Antigen-Mimic Nanoparticles in Ultrasensitive on-Chip Integrated Anti-p53 Antibody Quantification.

As a tumor-suppressing protein, p53 plays a crucial role in preventing cancer development. Its utility as an early cancer detection tool is significant, potentially enabling clinicians to forestall disease advancement and improve patient prognosis. In response to the pathological overexpression of this antigen in tumors, the prevalence of anti-p53 antibodies increases in serum, in a manner quantitatively indicative of cancer progression. This spike can be detected through techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation. In this study, we present an electrochemical approach that supports ultrasensitive and highly selective anti-p53 autoantibody quantification without the use of an immuno-modified electrode. We specifically employ antigen-mimicking and antibody-capturing peptide-coated magnetic nanoparticles, along with an AC magnetic field-promoted sample mixing, prior to the presentation of Fab-captured targets to simple lectin-modified sensors. The subfemtomolar assays are highly selective and support quantification from serum-spiked samples within minutes.

Potential clinical implications of molecular mimicry-induced autoimmunity.

BACKGROUND: Molecular mimicry is hypothesized to be a mechanism by which autoimmune diseases are triggered. It refers to sequence or structural homology between foreign antigens and self-antigens, which can activate cross-reactive lymphocytes that attack host tissues. Elucidating the role of molecular mimicry in human autoimmunity could have important clinical implications. OBJECTIVE: To review evidence for the role of molecular mimicry in major autoimmune diseases and discuss potential clinical implications. METHODS: Comprehensive literature review of clinical trials, observational studies, animal models, and immunology studies on molecular mimicry in multiple sclerosis, type 1 diabetes, rheumatoid arthritis, lupus, Guillain-Barre syndrome, autoimmune myocarditis, and primary biliary cirrhosis published from 2000-2023. RESULTS: Substantial indirect evidence supports molecular mimicry as a contributor to loss of self-tolerance in several autoimmune conditions. Proposed microbial triggers include Epstein-Barr virus, coxsackievirus, Campylobacter jejuni, and bacterial commensals. Key mechanisms involve cross-reactive T cells and autoantibodies induced by epitope homology between microbial and self-antigens. Perpetuation of autoimmunity involves epitope spreading, inflammatory mediators, and genetic factors. CONCLUSIONS: Molecular mimicry plausibly explains initial stages of autoimmune pathogenesis induced by infection or microbiota disturbances. Understanding mimicry antigens and pathways could enable improved prediction, monitoring, and antigen-specific immunotherapy for autoimmune disorders. However, definitive proof of causation in humans remains limited. Further research should focus on establishing clinical evidence and utility.

Virus-reactive T cells expanded in aplastic anemia eliminate hematopoietic progenitor cells by molecular mimicry.

ABSTRACT: Acquired aplastic anemia is a bone marrow failure syndrome characterized by hypocellular bone marrow and peripheral blood pancytopenia. Frequent clinical responses to calcineurin inhibition and antithymocyte globulin strongly suggest critical roles for hematopoietic stem/progenitor cell-reactive T-cell clones in disease pathophysiology; however, their exact contribution and antigen specificities remain unclear. We determined differentiation states and targets of dominant T-cell clones along with their potential to eliminate hematopoietic progenitor cells in the bone marrow of 15 patients with acquired aplastic anemia. Single-cell sequencing and immunophenotyping revealed oligoclonal expansion and effector differentiation of CD8+ T-cell compartments. We reexpressed 28 dominant T-cell receptors (TCRs) of 9 patients in reporter cell lines to determine reactivity with (1) in vitro-expanded CD34+ bone marrow, (2) CD34- bone marrow, or (3) peptide pools covering immunodominant epitopes of highly prevalent viruses. Besides 5 cytomegalovirus-reactive TCRs, we identified 3 TCRs that recognized antigen presented on hematopoietic progenitor cells. T cells transduced with these TCRs eliminated hematopoietic progenitor cells of the respective patients in vitro. One progenitor cell-reactive TCR (11A5) also recognized an epitope of the Epstein-Barr virus-derived latent membrane protein 1 (LMP1) presented on HLA-A∗02:01. We identified 2 LMP1-related mimotopes within the human proteome as activating targets of TCR 11A5, providing proof of concept that molecular mimicry of viral and self-epitopes can drive T cell-mediated elimination of hematopoietic progenitor cells in aplastic anemia.

IgG Antibody Responses to Epstein-Barr Virus in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Their Effective Potential for Disease Diagnosis and Pathological Antigenic Mimicry.

Background and Objectives: The diagnosis and pathology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) remain under debate. However, there is a growing body of evidence for an autoimmune component in ME/CFS caused by the Epstein-Barr virus (EBV) and other viral infections. Materials and Methods: In this work, we analyzed a large public dataset on the IgG antibodies to 3054 EBV peptides to understand whether these immune responses could help diagnose patients and trigger pathological autoimmunity; we used healthy controls (HCs) as a comparator cohort. Subsequently, we aimed at predicting the disease status of the study participants using a super learner algorithm targeting an accuracy of 85% when splitting data into train and test datasets. Results: When we compared the data of all ME/CFS patients or the data of a subgroup of those patients with non-infectious or unknown disease triggers to the data of the HC, we could not find an antibody-based classifier that would meet the desired accuracy in the test dataset. However, we could identify a 26-antibody classifier that could distinguish ME/CFS patients with an infectious disease trigger from the HCs with 100% and 90% accuracies in the train and test sets, respectively. We finally performed a bioinformatic analysis of the EBV peptides associated with these 26 antibodies. We found no correlation between the importance metric of the selected antibodies in the classifier and the maximal sequence homology between human proteins and each EBV peptide recognized by these antibodies. Conclusions: In conclusion, these 26 antibodies against EBV have an effective potential for disease diagnosis in a subset of patients. However, the peptides associated with these antibodies are less likely to induce autoimmune B-cell responses that could explain the pathogenesis of ME/CFS.

Molecular mimicry sweetens the deal for MANagement of host mRNA translation and stress responses by Legionella pneumophila.

The Legionella pneumophila effector SidI inhibits host mRNA translation and must be regulated for intracellular replication. Subramanian et al.1 reveal the mechanism of SidI translation inhibition and how stress signaling in response to sustained SidI activity drives host cell death.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.

Identification of potential molecular mimicry in pathogen-host interactions.

Pathogens have evolved sophisticated strategies to manipulate host signaling pathways, including the phenomenon of molecular mimicry, where pathogen-derived biomolecules imitate host biomolecules. In this study, we resurrected, updated, and optimized a sequence-based bioinformatics pipeline to identify potential molecular mimicry candidates between humans and 32 pathogenic species whose proteomes' 3D structure predictions were available at the start of this study. We observed considerable variation in the number of mimicry candidates across pathogenic species, with pathogenic bacteria exhibiting fewer candidates compared to fungi and protozoans. Further analysis revealed that the candidate mimicry regions were enriched in solvent-accessible regions, highlighting their potential functional relevance. We identified a total of 1,878 mimicked regions in 1,439 human proteins, and clustering analysis indicated diverse target proteins across pathogen species. The human proteins containing mimicked regions revealed significant associations between these proteins and various biological processes, with an emphasis on host extracellular matrix organization and cytoskeletal processes. However, immune-related proteins were underrepresented as targets of mimicry. Our findings provide insights into the broad range of host-pathogen interactions mediated by molecular mimicry and highlight potential targets for further investigation. This comprehensive analysis contributes to our understanding of the complex mechanisms employed by pathogens to subvert host defenses and we provide a resource to assist researchers in the development of novel therapeutic strategies.

Cross-regulation of antibody responses against the SARS-CoV-2 Spike protein and commensal microbiota via molecular mimicry.

The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

Bacteriophages suppress CRISPR-Cas immunity using RNA-based anti-CRISPRs.

Many bacteria use CRISPR-Cas systems to combat mobile genetic elements, such as bacteriophages and plasmids1. In turn, these invasive elements have evolved anti-CRISPR proteins to block host immunity2,3. Here we unveil a distinct type of CRISPR-Cas Inhibition strategy that is based on small non-coding RNA anti-CRISPRs (Racrs). Racrs mimic the repeats found in CRISPR arrays and are encoded in viral genomes as solitary repeat units4. We show that a prophage-encoded Racr strongly inhibits the type I-F CRISPR-Cas system by interacting specifically with Cas6f and Cas7f, resulting in the formation of an aberrant Cas subcomplex. We identified Racr candidates for almost all CRISPR-Cas types encoded by a diverse range of viruses and plasmids, often in the genetic context of other anti-CRISPR genes5. Functional testing of nine candidates spanning the two CRISPR-Cas classes confirmed their strong immune inhibitory function. Our results demonstrate that molecular mimicry of CRISPR repeats is a widespread anti-CRISPR strategy, which opens the door to potential biotechnological applications6.

Gardnerella Vaginolysin Potentiates Glycan Molecular Mimicry by Neisseria gonorrhoeae.

Bacterial vaginosis (BV) is a dysbiotic condition of the vaginal microbiome associated with higher risk of infection by Neisseria gonorrhoeae-the cause of gonorrhea. Here we test if one known facet of BV-the presence of bacterial cytolysins-leads to mobilization of intracellular contents that enhance gonococcal virulence. We cloned and expressed recombinant vaginolysin (VLY), a cytolysin produced by the BV-associated bacterium Gardnerella, verifying that it liberates contents of cervical epithelial (HeLa) cells, while vector control preparations did not. We tested if VLY mediates a well-known gonococcal virulence mechanism-the molecular mimicry of host glycans. To evade host immunity, N. gonorrhoeae caps its lipooligosaccharide (LOS) with α2-3-linked sialic acid. For this, gonococci must scavenge a metabolite made inside host cells. Flow cytometry-based lectin-binding assays showed that gonococci exposed to vaginolysin-liberated contents of HeLa cells displayed greater sialic acid capping of their LOS. This higher level of bacterial sialylation was accompanied by increased binding of the complement regulatory protein factor H, and greater resistance to complement attack. Together these results suggest that cytolytic activities present during BV may enhance the ability of N. gonorrhoeae to capture intracellular metabolites and evade host immunity via glycan molecular mimicry.

Two putative glutamate decarboxylases of Streptococcus pneumoniae as possible antigens for the production of anti-GAD65 antibodies leading to type 1 diabetes mellitus / Dos supuestas glutamato descarboxilasas de Streptococcus pneumoniae como posibles antígenos para la producción de anticuerpos anti-GAD65 que conducen a la diabetes mellitus tipo 1

Type 1 diabetes mellitus (T1DM) has been increasing in prevalence in the last decades and has become a global burden. Autoantibodies against human glutamate decarboxylase (GAD65) are among the first to be detected at the onset of T1DM. Diverse viruses have been proposed to be involved in the triggering of T1DM because of molecular mimicry, i.e., similarity between parts of some viral proteins and one or more epitopes of GAD65. However, the possibility that bacterial proteins might also be responsible for GAD65 mimicry has been seldom investigated. To date, many genomes of Streptococcus pneumoniae (the pneumococcus), a prominent human pathogen particularly prevalent among children and the elderly, have been sequenced. A dataset of more than 9000 pneumococcal genomes was mined and two different (albeit related) genes (gadA and gadB), presumably encoding two glutamate decarboxylases similar to GAD65, were found. The various gadASpn alleles were present only in serotype 3 pneumococci belonging to the global lineage GPSC83, although some homologs have also been discovered in two subspecies of Streptococcus constellatus (pharyngis and viborgensis), an isolate of the group B streptococci, and several strains of Lactobacillus delbrueckii. Besides, gadBSpn alleles are present in > 10% of the isolates in our dataset and represent 16 GPSCs with 123 sequence types and 20 different serotypes. Sequence analyses indicated that gadA- and gadB-like genes have been mobilized among different bacteria either by prophage(s) or by integrative and conjugative element(s), respectively. Substantial similarities appear to exist between the putative pneumococcal glutamate decarboxylases and well-known epitopes of GAD65. In this sense, the use of broader pneumococcal conjugate vaccines such as PCV20 would prevent the majority of serotypes expressing those genes that might potentially contribute to T1DM.(AU)

Microbes as triggers and boosters of Type 1 Diabetes - Mediation by molecular mimicry.

AIMS: Type 1 diabetes is characterized by steadily increasing incidence and largely obscured pathogenesis. Molecular mimicry is well-established as trigger for different autoimmune pathologies, but obscurely explored in the context of T1D. The presented study explores the underestimated role of molecular mimicry in T1D-etiology/progression in search for etiologic factors among human pathogens and commensals. METHODS: A comprehensive immunoinformatics analysis of T1D-specific experimental T-cell epitopes across bacterial, fungal, and viral proteomes was performed, coupled with MHC-restricted mimotope validation and docking of most potent epitopes/mimotopes to T1D-high-risk MHCII molecules. In addition, re-analysis of the publicly available T1D-microbiota dataset was performed, including samples at the pre-T1D disease stage. RESULTS: A number of bacterial pathogens/commensals were tagged as putative T1D triggers/boosters, including ubiquitous gut residents. The prediction of most likely mimicked epitopes revealed heat-shock proteins as most potent autoantigens for autoreactive T-cell priming via molecular mimicry. Docking revealed analogous interactions for predicted bacterial mimotopes and corresponding experimental epitopes. Finally, re-analysis of T1D gut microbiota datasets prompted pre-T1D as most significantly different/dysbiotic, compared to other explored categories (T1D stage/controls). CONCLUSIONS: Obtained results support the unrecognized role of molecular mimicry in T1D, suggesting that autoreactive T-cell priming might be the triggering factor of disease development.

Mimicking effects of cholesterol in lipid bilayer membranes by self-assembled amphiphilic block copolymers.

The effect of cholesterol on biological membranes is important in biochemistry. In this study, a polymer system is used to simulate the consequences of varying cholesterol content in membranes. The system consists of an AB-diblock copolymer, a hydrophilic homopolymer hA, and a hydrophobic rigid homopolymer C, corresponding to phospholipid, water, and cholesterol, respectively. The effect of the C-polymer content on the membrane is studied within the framework of a self-consistent field model. The results show that the liquid-crystal behavior of B and C has a great influence on the chemical potential of cholesterol in bilayer membranes. The effects of the interaction strength between components, characterized by the Flory-Huggins parameters and the Maier-Saupe parameter, were studied. Some consequences of adding a coil headgroup to the C-rod are presented. Results of our model are compared to experimental findings for cholesterol-containing lipid bilayer membranes.

Role of molecular mimicry in the SARS-CoV-2-human interactome for pathogenesis of cardiovascular diseases: An update to ImitateDB.

Mimicry of host proteins is a strategy employed by pathogens to hijack host functions. Domain and motif mimicry was explored in the experimental and predicted SARS-CoV-2-human interactome. The host first interactor proteins were also added to capture the continuum of the interactions. The domains and motifs of the proteins were annotated using NCBI CD Search and ScanProsite, respectively. Host and pathogen proteins with a common host interactor and similar domain/motif constitute a mimicry pair indicating global structural similarity (domain mimicry pair; DMP) or local sequence similarity (motif mimicry pair; MMP). 593 DMPs and 7,02,472 MMPs were determined. AAA, DEXDc and Macro domains were frequent among DMPs whereas glycosylation, myristoylation and RGD motifs were abundant among MMP. The proteins involved in mimicry were visualised as a SARS-CoV-2 mimicry interaction network. The host proteins were enriched in multiple CVD pathways indicating the role of mimicry in COVID-19 associated CVDs. Bridging nodes were identified as potential drug targets. Approved antihypertensive and anti-inflammatory drugs are proposed for repurposing against COVID-19 associated CVDs. The SARS-CoV-2 mimicry data has been updated in ImitateDB (http://imitatedb.sblab-nsit.net/SARSCoV2Mimicry). Determination of key mechanisms, proteins, pathways, drug targets and repurposing candidates is critical for developing therapeutics for SARS CoV-2 associated CVDs.

Molecular mimicry and autoimmunity in the time of COVID-19.

Infectious diseases are commonly implicated as potential initiators of autoimmune diseases (ADs) and represent the most commonly known factor in the development of autoimmunity in susceptible individuals. Epidemiological data and animal studies on multiple ADs suggest that molecular mimicry is one of the likely mechanisms for the loss of peripheral tolerance and the development of clinical disease. Besides molecular mimicry, other mechanisms such as defects in central tolerance, nonspecific bystander activation, epitope-determinant spreading, and/or constant antigenic stimuli, may also contribute for breach of tolerance and to the development of ADs. Linear peptide homology is not the only mechanism by which molecular mimicry is established. Peptide modeling (i.e., 3D structure), molecular docking analyses, and affinity estimation for HLAs are emerging as critical strategies when studying the links of molecular mimicry in the development of autoimmunity. In the current pandemic, several reports have confirmed an influence of SARS-CoV-2 on subsequent autoimmunity. Bioinformatic and experimental evidence support the potential role of molecular mimicry. Peptide dimensional analysis requires more research and will be increasingly important for designing and distributing vaccines and better understanding the role of environmental factors related to autoimmunity.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

Intricate 3D architecture of a DNA mimic of GFP.

Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures1-3. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates4 conditional fluorophores derived from GFP. To extend previous structural studies5,6 of fluorogenic RNAs, GFP and other fluorescent proteins7 to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen8 in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.