Generation of the induced pluripotent stem cell line IOCVi001-A from a patient with the MFRP-related retinitis pigmentosa-nanophthalmos syndrome.

Retinitis pigmentosa (RP) is the most common retinal degeneration in humans and is characterized by the progressive degeneration of rods and cones and retinal pigment epithelium. We generated the IOCVi001-A induced pluripotent stem cell (iPSC) line from dermal fibroblast of a patient with a homozygous c.498_499insC (p.(Asn167Glnfs⁎34) variant in the Membrane-type frizzled related protein (MFRP) gene, a genetic defect causing a syndrome characterized by RP and small eye size (nanophthalmos). IOCVi001-A displayed normal stemness, expressed pluripotent stem cell markers and displayed a normal karyotype. This iPSC line can be used for in vitro disease modeling for complex forms of RP.

Identification of Genetic Variants for Diabetic Retinopathy Risk Applying Exome Sequencing in Extreme Phenotypes.

Background: Diabetic retinopathy (DR) risk has been shown to vary depending on ethnic backgrounds, and thus, it is worthy that underrepresented populations are analyzed for the potential identification of DR-associated genetic variants. We conducted a case-control study for the identification of DR-risk variants in Mexican population. Methods: We ascertained 60 type 2 diabetes mellitus (T2DM) patients. Cases (n = 30) were patients with advanced proliferative DR (PDR) with less than 15 years after a T2DM diagnosis while controls (n = 30) were patients with no DR 15 years after the diagnosis of T2DM. Exome sequencing was performed in all patients, and the frequency of rare variants was compared. In addition, the frequency of variants occurring in a set of 169 DR-associated genes were compared. Results: Statistically significant differences were identified for rare missense and splice variants and for rare splice variants occurring more than once in either group. A strong statistical difference was observed when the number of rare missense variants with an aggregated prediction of pathogenicity and occurring more than once in either group was compared (p = 0.0035). Moreover, 8 variants identified more than once in either group, occurring in previously identified DR-associated genes were recognized. The p.Pro234Ser KIR2DS4 variant showed a strong protective effect (OR = 0.04 [0.001-0.36]; p = 0.04). Conclusions: Our study showed an enrichment of rare splice acceptor/donor variants in patients with PDR and identified a potential protective variant in KIR2DS4. Although statistical significance was not reached, our results support the replication of 8 previously identified DR-associated genes.

A new missense variant in RAB3GAP2 in a family with muscular dystrophy-short stature and defective autophagy: An expansion of the micro/Martsolf spectrum or a new phenotype?

We describe a sibling pair of Mennonite origin born from consanguineous parentage with a likely new phenotype of limb-girdle muscular dystrophy, short stature, ptosis, and tracheomalacia. Exome sequencing in the affected subjects identified a novel homozygous RAB3GAP2 missense variant as the potential causal variant. As RAB3GAP2 has been recently shown to be involved in the autophagy process, we analyzed patient-derived fibroblasts by fluorescence microscopy and demonstrated defective autophagic flux under rapamycin and serum starvation conditions when compared with wild-type cells. The phenotype in the siblings described here is distinct from Martsolf and Warburg's micro syndromes, the currently known diseases arising from RAB3GAP2 pathogenic variants. Thus, this work describes a potentially novel recessive phenotype associated with a RAB3GAP2 defect and manifesting as a muscular dystrophy-short stature disorder with no ocular anomalies. Functional analyses indicated defective autophagy in patient-derived fibroblasts, supporting the involvement of RAB3GAP2 in the etiology of this disorder. Our results contribute to a better characterization of the Martsolf/micro spectrum phenotype.

Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems.

BACKGROUND: In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. AIM OF THE STUDY: To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. METHODS: We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. RESULTS: We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. CONCLUSIONS: This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells' intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/ß-catenin signaling.

A Three-Dimensional Alzheimer's Disease Cell Culture Model Using iPSC-Derived Neurons Carrying A246E Mutation in PSEN1.

Alzheimer's disease (AD) is a chronic brain disorder characterized by progressive intellectual decline and memory and neuronal loss, caused mainly by extracellular deposition of amyloid-ß (Aß) and intracellular accumulation of hyperphosphorylated tau protein, primarily in areas implicated in memory and learning as prefrontal cortex and hippocampus. There are two forms of AD, a late-onset form that affects people over 65 years old, and the early-onset form, which is hereditable and affect people at early ages ~45 years. To date, there is no cure for the disease; consequently, it is essential to develop new tools for the study of processes implicated in the disease. Currently, in vitro AD three-dimensional (3D) models using induced pluripotent stem cells (iPSC)-derived neurons have broadened the horizon for in vitro disease modeling and gained interest for mechanistic studies and preclinical drug discovery due to their potential advantages in providing a better physiologically relevant information and more predictive data for in vivo tests. Therefore, this study aimed to establish a 3D cell culture model of AD in vitro using iPSCs carrying the A246E mutation. We generated human iPSCs from fibroblasts from a patient with AD harboring the A246E mutation in the PSEN1 gene. Cell reprogramming was performed using lentiviral vectors with Yamanaka's factors (OSKM: Oct4, Sox2, Klf4, and c-Myc). The resulting iPSCs expressed pluripotency genes (such as Nanog and Oct4), alkaline phosphatase activity, and pluripotency stem cell marker expression, such as OCT4, SOX2, TRA-1-60, and SSEA4. iPSCs exhibited the ability to differentiate into neuronal lineage in a 3D environment through dual SMAD inhibition as confirmed by Nestin, MAP2, and Tuj1 neural marker expression. These iPSC-derived neurons harbored Aß oligomers confirmed by Western Blot (WB) and immunostaining. With human iPSC-derived neurons able to produce Aß oligomers, we established a novel human hydrogel-based 3D cell culture model that recapitulates Aß aggregation without the need for mutation induction or synthetic Aß exposure. This model will allow the study of processes implicated in disease spread throughout the brain, the screening of molecules or compounds with therapeutic potential, and the development of personalized therapeutic strategies.

Human Dental Pulp Stem Cells Display a Potential for Modeling Alzheimer Disease-Related Tau Modifications.

We present here the first description of tau in human dental pulp stem cells (DPSCs) evidenced by RT-PCR data on expression of the gene MAPT and by immunocytochemical detection of epitopes by 12 anti-tau antibodies. The tau specificity of eight of these antibodies was confirmed by their affinity to neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) postmortem brain samples. We therefore used DPSCs and AD brain samples as a test system for determining the probability of the involvement of tau epitopes in the mechanisms converting tau into NFT in AD. Three antibodies to non-phosphorylated and seven antibodies to phosphorylated epitopes bound tau in both DPSCs and AD NFTs, thus suggesting that their function was not influenced by inducers of formation of NFTs in the AD brain. In contrast, AT100, which recognizes a hyperphosphorylated epitope, did not detect it in the cytoplasm of DPSCs but detected it in AD brain NFTs, demonstrating its AD diagnostic potential. This indicated that the phosphorylation/conformational events required for the creation of this epitope do not occur in normal cytoplasm and are a part of the mechanism (s) leading to NFT in AD brain. TG3 bound tau in the cytoplasm and in mitotic chromosomes but did not find it in nuclei. Collectively, these observations characterize DPSCs as a novel tau-harboring neuronal lineage long-term propagable in vitro cellular system for the normal conformational state of tau sites, detectable by antibodies, with their state in AD NFTs revealing those involved in the pathological processes converting tau into NFTs in the course of AD. With this information, one can model the interaction of tau with inducers and inhibitors of hyperphosphorylation toward NFT-like aggregates to search for drug candidates. Additionally, the clonogenicity of DPSCs provides the option for generation of cell lineages with CRISPR-mutagenized genes of familial AD modeling.

Wnt/ß-Catenin/TCF Pathway Is a Phase-Dependent Promoter of Colony Formation and Mesendodermal Differentiation During Human Somatic Cell Reprogramming.

Somatic cell reprogramming is a biphasic phenomenon that goes through a mesenchymal-to-epithelial transition, called initiation phase, followed by a maturation phase wherein reprogramming cells acquire pluripotency. Here, we show that these phases display a differential response to Wnt signaling activation. Wnt signaling increases colony formation by promoting cellular epithelialization during the initiation phase in a TCF7-dependent manner. However, during maturation phase, it is also responsible for inducing mesendodermal differentiation, which is negatively regulated by TCF7L1. Thus, Wnt signaling inhibition or TCF7L1 overexpression downregulates mesendodermal gene expression without perturbing pluripotency. Together, our results demonstrate that a phase-specific modulation of Wnt signaling leads to an improved reprogramming efficiency in terms of colony output and pluripotency acquisition. This work provides new insights into the cell context-dependent roles of Wnt signaling during human somatic cell reprogramming. Stem Cells 2018;36:683-695.

Human Deciduous Teeth Stem Cells (SHED) Display Neural Crest Signature Characters.

Human dental tissues are sources of neural crest origin multipotent stem cells whose regenerative potential is a focus of extensive studies. Rational programming of clinical applications requires a more detailed knowledge of the characters inherited from neural crest. Investigation of neural crest cells generated from human pluripotent stem cells provided opportunity for their comparison with the postnatal dental cells. The purpose of this study was to investigate the role of the culture conditions in the expression by dental cells of neural crest characters. The results of the study demonstrate that specific neural crest cells requirements, serum-free, active WNT signaling and inactive SMAD 2/3, are needed for the activity of the neural crest characters in dental cells. Specifically, the decreasing concentration of fetal bovine serum (FBS) from regularly used for dental cells 10% to 2% and below, or using serum-free medium, led to emergence of a subset of epithelial-like cells expressing the two key neural crest markers, p75 and HNK-1. Further, the serum-free medium supplemented with neural crest signaling requirements (WNT inducer BIO and TGF-ß inhibitor REPSOX), induced epithelial-like phenotype, upregulated the p75, Sox10 and E-Cadherin and downregulated the mesenchymal genes (SNAIL1, ZEB1, TWIST). An expansion medium containing 2% FBS allowed to obtain an epithelial/mesenchymal SHED population showing high proliferation, clonogenic, multi-lineage differentiation capacities. Future experiments will be required to determine the effects of these features on regenerative potential of this novel SHED population.

HIV-1 V3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features.

The crown region of the V3 loop in HIV-1 that contains the conserved amino acid sequence GPGR/G is known as the principal neutralizing determinant due to the extraordinary ability of antibodies to this region to neutralize the virus. To complement the existing peptide models of this epitope, we describe a family of 18 phage-displayed peptides, which include linear 12mer and constrained 7mer peptides that was selected by screening random libraries with serum from HIV-1 subtype B-infected patients. The 7mer constrained peptides presented two conserved amino acid sequences: PR-L in N-terminus and GPG in the C-terminus. On the basis of these peptides we propose a mimotope model of the V3 crown epitope in which the PR-L and GPG sequences represent the two known epitope binding sites. The GPG, has the same function as the V3 crown GPGR sequence but without the involvement of the "R" despite its being considered as the signature of the epitope in B-subtype viruses. The PR-L contains a proline not existing in the epitope that is postulated to induce kinks in the backbones of all peptides and create a spatial element mimicking the N-terminal conformationally variable binding site. Rabbit serum to these mimotopes recognized the V3 peptides and moderately decreased the fusion between HIV-1 Env- and CD4-expressing Jurkat cells. This study proposes the efficient generation by means of patient sera of V3 epitope mimics validated by interaction with the antibodies to contemporary viruses induced in patients. The serum antibody-selectable mimotopes are sources of novel information on the fine structure-function properties of HIV-1 principal neutralizing domain and candidate anti-HIV-1 immunogens.

Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis.

Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.

Synthetic peptide-targeted selection of phage display mimotopes highlights immunogenic features of α-helical vs non-helical epitopes of Taenia solium paramyosin: implications for parasite- and host-protective roles of the protein.

Paramyosin of the pig-human parasite Taenia solium (TPmy) is a α-helical protein located on the worm surface that is suggested to fulfill an immunomodulatory role protecting the parasite against host immune system. Besides, in challenging experiments the protein shows a vaccine potential. These observations imply that TPmy harbors antigenic determinants for each of these contrasting actions. However the suggestion was not given a support from experimental data because respective epitopes have not been described thus far. To circumvent this difficulty, we use synthetic peptides with sequences of regions composed of α-helical or linear structure to induce rabbit antibody responses for phage-display mapping of epitope core amino-acid sets. Antibodies to α-helical regions were weak binders and M13 phage-displayed peptides selected by them from two different libraries exhibited no amino-acid similarities with the original protein site. In contrast, the antibodies produced in response to non-helical segment within α-helical structure were better binders and selectors of perfect structural mimics of the protein site. This first phage display epitope analysis of TPmy supports the notion that the rod-like α-helix, which encompasses over 90% of the total amino acids, may serve as an immunomodulatory shield that protects the parasite. Further, the seven non-helical segments of the TPmy molecule may represent the only anti-parasite discrete immunogenic epitopes whose representative mimotopes can be utilized in development of pure epitope vaccines.

Phage display identifies two Caprine Arthritis Encephalitis Virus env epitopes.

Using phage display and IgG of a goat infected with Caprine Arthritis Encephalitis Virus (CAEV) we obtained families of 7 mer constrained peptides with consensus motifs LxSDPF/Y and SWN/KHWSY and mapped the epitopes mimicked by them at the Env 6-LISDPY-11 and 67-WNTYHW-72 sites of the mature gp135 amino acid sequence. The first epitope fell into the N-terminal immunogenic aa1-EDYTLISDPYGFS- aa14 site identified previously with a synthetic peptide approach; the second epitope has not been described previously. The first epitope is mostly conserved across CAEV isolates whereas the second newly described epitope is extremely conserved in Small Ruminant Lentiviruses env sequences. As being immunodominant, the epitopes are candidate targets for mimotope-mediated diagnosis and/or neutralization.

Immunogenic peptides from phage display libraries with potential of protecting mice against the Pseudorabies virus.

Phage display selection of combinatorial peptide libraries has demonstrated its almost unlimited potential in identifying binding ligands for many targets. The method shows promise for selection of immunogenic peptides against pathogens by antibodies. We have undertaken a study designed to select such mimics for one of the representatives of Herpesviridae, the Pseudorabies virus (PrV), infecting pigs and causing severe neurological complications known as Aujeszky's disease. By screening a 12mer linear and a 7mer cysteine-constrained libraries with immunoglobulins of a rabbit immunized with the virus, a family of 10 antigenic and immunogenic peptides was derived sharing a sequence motif K(L/P/V)GDP(R/K/L). Groups of six C57BL/6 mice were immunized with bacteriophages expressing peptides with this motif sequences. Some of the mice were found to be positive in seroneutralization assay; in a challenge setting, all but two immunized mice survived, albeit presenting some disease symptoms. We discuss the perspectives and limits of generating peptide leads by library screening with immune polyclonal antiserum for designing pure epitope-based vaccines to PrV in the future.

Constrained peptide models from phage display libraries highlighting the cognate epitope-specific potential of the anti-HIV-1 mAb 2F5.

The monoclonal antibody 2F5 (mAb 2F5), one of the most potent broadly neutralizing mAbs targeted to the HIV-1 gp41 membrane proximal exterior region (MPER), displays an unusually wide antigenic specificity, tolerating amino acid substitutions at virtually all positions of the 662-ELDKWAS-668 epitope sequence when presented by peptides. Investigating this phenomenon, Menendez et al. [22] concluded that the paratope of 2F5 contains two distinct binding compartments. One is specific and binds the DKW epitope core; the other is multi-specific and binds to the flanking DKW regions that can be distinct from the epitope sequence. Because the DKW-flanking amino acids are strongly conserved in viruses, it is not clear whether the DKW only satisfies the 2F5 epitope recognition demand. In this study, we demonstrate that the specificity of recognition of the epitope depends on the structural context in which the cognate epitope sequence is presented. The antibody does not tolerate any replacements of the DKW-flanking epitope amino acids and binds exclusively to the (L)DKWA sequence provided that it is presented by a 7-mer constrained peptide exposed by the M13 phage pIII protein. Our data propose a novel epitope recognition model in which the 2F5 mAb requires a sequence longer than DKW and no substitution of flanking amino acids for specific recognition of the peptide. Additionally, immunization data supports the notion that the binding and neutralizing immunogenic structural features of the described epitope model do not coincide.

The CD271 expression could be alone for establisher phenotypic marker in Bone Marrow derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are of great interest for their potential use in cellular therapies. To define the population more precisely, diverse surface markers have been used. We propose here to use CD271 as the sole marker for MSCs in fresh bone marrow. We compared CD271+ populations to the presence or absence of five defined markers for MSCs: CD90+, CD105+, CD45-, CD34- and CD79. The correlations between markers were evaluated and analyzed with a Pearson's correlation test. We found that the average percentage of cells expressing the combination of markers CD90+, CD105+, CD45-, CD34- and CD79- was 0.54%, and that the average percentage average of CD271+ cells was 0.53%. The results were significant (p<0.05). The exclusive use of CD271 as a marker for MSCs from fresh samples of bone marrow appears to be highly selective. Using CD271 as the sole identification marker for MSCs could reduce costs and accelerate the process of identifying MSCs for the field of cellular therapy.

Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

Immunology of scorpion toxins and perspectives for generation of anti-venom vaccines.

Scorpions and other venomous animals contain concentrates of biologically active substances developed to block vital physiological and biochemical functions of the victims. These have contrasting human health concerns, provide important pharmacological raw material and pose a serious threat to human life and health in tropical and subtropical regions. Because only occasional and minor quantities of venom are introduced into the human organism with a scorpion sting and their mortal effect is an acute phenomenon these substances are unknown to the immune defense system and thus no immunity has appeared against them during evolution. Antidotes prepared from animal anti-sera are effective against some species of scorpions but depend on the manufacturer and the availability of product to the medical community. Although significant progress has been made in immunological studies of certain groups of toxins, few centers are dedicated to this research. Information is still insufficient to generate a comprehensive picture of the subject and to propose vaccines against venoms. A novel approach based on mimotopes selected from phage-displayed random peptide libraries show potential to impel further progress of toxin immunological studies and to provide putative vaccine resources. In this report we revise the "state of the art" in the field.

Array assessment of phage-displayed peptide mimics of Human Immunodeficiency Virus type 1 gp41 immunodominant epitope: binding to antibodies of infected individuals.

An array of phage-displayed mimetic peptides representing 18 sequences from the HIV-1 gp41 immunodominant epitope was fabricated on a nitrocellulose membrane. It used to compare the antibody-binding affinity of the peptide and to monitor the immune response of four patients prior to and after the initiation of Highly Active Anti-Retroviral Therapy in parallel assays with several probes using near-infrared fluorescence detection. The proposed multiplexed approach is highly sensitive, consumes less sample and can be used to analyze the immune repertoires of virus-infected individuals.

Potential of peptides selected from random phage-displayed libraries to mimic conformational epitopes: a study on scorpion toxin Cn2 and the neutralizing monoclonal antibody BCF2.

Many conformational epitopes cannot be mapped by the use of a phage display approach due to the lack of amino acid similarity with the selected peptides. Exploring the potential of the method, we selected mimotopes of the discontinuous, highly conformational epitope of scorpion neurotoxin Cn2, whose 3D structure is known, using its generic neutralizing monoclonal antibody BCF2. With an exhaustive selection procedure, we isolated from a 12-mer phage library a large collection of mimotopes that reproduce the antigenic and immunogenic specificity of the Cn2-epitope. The selected peptides presented three sequence motifs, the most abundant of which, RD(N)XXGF, appeared in 15 different sequence contexts displayed by 97 out of 206 clones. In the most reactive mimotope, displayed by 24 (25%) clones, the motif was flanked by two Cys residues allowing the adoption of a cyclic conformation. Motifs QL(H,M)L(M) and (S/T)WHLP were selected with less efficiency. Comparison of the motifs with the primary and three-dimensional structure of Cn2 as well as with a model of the Cn2-BCF2(Fv) complex suggests that RD(N)XXGF, which does not share sequence similarity with the epitope, mimics its central structural element, turn 7-11, by using an alternative amino acid combination nevertheless keeping the nature of its interactions with BCF2. The QL(H,M)L(M) is assumed to mimic the hydrophobic part of the epitope. The principles of the conformational mimicry by phage-displayed peptides are discussed.

Molecular localization and crossreactivity of two epitopes of noxiustoxin from scorpion Centruroides noxius, identified by a panel of monoclonal antibodies and peptide mimotopes.

Mimotopes derived from peptide phage display libraries may reproduce basic functions of epitopes including their antigenicity. In case of toxins, this property makes phage displayed mimotopes highly specific vaccine components free of the toxicity. To explore the potential of mimotopes for vaccine development, their ability of substituting the whole toxin molecule deserves a detailed characterization. We used mimotopes of noxiustoxin (NTX), a neurotoxin from scorpion Centruroides noxius, for studying its epitopes recognized by a panel of six monoclonal antibodies (mAbs), as well as their crossreactivity with homologous toxins from other species of the Centruroides genus. Although competitive (displacement) immunoassay showed that all six mAbs inhibit each other for binding to whole NTX molecule, the mimotopes used as specific probes allowed separation of the mAbs into two functional groups recognizing distinct non-overlapping epitopes mapped on the opposite sites of the three-dimensional structure of the toxin. The use of mimotopes permitted a precise specificity analysis of a panel of antibodies raised against this toxin, that may be very important for immunological characterization of other scorpion toxins and for vaccine development.