Search Results (696)

Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16, is a major driver of HPV-associated cancers; however, strategies for treating established HPV-induced tumors remain scarce. Here, we developed a DNA-based vaccine linking the SARS-CoV-2 spike (S) protein with an HPV16 E7 epitope (aa 49-57) to simultaneously induce antiviral humoral immunity and antitumor cellular responses. We generated 2 constructs, S-E7 and S-RE7, with the latter incorporating a furin cleavage site (R) to enhance antigen processing. In vitro, S-RE7 significantly enhanced E7-specific CD8⁺ T cell activation compared to S-E7, highlighting the importance of the furin sequence. In vivo, both S-linked vaccines elicited robust E7-specific CD8⁺ T cell responses and provided complete protection against TC-1 tumor challenge in a prophylactic murine model, with long-lasting immunity upon tumor rechallenge. In therapeutic settings, vaccination with S-E7 or S-RE7 significantly suppressed tumor growth, extended survival, and reduced circulating myeloid-derived suppressor cells (MDSCs), indicating alleviation of systemic immunosuppression. Notably, S-RE7 demonstrated faster antitumor effects overall in early tumor progression. In addition to cellular immunity, both constructs induced high levels of anti-spike antibodies, with S-RE7 eliciting approximately fourfold higher responses than S-E7. Furthermore, S-RE7 effectively boosted pre-existing anti-spike immunity in mice that were previously vaccinated. This “two-in-one” strategy represents a promising and versatile platform for the prevention and treatment of HPV-associated cancers while maintaining preparedness against potential SARS-CoV-2. Full article

Identifying antigens that elicit protective immunity is the key step for vaccine development. Here, we describe the use of the MS2 VLP platform to identify epitopes of SARS-CoV-2 structural proteins recognized by antibodies from COVID-19 patients, and to present those epitopes to the immune system as vaccines. We constructed an MS2 virus-like particle (VLP) library covering all four structural proteins of SARS-CoV-2 and affinity-selected vaccine candidates by biopanning on antibodies from infected humans. We focused on the structural proteins, reasoning that they are the most likely targets of a protective antibody response. The epitopes we found map almost entirely to the spike and nucleocapsid proteins. The VLPs displaying such epitopes were produced individually in E. coli and then tested for their potential as vaccines. While none of the affinity-selected spike-specific VLPs elicited neutralizing antibodies, VLPs displaying nucleocapsid epitopes induced protective immunity in a hamster model. This work illustrates the MS2 VLP platform’s capacity for the identification of new vaccine candidates and raises the possibility that VLPs displaying nucleocapsid epitopes could provide long-lasting protection against a range of virus variants. Full article

Background: Despite the circulation of evolutionarily related cold-causing coronaviruses (CCCs) in the pre-COVID era, most individuals lacked pre-existing serum IgG and/or class-switched memory B cell (B_mem) reactivity for the SARS-CoV-2 Spike (S) glycoprotein expressed by the ancestral Wuhan-Hu-1 (WH1) strain. Subsequent priming of the immune system through natural infection or prophylactic COVID-19 mRNA vaccination successfully generated robust B_mem responses against the WH1-S antigen, along with eliciting cross-reactivity for the future Omicron (BA.1) variant responsible for breakthrough infections (BTIs). However, to what extent immunological imprinting of B_mem towards the WH1-S antigen detrimentally constrains the elicitation of variant-specific antibody responses following subsequent booster vaccinations or BTIs—a phenomena referred to as “original antigenic sin”—remains an unresolved and open question. Methods: Using ImmunoSpot^®, we evaluated peripheral blood mononuclear cells (PBMCs) from defined human cohorts for IgG⁺ ASC reactivity against Spike proteins representing CCCs and SARS-CoV-2. Additionally, we developed a novel dual-label inverted FluoroSpot assay to distinguish between strain-specific and cross-reactive IgG⁺ ASCs recognizing epitopes in the receptor binding domain (RBD) of SARS-CoV-2 Omicron variants. Results: Our data demonstrate a lack of appreciable back-boosting of IgG⁺ B_mem recognizing structurally conserved epitopes shared between CCCs and SARS-CoV-2. Moreover, we found evidence for immunological imprinting and the preferential expansion of B_mem recognizing cross-reactive epitopes in the RBD following BTI. Nevertheless, Omicron strain-specific B_mem were detected in PBMC donors collected in 2025. Conclusions: Our novel inverted dual-label FluoroSpot methodology evidenced preferential expansion of cross-reactive B_mem following breakthrough SARS-CoV-2 infection and supports the influence of original antigenic sin shaping the recall response. Moreover, the inverted dual-label assay provides a highly flexible and easily implementable technique for distinguishing between strain-specific and cross-reactive B cell responses and has broad applications in translational vaccine research against pathogens that undergo antigenic drift. Full article

Porcine epidemic diarrhea virus (PEDV) continues to undergo genetic evolution and remains a major etiological agent of enteric disease in swine, causing significant economic losses worldwide. This study investigated the molecular epidemiology and genetic characteristics of PEDV circulating in Chongqing, China, between 2022 and 2024. A total of 296 diarrheic piglet samples collected from nine regions were screened using RT-qPCR, of which 48.31% (143/296) tested positive for PEDV. A subset of positive samples was subjected to S gene amplification and sequencing, yielding 15 complete sequences. Phylogenetic analysis revealed that all sequenced strains clustered within the G2c lineage and showed high nucleotide similarity (93.37–94.09%) to the classical CV777 strain. Recombination analysis indicated potential recombination events among field strains involving S-INDEL and G2b-like parental lineages, although these findings are based on a limited number of sequences. Sequence analysis identified multiple amino acid substitutions within the COE antigenic region, while other neutralizing epitopes (SS2, SS6, and 2C10) remained largely conserved. In addition, variation in predicted N-glycosylation sites was observed among some strains. Structural modelling suggested that these changes may influence spike protein conformation and antigenic properties; however, these interpretations are based on in silico analysis and require experimental validation. Overall, the findings indicate ongoing genetic evolution of PEDV in Chongqing and suggest circulation of G2c-associated variants in diarrheic piglets. However, given the limited and non-random nature of sequencing, these results may not fully represent the broader viral population. Continued large-scale molecular surveillance and functional studies are needed to better understand PEDV evolution and to support the development of improved control strategies and vaccines. Full article

Aflatoxin B1 (AFB1), a highly carcinogenic mycotoxin, has been the focus of research for the development of efficient detection methods. In this study, a novel magnetic immuno-ratiometric fluorescent sensing system was constructed for the quantitative detection of AFB1. Green-emitting carbon quantum dots were conjugated with AFB1 monoclonal antibody to obtain GCDs@AFB1 mAb, and AFB1 oxime was immobilized on Fe₃O₄ magnetic microspheres to prepare AFB1-Ox@Fe₃O₄ NPs. After the immune-competitive adsorption of GCDs@AFB1 mAb by AFB1-Ox@Fe₃O₄ NPs and free AFB1, magnetic separation was performed. Red fluorescent silver nanoclusters were introduced as an internal reference to construct a GCDs-AgNCs ratiometric fluorescent system. The sensor exhibited a good linear response in the range of 0~240 pg/mL with a low limit of detection of 18 pg/mL and excellent selectivity. The spiked recoveries in real samples ranged from 92.14% to 110.02%, with a relative standard deviation of 0.57% to 4.58%. Combining the specific antigen–antibody recognition with magnetic separation technology, this method addresses the issues of poor stability and high environmental interference of traditional fluorescent sensors, and provides a new strategy for the sensitive and stable detection of AFB1. Full article

Convergent evolution in protein antigens is common across pathogens, including SARS-CoV-2; the most likely reason is the need to evade the selective pressure exerted by previous infection- or vaccine-elicited immunity. There is a pressing need for automated analysis of convergent mutations. We developed ConvMut, a tool to identify patterns of recurrent mutations in SARS-CoV-2 evolution; we exploited the granular phylogeny-based lineage hierarchy developed by PANGO, allowing us to observe deltas, i.e., groups of mutations that are acquired with respect to the immediately upstream tree nodes. Deltas comprise amino acid substitutions, insertions, and deletions. ConvMut can perform individual protein analysis to identify the most common single mutations acquired independently in a given subtree. Lineages are then gathered into clusters according to user-selected sets of shared mutations. An interactive graph orders the evolutionary steps of clusters, details the acquired amino acid change for each sublineage, and allows us to trace the evolutionary path until a selected lineage. ConvMut also supports frequency analysis for a given nucleotide or amino acid changes at a given residue across a selected phylogenetic subtree. ConvMut facilitates the exploration of convergent evolutionary trends in SARS-CoV-2, providing insights that could support the development of broadly effective anti-Spike monoclonal antibodies and Spike-based vaccines. Full article

Background: The COVID-19 pandemic exposed critical gaps in pandemic preparedness and highlighted the need for vaccine platforms capable of rapid adaptation. Outer membrane vesicle (OMV)-based platforms utilizing vesicles derived from genetically detoxified Neisseria meningitidis serogroup B (Nm-nOMV) represent a promising plug-and-play approach. Methods: This Phase I, first-in-human, randomized, double-blind, placebo- and OMV-controlled trial, evaluated safety, tolerability, and immunogenicity of intranasally administered OMVs combined with SARS-CoV-2 Spike protein in healthy SARS-CoV-2 seropositive adults aged 18–55 years. Forty participants were enrolled across two cohorts: a low-dose cohort receiving 140 μg OMV/70 μg Spike (OMV + Spike, n = 13; OMV alone, n= 3; Placebo, n = 5) and a high-dose cohort receiving 280 μg of OMV/140 μg of Spike (OMV + Spike, n = 13; OMV alone, n = 3; Placebo, n = 3), administered on Days 1 and 22. Safety was assessed through adverse events, vital signs, laboratory parameters, ECG, and pulse oximetry. Immunogenicity was evaluated via systemic SARS-CoV-2 neutralizing antibodies, antigen-specific antibodies (IgG and IgA), and mucosal antibodies (IgA in nasal wash). Results: Intranasal administration of OMVs combined with SARS-CoV-2 Spike protein was safe, well-tolerated, and immunogenic. No serious adverse events were reported, and adverse events were predominantly mild and transient. Dose-dependent increases in systemic and mucosal immune responses were observed, with statistically significant enhanced serum IgG and nasal wash IgA antibodies in the high-dose group. Conclusions: The current clinical data confirm key aspects of the preclinical profile, which demonstrate the potential of the Nm-nOMV platform as a strong adjuvant for mucosal vaccines. These findings support the broader application of the Nm-nOMV vaccine platform in pandemic preparedness. Full article

Background: While vaccines are designed to elicit targeted immune responses, in some cases, the immunogenic molecules employed can inherently interact with broader host cellular pathways as a secondary consequence. This phenomenon can be exemplified by COVID-19 vaccines. COVID-19 vaccines, including mRNA platforms, use the SARS-CoV-2 spike protein as an immunogen to induce the production of neutralizing antibodies. The spike protein binds the ACE2 (angiotensin-converting enzyme 2) receptor on human cells, mediating viral entry and infection. ACE2 is widely expressed across multiple tissues and is a key component of the renin–angiotensin–aldosterone system (RAAS) that acts as a homeostatic regulator of systemic and local blood flow, blood pressure, cardiac function, fluid balance and immunity. Some studies have proposed the interaction between the spike protein and ACE2 as a possible contributing factor to rare adverse effects observed following COVID-19 vaccination, including myocarditis, pericarditis, thrombosis, and reported alterations in blood pressure, though these mechanisms remain to be fully elucidated. Objectives: As a proof-of-concept approach in vaccine antigen development, we engineered SARS-CoV-2 spike mutants with impaired binding to the host receptor ACE2. Methods: By rational design, we produced and validated in vitro and in vivo spike point mutants that do not effectively bind ACE2. Results: The engineered spike mutants do not effectively bind the human entry receptor ACE2 while retaining the immunogenic properties equal to or better than the wild type spike and thus generate a protective response in animals when used as a vaccination agent. Conclusions: By establishing a straightforward molecular strategy for rational vaccine design, this work demonstrates the feasibility of limiting specific antigen–host receptor interactions while maintaining immunogenicity. This approach may be applicable to future vaccination strategies where antigen interaction with host cells could potentially interfere with physiological pathways. Full article

The 2019 coronavirus disease pandemic (COVID-19) showed how genetic mutations can alter coronavirus characteristics. However, the evolution of livestock coronaviruses remains understudied. We analyzed 15 bovine coronavirus (BCoV), three porcine hemagglutinating encephalomyelitis virus (PHEV) and 18 porcine respiratory coronavirus (PRCV) isolates, mainly from Belgian livestock collected between 2020 and 2023. Spike gene phylogenetic analysis showed nucleotide substitution rates comparable between BCoV and PRCV, while PHEV appeared slower. Unlike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), synonymous substitutions were preferred, limiting amino acid variation across decades in the animal coronaviruses. Virus neutralization assays with swine antisera indicated minimal antigenic change in PHEV and PRCV. Recent BCoV isolates showed antigenic divergence from the classical Mebus vaccine strain. The impact of this divergence on vaccine efficacy may warrant further research. Our findings underscore the need for periodic surveillance, as changes in surface proteins may affect pathogenicity, tissue tropism, host range and vaccine efficacy. Full article

The continuous evolution of the SARS-CoV-2 virus, marked by the emergence of new variants, poses a significant threat to the efficacy of existing vaccines. However, a promising approach to addressing vaccine failure caused by viral mutations (particularly in the spike protein) is the development of a variant-proof (conserved), non-spike, multiepitope universal nanostructure vaccine with multifunctionality, biocompatibility, self-adjuvanticity, and structural similarity to pathogens in terms of size and shape. This study aimed to design a self-assembled nanostructure vaccine (SANV) featuring pentameric and trimeric coiled-coil peptide motifs, as well as other functional motifs, including epitopes, TAT, PADRE, and adjuvant. The cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B lymphocyte (BL) epitopes of SANV were screened from the IEDB with more than 50% individual predicted population coverage (PPC) and fused using linkers to enable self-assembly. The multimerization of the 24 SANV monomers was modeled using the GalaxyHomomer and AlphaFold web servers. Subsequently, the leading SANV constructs with (SANVa9) and without (SANVb6) adjuvant were analyzed for their physicochemical profiles and assessed for antigenicity, allergenicity, solubility, and antioxidant potential. Furthermore, the molecular interactions, specificity, and stability of SANVa9 and SANVb6 with the broadly neutralizing sarbecovirus antibody 5817 and toll-like receptors (TLR2, TLR3, and TLR7) were analyzed using molecular docking and simulation over a 100-nanosecond time scale. Finally, the comparative immune simulation profiles of SANVa9 and SANVb6 with controls indicated stronger, broad-spectrum immune responses that could be translated into in vitro and in vivo studies and warrant further evaluation before clinical use. Full article

Introduction: From 12 March 2020, when the first cases of COVID-19 were registered in Kosovo, to 9 March 2023, there were a total of 273,310 reported cases of COVID-19 and 3211 reported deaths in Kosovo (CFR: 1.17%). Health care workers (HCWs) have been at a higher risk of contracting SARS-CoV-2 infection; nevertheless, data on seroprevalence of SARS-CoV-2 antibodies among HCWs in Kosovo are very limited. Methodology: A cross-sectional serology study with 1654 healthcare professionals throughout Kosovo was conducted to determine the presence of antibodies against the spike antigen of SARS-CoV-2. In addition, a structured questionnaire was administered to study participants to obtain basic demographic data, and information on prior infection and COVID-19 vaccination status. Results: Antibodies against the spike antigen of SARS-CoV-2 were detected in almost all (99.8%) HCWs that participated in the study. The average antibody titer was 8030.8 AU/mL in women and 9533.7 AU/mL in men. Sixty-four percent of HCWs in this study reported prior infection with SARS-CoV-2, 6% of whom were hospitalized. Over 98% of study participants had received SARS-CoV-2 vaccination. Conclusions: Almost all HCWs participating in the study had antibodies against the spike antigen of SARS-CoV-2. This is most probably the result of the high COVID-19 vaccination rate in Kosovo as well as infection with SARS-CoV-2. Full article

A capacitive screen-printed electrode immunosensor operating in non-faradaic mode by dispensing redox probes was developed for the Coronavirus 2 Spike (S) protein. This new strategy enabled direct detection of the S protein by measuring changes in the electrochemical capacitance resulting from antigen–antibody interactions on the electrode surface, altering interfacial dielectric properties. To enhance analytical sensitivity and provide an electrode surface with attractive capacitive and conductive properties, an in-house graphite ink-based screen-printed electrode was developed and subsequently modified with a polypyrrole (PPy) layer in bulk-synthesized in the presence of Cetyltrimethylammonium bromide (CTAB). CTAB acted as a dispersing and structure-directing agent, promoting homogeneous distribution and guiding the PPy polymerization, resulting in a composite with improved charge density storage and high conductivity. Analytical signals of the S proteins in spiked serum were detected by measuring the Specific Capacitances taken from cyclic voltammograms. This capacitive immunosensor achieved a linear range from 1 to 100 µg/mL (R² = 0.989, p < 0.05), with a limit of detection of 0.45 µg/mL of S protein, which falls within the clinical range for COVID-19 diagnostics. Probe-free detection without ferri/ferrocyanide steps minimizes errors by probe adsorptions and is easy to use as a point-of-care, unlike conventional immunosensors. Full article

Antigen detection provides rapid and convenient diagnosis of respiratory infections. This study develops an innovative dual-fluorescence aptasensing method based on polydopamine-functionalized MXene (PDA-MXene) for the simultaneous detection of spike protein and hemagglutinin protein. The method employs green- and red-emitting quantum dot (QD) probes as fluorescence reporters, and the PDA-MXene as an effective adsorption and separation substrate. Coupled with a centrifugation-assisted separation strategy, this design method reduces background interference and enhances detection reliability. The method demonstrates good analytical performance, with detection limits of 0.82 ng/mL for spike protein and 2.11 ng/mL for hemagglutinin protein in single-channel mode. The dual-channel mode enables reliable and simultaneous quantification of both target proteins with minimal spectral cross-talk. Furthermore, this method exhibits high specificity against interferents including ions, proteins, and toxins. Artificial saliva, chosen as real sample, is spiked with target proteins to investigate the practical applicability of the method, showing recovery rates for both target proteins between 100 and 114 sensing strategy is simple to operate and allows the detection of new targets by simply replacing the azide-modified aptamer lyophilized powder. It therefore holds promising application for the simultaneous detection of multiple proteins in point-of-care testing and health monitoring fields. Full article

Background: Monoclonal antibodies (mAbs) initially played a major role in outpatient COVID-19 management by providing rapid passive immunity and reducing progression to severe disease. However, continuous SARS-CoV-2 evolution progressively compromised the effectiveness of several anti-spike products. This narrative review summarizes the trajectory of COVID-19 mAbs across three phases: early clinical efficacy, loss of efficacy due to immune escape, and future directions. Methods: We conducted a narrative review focusing on mechanisms of action, pivotal clinical trials, and real-world effectiveness of neutralizing anti-spike mAbs and host-directed immunomodulatory mAbs. Emphasis was placed on the impact of variants—especially Omicron—on susceptibility and clinical use, as well as on emerging next-generation platforms. Results: First-generation neutralizing mAbs substantially reduced the hospitalization rates during the Alpha and Delta waves, while immunomodulatory mAbs became standard options for the hyperinflammatory phase in hospitalized patients. With the emergence of Omicron and its sub-lineages, extensive immune escape led to marked reductions in neutralization for many earlier anti-spike agents and consequent restrictions in use. Later-generation approaches targeting more conserved epitopes provided temporary solutions but were also challenged by ongoing antigenic drift. Host-directed immunomodulators retained clinical relevance because their mechanism is independent of viral spike mutations. Conclusions: The clinical role of monoclonal antibodies in COVID-19 has been dynamic and increasingly constrained by viral evolution. Future strategies should prioritize broadly neutralizing antibodies targeting conserved epitopes, innovative delivery platforms, and integration with real-time surveillance to preserve clinical utility in the endemic phase and improve preparedness for future outbreaks. Full article

Background/Objectives: In the first months of their life, infants rely on maternal antibodies for immune protection. Breastmilk is a major source of these defenses, supplying secretory IgA, IgG, and IgM that help guard mucosal surfaces against pathogens such as SARS-CoV-2. Most studies on breastmilk immunity in the context of COVID-19 have emphasized circulating monomeric IgA, rather than the multimeric secretory IgA (sIgA) that is active at mucosal barriers. This study assessed in-depth the contribution of breastmilk antibody subtypes to SARS-CoV-2 neutralization capacity and how these profiles differ following maternal COVID-19 infection versus vaccination during pregnancy or postpartum. Methods: In this prospective cohort study, breastmilk samples were collected longitudinally from individuals who had COVID-19 during pregnancy or received COVID-19 mRNA vaccination during pregnancy or postpartum. Serological assays measured IgG, IgM, systemic IgA, and secretory IgA against SARS-CoV-2 spike and nucleocapsid antigens. Results: COVID-19 infection during pregnancy resulted in significantly higher systemic and secretory IgA levels compared to vaccination. Secretory IgA demonstrated a strong correlation with neutralization capacity. Principal component analysis revealed distinct antibody profiles in COVID-19-exposed individuals versus vaccinated cohorts, with significant overlap between pregnancy and postpartum vaccination groups. Conclusions: Although both COVID-19 vaccination and disease elicit sustained COVID-19-related antibodies in breastmilk, COVID-19 infection elicits a broader and more diverse antibody response in breastmilk, specifically with a greater secretory IgA generation. These findings support the value of maternal vaccination to safely confer mucosal immunity to neonates and the need for optimized vaccine formulations for mucosal immunity. Full article