibodies in sera is unknown. Objectives: The aim of the study was to determine conversion factors for concentrations of IgG against hepatitis B surface antigen (HBs), SARS-CoV-2 receptor binding domain (RBD) and nucleoprotein (NP) as well as tetanus toxin (Ttx) in serum and to compare antigen-specific IgG concentrations in serum samples. Methods: Absorption equivalence ELISAs were used to determine conversion factors for international units (IU) for anti-HBs, anti-SARS-CoV-2-RBD and NP and for anti-Ttx immunoglobulin G. The antigen-specific IgG concentrations in serum samples were then measured in units/mL and the ratio of IgG concentrations in the sera was determined using the conversion factors. Results: One IU of anti-HBs IgG corresponded to 24.4 BAU of anti-CoV-2 RBD IgG, 6.87 BAU of anti-CoV-2 NP and 14 mIU of anti-Ttx IgG. One BAU anti-SARS-CoV-2 NP-specific IgG is equivalent to 3.5 BAU SARS-CoV-2 RBD-specific IgG. Conversion of international units showed that median serum anti-Ttx-IgG concentrations were 50 times higher and anti-CoV-2-RBD-IgG concentrations were 390 times higher than median anti-HBs-IgG concentrations. In addition, after SARS-CoV-2 infection, the concentration of NP-specific IgG in serum was generally higher than that of RBD-specific IgG. Conclusions: The study provides conversion factors for serum concentrations of IgG against HBs, SARS-CoV-2 RBD and NP, as well as Ttx-IgG. This offers new insights into serum IgG concentrations and allows conclusions to be drawn about plasma cell pools. Full article

Background: COVID-19 vaccination has been a key tool in protecting healthcare workers (HCWs), but breakthrough infections have occurred. The durability of vaccine-induced immunity and its impact on HCWs remain critical for public health strategies. Methods: In this small cohort study (n = 32), we assessed antibody levels and breakthrough infection rates in HCWs over 12 months post-vaccination, providing insights for booster strategies and infection control. A cohort of 32 HCWs was screened for SARS-CoV-2 infection using weekly self-administered swabs and blood samples collected at baseline, 6 months, and 12 months. SARS-CoV-2 antibodies (IgG, IgM) targeting spike proteins and nucleocapsids were analyzed using a multi-antigen serology panel. Pooled nucleic acid testing was employed for infection detection. Results: Nine participants showed breakthrough infections, with nucleocapsid antibodies indicating prior infection. Eight of these cases occurred after the third vaccine dose during the Omicron-dominant period. Anti-spike antibody levels declined significantly in participants without prior infection, while those with breakthrough infections exhibited increased levels. The half-life of S1 and S1 receptor-binding domain (RDB) vaccine-induced antibodies was 144 and 166 days, respectively, which aligns with CDC data. These findings provide valuable insights for determining the optimal timing of booster doses. Conclusions: Our findings highlight the waning antibody levels over time and the occurrence of breakthrough infections. Although based on a small sample, these data support the need for ongoing monitoring and timely boosters. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article

Background/Objectives: Vaccine immunogenicity is often suboptimal in vulnerable populations such as the elderly, infants, and individuals in low- and middle-income countries. One contributing factor may be pre-existing immunomodulatory conditions, including helminth infections. This study investigates the impact of Fasciola hepatica (F. hepatica) derived molecules on the early humoral response to the COVID-19 mRNA vaccine Comirnaty^® in a mouse model. Methods: BALB/c mice were pretreated with a F. hepatica protein extract (FH) or complete Freund’s adjuvant (CFA) prior to vaccination. Cytokine production and antibody responses were assessed at 0, 14, and 21 days post-vaccination (dpv) through serum analysis and ex vivo splenocyte stimulation with the SARS-CoV-2 receptor-binding domain (RBD) or LPS. Results: At 0 dpv, FH-treated mice showed increased serum IL-10, while CFA treatment induced IL-12. FH- but not CFA-treated splenocytes secreted IL-10 upon RBD or LPS stimulation. At 21 dpv, FH-treated mice lacked IFN-γ production but maintained IL-10 and showed elevated IL-4, consistent with a Th2-skewed profile. Although total anti-RBD IgG levels were similar between groups, FH-treated mice exhibited reduced IgG avidity and a higher IgG1/IgG2 ratio. CFA-treated mice showed delayed avidity maturation. Conclusions: Prior exposure to F. hepatica antigens can modulate the early immune response to Comirnaty^®, affecting both cellular activation and antibody quality. This altered response may reflect a reduced early protective capacity of the vaccine, which might need to be considered when designing or evaluating vaccination strategies using mRNA vaccines in helminth-endemic regions. Full article

Aim. This study aimed to assess the SARS-CoV-2 herd immunity in the Republic of Armenia (RA) by late 2022. Materials and Methods. A randomized study was conducted from 28 November to 2 December (2022) by the Saint Petersburg Pasteur Institute (Russia) in collaboration with the National Center for Disease Control and Prevention (Armenia). This study was approved by the ethics committees at both organizations. A volunteer cohort (N = 2974) was formed and grouped by participant age, region, or activity. Antibodies (Abs) to viral nucleocapsid antigen (Nc) and receptor-binding domain (RBD) in plasma were determined by ELISA. The statistical significance of differences was calculated using a p < 0.05 threshold, unless noted. Results. At the end of 2022, estimated SARS-CoV-2 seroprevalence (Nc and/or RBD Abs) among the Armenian population was 99% (95%CI: 98.5–99.3). It was evenly distributed throughout the cohort without any significant differences by age, region, or activity. Volunteers with low (32–124 BAU/mL) or medium (125–332 BAU/mL) anti-Nc Ab levels prevailed: 32.4% (95%CI: 30.7–34.1) and 25.5% (95% CI: 24.0–27.1), respectively. Regarding anti-RBD Abs, maximum levels (>450 BAU/mL) were detected in 40% of children. The share of individuals with high anti-RBD Abs levels increased with age, reaching 65% among those aged 70⁺ years. The important contribution to the formation of herd immunity to coronavirus infection was made by vaccination in the preceding period (1 April 2021 to 1 May 2022). The contribution from individuals with post-vaccination immunity was estimated to be above 80%. Hybrid immunity, formed after vaccination of those who had earlier experienced COVID-19, was characterized by greater effectiveness than post-vaccination immunity alone. Conclusions. Within the context of mass prophylactic vaccination, effective herd immunity to SARS-CoV-2 was formed, which helped to stop epidemic spread in the Republic. Full article