15-mer Peptides Research Articles

Five-mer peptides prevent short-term spatial memory deficits in Aβ25-35-induced Alzheimer’s model mouse by suppressing Aβ25-35 aggregation and resolving its aggregate form

BackgroundThe development of drugs for Alzheimer’s disease (AD), which is related to the misfolding and aggregation of amyloid-β (Aβ), is high in demand due to the growing number of AD patients. In this study, we screened 22 kinds of 5-mer synthetic peptides derived from the Box A region of Tob1 protein to find a peptide effective against Aβ aggregation.MethodsA Thioflavin T (ThT) assay was performed to evaluate aggregation and screen aggregation inhibitors. Male ICR mice (6 weeks old) were administered saline, 9 nmol Aβ25-35, or a mixture of 9 nmol Aβ25-35 and 9 nmol GSGFK in the right lateral ventricle. Short-term spatial memory was assessed through Y-maze. Microglia cells (BV-)2 cells were plated on 24-well plates (4 × 104 cells/well) and incubated for 48 h, and then, the cells were treated with 0.01, 0.05, 0.1, 0.2, or 0.5 mM GSGFK. After incubation for 24 h, bead uptake was evaluated using a laser confocal microscope and Cytation 5.ResultsWe found two kinds of peptides, GSGNR and GSGFK, that were not only suppressed by aggregation of Aβ25-35 but also resolved the aggregated Aβ25-35. Results obtained from the Y-maze test on an Aβ25-35-induced AD model mouse indicated that GSGFK prevents the deficits in short-term memory induced by Aβ25-35. The effect of GSGFK on phagocytosis in BV-2 cells proved that GSGFK activates the phagocytic ability of microglia.ConclusionsIn conclusion, 5-mer peptides prevent short-term memory deficit in Aβ25-35 induced AD model mouse by reducing the aggregated Aβ25-35. They may also upregulate the phagocytic ability of microglia, which makes 5-mer peptides suitable candidates as therapeutic drugs against AD.

Abstract 5153: The role of CCL22-specific CD4 T cells in modulating the immunosuppressive tumor microenvironment in ovarian cancer

Abstract Background: CCL22, a macrophage-derived chemokine, is overexpressed in ovarian cancer and exerts immunosuppressive functions by the recruitment of regulatory T cells (Tregs) to the tumor microenvironment (TME). Our research group described naturally occurring T cells specific for an MHC class I epitope derived from CCL22. CD8+ CCL22-specific T cells isolated from cancer patients could recognize CCL22-expressing cells and decrease the levels of CCL22 in ovarian ascites in vitro. Expansion of CCL22-specific CD8+ T cells in animal models of cancer by peptide vaccination led to a therapeutic anti-tumor effect by reducing Treg recruitment into the TME. In this study we aimed at characterizing CD4+ CCL22-specific T cells and their potential for modulating the tumor microenvironment in ovarian cancer. Methods: CCL22-responses were screened with a library of overlapping 20mer peptides covering the entire sequence of CCL22 in PBMCs from healthy donors and ovarian cancer patients. CCL22-specific T cells were isolated from PBMCs and ovarian ascites for functional characterization through IFNγ ELISpot and ICS against peptides and CCL22-expressing target cells. Cytokine secretion was measured with protein multiplex immunoassays. Vaccination and therapeutic efficacy were evaluated in BALB/c CT26 and 4T1 syngeneic models. Results: We identified an immunogenic region in the CCL22 protein sequence and detected CD4 T-cell responses against CCL22-derived peptides in healthy donors and ovarian cancer patients through IFNγ ELISpot. CCL22-specific CD4+ cells have also been identified ex vivo in the ascites fluid from patients with ovarian cancer. We isolated and expanded CCL22-specific CD4+ T-cell clones from the ovarian ascites and demonstrated their reactivity against CCL22-producing cell lines. Furthermore, the stimulation of CCL22-specific CD4+ T cells in the PBMC culture and ascites samples was associated with a modulation of the cytokine environments. Additionally, vaccination with surrogate murine 20mer and MHC-II restricted CCL22-derived peptides lead to successful induction of specific T-cell responses in the spleens and draining lymph nodes of vaccinated animals. Activation of such CCL22-specific CD4 T cell responses was associated with a therapeutic effect seen as reduction of the tumor growth in different models of cancer. Conclusion: This study provides evidence for the ability of pro-inflammatory CD4+ CCL22-specific T cells to modulate the tumor microenvironment, particularly in ovarian cancer. Altogether we provide the rationale for the development of a CCL22-based immune modulatory vaccination for ovarian cancer. Citation Format: Ines Lecoq, Evelina Martinenaite, Marie C. Westergaard, Inge Marie Svane, Mads H. Andersen. The role of CCL22-specific CD4 T cells in modulating the immunosuppressive tumor microenvironment in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5153.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Tumor-Specific CircRNA-Derived Antigen Peptide Identification for Hepatobiliary Tumors

The application of tumor antigen-based immunotherapy is hindered by the rarity of validated immunogenic peptides. In this study, we aimed to investigate the potential of circular RNAs (circRNAs) as a novel source of tumor antigen peptides in hepatobiliary tumor organoids. Using RNA-sequencing (RNA-seq) with an algorithm-based score tool, 3950 translated tumor-specific circRNAs were predicted to generate 18 971 antigen peptides in 27 organoids. In view of the antigen landscape, 11 amino acid length (mer) peptides and human leukocyte antigen (HLA)-A binding peptides harbored the highest immunogenicity-related scores. In three out of five analyzed organoids, 13 predicted antigen peptides were directly confirmed as HLA-A, -B, and -C (HLA-ABC) binding peptides with mass spectrometry (MS)-based immunopeptidomics. CircRNA-derived tumor-specific peptides presented by the HLA-ABC molecules stimulated cluster of differentiation 8 (CD8) T cells to exhibit increased CD107a interferon γ (IFNγ) co-expressions and IFNγ secretion in flow cytometry and enzyme-linked immunosorbent assay (ELISA). Cytotoxic T cell activity targeting the organoids, induced by the immunogenic circRNA-derived peptides, was verified in a killing assay. Notably, the antigen peptide YGFNEILKK from circTBC1D15 was not only recognized as an HLA-ABC-presented peptide of the organoids but also drastically reduced the tumor organoid survival rate. Our findings highlight a crucial subset for generating tumor antigens, which has implications for targeting tumor-specific circRNAs in cancers.

Computational Design for Identification of Human Anti-MUC1 Heteroclitic Peptides in the Treatment of HER2-Positive Breast Cancer through Neural Network Training and Monomeric based Design.

Generation of the human anti-MUC1 peptide through neural network training and monomeric design method. Analyzing 9-mer peptide potential computationally for treatment of HER2-positive breast cancer. With the advancements of cancer genome atlas project (TCGA), cancer dependancy project (DepMap) and human protein atlas (HPA), large-scale datasets are generated for oncology studies. However, after development of redefined breast cancer drug targets, there are key issues in successful breast cancer treatments that needed to be pursued which paved the pathway for new approaches or strategies. In that respect, our research data aimed to represent a new aspect of breast cancer drug development studies. Extract human MUC1 sequences from various databases. Perform neural networking method for novel peptides sequences. Analyze the potentiality of generated heteroclitic peptide sequences for suitable vaccine candidate for breast cancer treatment. Input scaffolds of protein database (PDB) files for human MUC1 were retrieved and loaded into Evo design server with monomeric based design option. Further, neural network training approaches were followed and other computational tools were used for alignment-independent prediction of protective antigens and subunit vaccines potency of designed heteroclitic peptides. Study findings revealed two human anti-MUC1 heteroclitic peptides of 9mers (WAVWTYVSV, FMSFYIMNL), which showed the lowest energy cluster and sequence identity, normalized relative error rate of secondary structure, solvent accessibility, backbone torsion angles for neural networking and RMSD values in evolutionary profiling, and online MHCPred IC50 interaction values. VaxiGen v2.0 server revealed subunit vaccine potency values of in-silico designed two heteroclitic peptides were 0.1551 (WAVWTYVSV) and 0.3508 (FMSFYIMNL) with a threshold value of 0.5 followed by AllerTOP v2.0 for their allergenicity nature in immunogenic reactions. Computationally designed heteroclitic peptide WAVWTYVSV indicated promising values which can be utilised as drug delivery or tumour marker candidate in the treatment of human breast cancer by eliciting lyse of tumor cells.

IgE and IgG4 Epitopes of Dermatophagoides and Blomia Allergens before and after Sublingual Immunotherapy

Sublingual immunotherapy (SLIT) is used worldwide to treat house dust mites (HDM) allergy. Epitope specific immunotherapy with peptide vaccines is used far less, but it is of great interest in the treatment of allergic reactions, as it precludes the drawbacks of allergen extracts. The ideal peptide candidates would bind to IgG, blocking IgE-binding. To better elucidate IgE and IgG4 epitope profiles during SLIT, sequences of main allergens, Der p 1, 2, 5, 7, 10, 23 and Blo t 5, 6, 12, 13, were included in a 15-mer peptide microarray and tested against pooled sera from 10 patients pre- and post-1-year SLIT. All allergens were recognized to some extent by at least one antibody isotype and peptide diversity was higher post-1-year SLIT for both antibodies. IgE recognition diversity varied among allergens and timepoints without a clear tendency. Der p 10, a minor allergen in temperate regions, was the molecule with more IgE-peptides and might be a major allergen in populations highly exposed to helminths and cockroaches, such as Brazil. SLIT-induced IgG4 epitopes were directed against several, but not all, IgE-binding regions. We selected a set of peptides that recognized only IgG4 or were able to induce increased ratios of IgG4:IgE after one year of treatment and might be potential targets for vaccines.

Toxic and antigenic characterization of Peruvian Micrurus surinamensis coral snake venom

Micrurus surinamensis is a semi-aquatic coral snake found in primary forest region and can cause relevant human accidents. In this work we investigated the toxic and antigenic activities of the Peruvian Micrurus surinamensis venom (MsV). We found that MsV show hyaluronidase activity but lack LAAO and PLA2 enzymatic activities. Interestingly, MsV induce edematogenic responses but cannot cause nociceptive effects. Furthermore, MsV can reduce in vitro cell viability in MGSO-3 cell line derived from human breast cancer tissue. To evaluate its antigenic potential, rabbits were immunized with MsV, which proved to be immunogenic. ELISA, immunobloting and in vivo neutralization assays demonstrated that the specific rabbit anti-MsV antivenom is more efficient than the therapeutic Brazilian antivenom in recognizing and neutralizing the lethal activity of MsV. MsV differs in protein profile and biological activities from M. frontalis venom (MfV), used as control, which impairs its recognition and neutralization by Brazilian therapeutic anti-elapidic antivenom. We performed a SPOT immunoassay for the identification of B-cell linear epitopes in the main toxins described for MsV targeted by the elicited neutralizing antibodies previously produced. A membrane containing 15-mer peptides representing the sequences of five 3TFxs and five PLA2s was produced and probed with anti- MsV antibodies. Results revealed important regions in 3FTx toxins for venom neutralization. Identifying the main MsV components and its biological activities can be helpful in guiding the production of antivenoms and in the optimization of treatment for coral snake envenomation in Brazil.

Identification of a short ACE2-derived stapled peptide targeting the SARS-CoV-2 spike protein

The design and synthesis of a series of peptide derivatives based on a short ACE2 α-helix 1 epitope and subsequent [i - i+4] stapling of the secondary structure resulted in the identification of a 9-mer peptide capable to compete with recombinant ACE2 towards Spike RBD in the micromolar range. Specifically, SARS-CoV-2 Spike inhibitor screening based on colorimetric ELISA assay and structural studies by circular dichroism showed the ring-closing metathesis cyclization being capable to stabilize the helical structure of the 9-mer 34HEAEDLFYQ42 epitope better than the triazole stapling via click chemistry. MD simulations showed the stapled peptide being able not only to bind the Spike RBD, sterically interfering with ACE2, but also showing higher affinity to the target as compared to parent epitope.

CoV2-TCR: A web server for screening TCR CDR3 from TCR immune repertoire of COVID-19 patients and their recognized SARS-CoV-2 epitopes

Although multiple vaccines have been developed and widely administered, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been reported to evade immune responses and spread diffusely. Here, 108 RNA-seq files from coronavirus disease 2019 (COVID-19) patients and healthy donors (HD) were downloaded to extract their TCR immune repertoire by MiXCR. Those extracted TCR repertoire were compared and it was found that disease progression was related negatively with diversity and positively with clonality. Specifically, greater proportions of high-abundance clonotypes were observed in active and severe COVID-19 samples, probably resulting from strong stimulation of SARS-CoV-2 epitopes and a continued immune response in host. To investigate the specific recognition between TCR CDR3 and SARS-CoV-2 epitopes, we constructed an accurate classifier CoV2-TCR with an AUC of 0.967 in an independent dataset, which outperformed several similar tools. Based on this model, we observed a huge range in the number of those TCR CDR3 recognizing those different peptides, including 28 MHC-I epitopes from SARS-CoV-2 and 22 immunogenic peptides from SARS-CoV-2 variants. Interestingly, their proportions of high-abundance, low-abundance and rare clonotypes were close for each peptide. To expand the potential application of this model, we established the webserver, CoV2-TCR, in which users can obtain those recognizing CDR3 sequences from the TCR repertoire of COVID-19 patients based on the 9-mer peptides containing mutation site(s) on the four main proteins of SARS-CoV-2 variants. Overall, this study provides preliminary screening for candidate antigen epitopes and the TCR CDR3 that recognizes them, and should be helpful for vaccine design on SARS-CoV-2 variants.

Synchronization of opening and closing of two gramicidin A channels pulled together by a linker: possible relevance to channel clustering.

The linear 15-mer peptide gramicidin A (gA) produced by Bacillus brevis is known to form the simplest natural ion channel in lipid membranes representing a head-to-head transmembrane dimer. Its incorporation into a planar lipid bilayer manifests itself in regular electrical current transitions. If two gA subunits are tightly connected by a water-soluble, flexible linker of a certain length, the current transitions become heterogeneous: in a part of them, the amplitude is almost twofold higher than that of a single channel, thereby demonstrating the synchronous opening of two single channels. The lifetime, i.e. the open-state duration, of this dual channel is by several orders of magnitude longer than that of the single channel. Here, we used the ideas of the theory of excitons to hypothesize about the mechanism of synchronous opening and closing of two adjacent channels. Two independent (uncoupled) single channels can be described by two independent conformational coordinates q1 and q2, while two closely located channels can exhibit collective behavior, if the coupling between them produces mixing of the individual states (q1,0) and (0,q2). We suppose that a similar phenomenon can occur not only with synthetic derivatives of gA, but also with such natural channel-forming peptide antibiotics and toxins as alamethicin and syringomycin. In particular, channel clustering observed with these peptides may be also associated with formation of collective conductance states, resulting from mixing of their monomeric states, which allows us to explain the fact that clusters of these channels transmit ions and nonelectrolytes of the same size as the original single channels.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

Epitope identification for p53R273C mutant.

With the rise of immunotherapy based on cancer neoantigen, identification of neoepitopes has become an urgent problem to be solved. The TP53 R273C mutation is one of the hotspot mutations of TP53, however, the immunogenicity of this mutation is not yet clear. The aim of this study is to identify potential epitopes for p53R273C mutant. In this study, bioinformatic methods, peptide exchange assay, and peptide-immunized human leukocyte antigen (HLA) transgenic mouse model were used to explore the immunogenicity of this mutation. Peptides with higher affinity to common HLA-A alleles (A*11:01, A*02:01) were discovered by computational prediction. All the 8-11 mer peptides contain the mutation site were synthesized and soluble peptides were used in the peptide exchange assay. However, the exchange efficiencies of these predicted peptides to HLAs were lower. Fortunately, other peptides with higher exchange efficiency were discovered. Then, the immunogenicity of these peptides was validated with the HLA-A2 transgenic mice model. We identified three potential neoepitopes of p53R273C for HLA-A*02:01, one potential neoepitope for HLA-A*11:01 and no neoepitope for HLA-A*24:02.

Combination TIGIT/PD-1 blockade enhances the efficacy of neoantigen vaccines in a model of pancreatic cancer.

Cancer neoantigens are important targets of cancer immunotherapy and neoantigen vaccines are currently in development in pancreatic ductal adenocarcinoma (PDAC) and other cancer types. Immune regulatory mechanisms in pancreatic cancer may limit the efficacy of neoantigen vaccines. Targeting immune checkpoint signaling pathways in PDAC may improve the efficacy of neoantigen vaccines. We used KPC4580P, an established model of PDAC, to test whether neoantigen vaccines can generate therapeutic efficacy against PDAC. We focused on two immunogenic neoantigens associated with genetic alterations in the CAR12 and CDK12 genes. We tested a neoantigen vaccine comprised of two 20-mer synthetic long peptides and poly IC, a Toll-like receptor (TLR) agonist. We investigated the ability of neoantigen vaccine alone, or in combination with PD-1 and TIGIT signaling blockade to impact tumor growth. We also assessed the impact of TIGIT signaling on T cell responses in human PDAC. Neoantigen vaccines induce neoantigen-specific T cell responses in tumor-bearing mice and slow KPC4580P tumor growth. However, KPC4580P tumors express high levels of PD-L1 and the TIGIT ligand, CD155. A subset of neoantigen-specific T cells in KPC4580P tumors are dysfunctional, and express high levels of TIGIT. PD-1 and TIGIT signaling blockade in vivo reverses T cell dysfunction and enhances neoantigen vaccine-induced T cell responses and tumor regression. In human translational studies, TIGIT signaling blockade in vitro enhances neoantigen-specific T cell function following vaccination. Taken together, preclinical and human translational studies support testing neoantigen vaccines in combination with therapies targeting the PD-1 and TIGIT signaling pathways in patients with PDAC.

In-Plate Chemical Synthesis of Isopeptide-Linked SUMOylated Peptide Fluorescence Polarization Reagents for High-Throughput Screening of SENP Preferences.

Small ubiquitin-like modifiers (SUMOs) are conjugated to protein substrates in cells to regulate their function. The attachment of SUMO family members SUMO1-3 to substrate proteins is reversed by specific isopeptidases called SENPs (sentrin-specific protease). Whereas SENPs are SUMO-isoform or linkage type specific, comprehensive analysis is missing. Furthermore, the underlying mechanism of SENP linkage specificity remains unclear. We present a high-throughput synthesis of 83 isopeptide-linked SUMO-based fluorescence polarization reagents to study enzyme preferences. The assay reagents were synthesized via a native chemical ligation-desulfurization protocol between 11-mer peptides containing a γ-thiolysine and a SUMO3 thioester. Subsequently, five recombinantly expressed SENPs were screened using these assay reagents to reveal their deconjugation activity and substrate preferences. In general, we observed that SENP1 is the most active and nonselective SENP while SENP6 and SENP7 show the least activity. Furthermore, SENPs differentially process peptides derived from SUMO1-3, who form a minimalistic representation of diSUMO chains. To validate our findings, five distinct isopeptide-linked diSUMO chains were chemically synthesized and proteolysis was monitored using a gel-based read-out.

Detection of the peptidyl epitope for vaccine development against MPV

BackgroundMonkeypox Virus (MPV) is the cause of zoonotic disease characterized by skin-eruption with pus cell formation and lymphadenopathy. This virus belongs to the Orthopoxvirus genus with DNA as its genetic material. Previously, this infection was reported from Africa and occasionally from USA and UK. However, recently there is a sudden surge of infection in non-epidemic countries and a new strain of MPVhas been discovered. Therefore it is important to revisit the phylogeny of MPV with the addition of new strains. Recently WHO also stressed the need of developing vaccines for new strains. In this scenario we have two objectives for this study -first, to reveal the exact phylogenetic position of the 2022 strain and second, to identify specific peptides which may be used for vaccine development in the future. MethodsThe phylogenetic analysis was done with the help of Bayesian phylogeny. The dN/dS calculation was performed based on DNA polymerase genes of selected MPV strains. The peptidyl-epitope was searched in MPV2022/2 SLO strain with the help of several algorithms implemented in Allergen FP v.1.0, NetMHCII 2.3, MHCpred and Toxin Pred. The structure prediction of the proteins and peptides was performed through Hpepdock. The quality of the structures was validated through the Ramachandran plot. The molecular dynamics and simulation were performed through Gromacs software. The interaction between peptide and protein was assessed through Ligplot software. ResultsThe phylogenetic analysis revealed that the considered 2022 MPVstrains were close to the USA strains. The evolutionary analysis showed the volatile nature of the genome. Moreover, 9-mer peptide sequence was identified as an epitope for vaccine development. ConclusionsThe emergence of more virulent strains in near future may not be ruled out. Immunocompromised patients are more susceptible to this virus hence sub-unit vaccine is a better choice than a recombinant or attenuated vaccine against monkeypox. We have identified a small stretch of specific peptide which may be used for developing a subunit vaccine against this virus.

Peptide-based direct electrochemical detection of receptor binding domains of SARS-CoV-2 spike protein in pristine samples

RNA isolation and amplification-free user-friendly detection of SARS-CoV-2 is the need of hour especially at resource limited settings. Herein, we devised the peptides of human angiotensin converting enzyme-2 (hACE-2) as bioreceptor at electrode interface for selective targeting of receptor binding domains (RBD) of SARS-CoV-2 spike protein (SP). Disposable carbon-screen printed electrode modified with methylene blue (MB) electroadsorbed graphene oxide (GO) has been constructed as cost-efficient and scalable platform for hACE-2 peptide-based SARS-CoV-2 detection. In silico molecular docking of customized 25 mer peptides with RBD of SARS-CoV-2 SP were validated by AutoDock CrankPep. N-terminal region of ACE-2 showed higher binding affinity of − 20.6 kcal/mol with 15 H-bond, 9 of which were < 3 Å. Electrochemical biosensing of different concentrations of SPs were determined by cyclic voltammetry (CV) and chronoamperometry (CA), enabling a limit of detection (LOD) of 0.58 pg/mL and 0.71 pg/mL, respectively. MB-GO devised hACE-2 peptide platform exert an enhanced current sensitivity of 0.0105 mA/pg mL−1 cm−2 (R2 = 0.9792) (CV) and 0.45 nA/pg mL−1 (R2 = 0.9570) (CA) against SP in the range of 1 pg/mL to 1 µg/mL. For clinical feasibility, nasopharyngeal and oropharyngeal swab specimens in viral transport medium were directly tested with the prepared peptide biosensor and validated with RT-PCR, promising for point-of-need analysis.

357 Cutibacterium acnes CAMP factor 1-related peptides as Toll-Like Receptor-2 modulators

Acne vulgaris is a disorder affecting the pilosebaceous unit resulting in both inflammatory and non-inflammatory clinical lesions. Cutibacterium acnes (C. acnes) of a specific lineage, plays a key role in inflammatory acne by producing several virulence factors thereby increasing their inflammatory capacity. The C. acnes mutated CAMP1 factor display strong TLR-2 binding property in highly inflammatory C. acnes strains. CAMP1 peptide array analysis of 116 15-mer peptides sequences with an overlap of 11 amino acids, were immobilized onto glass plate and showed that seven peptides (A14, A15, B1, B2, B3, C1 and C3) were specifically recognized by TLR-2 and located on the same side of the in silico 3D-CAMP1 structure.

10-mer and 9-mer WALK Peptides with Both Antibacterial and Anti-Inflammatory Activities.

Natural antimicrobial peptides (AMPs) are multifunctional host defense peptides (HDPs) that are valuable for various therapeutic applications. In particular, natural and artificial AMPs with dual antibacterial immunomodulatory functions emerged as promising candidates for the development of therapeutic agents to treat infectious inflammation. In an effort to develop useful AMP variants with short lengths and simple amino acid composition, we devised a de novo design strategy to generate a series of model peptide isomer sequences, named WALK peptides, i.e., tryptophan (W)-containing amphipathic-helical (A) leucine (L)/lysine (K) peptides. Here, we generated two groups of WALK peptide isomers: W2L4K4 (WALK244.01~WALK244.10) and W2L4K3 (WALK243.01~WALK243.09). Most showed apparent antibacterial activities against both Gram-positive and Gram-negative bacteria at a concentration of approximately 4 μg/mL along with varied hemolytic activities against human red blood cells. In addition, some exhibited significant anti-inflammatory activities without any significant cytotoxicity in macrophages. Collectively, these results suggest that the two selected peptides, WALK244.04 and WALK243.04, showed promise for the development of antibacterial and anti-inflammatory agents.

Hydrophobicity identifies false positives and false negatives in peptide-MHC binding.

Major Histocompability Complex (MHC) Class I molecules allow cells to present foreign and endogenous peptides to T-Cells so that cells infected by pathogens can be identified and killed. Neural networks tools such as NetMHC-4.0 and NetMHCpan-4.1 are used to predict whether peptides will bind to variants of MHC molecules. These tools are trained on data gathered from binding affinity and eluted ligand experiments. However, these tools do not track hydrophobicity, a significant biochemical factor relevant to peptide binding, in their predictions. A previous study had concluded that the peptides predicted to bind to HLA-A*0201 by NetMHC-4.0 were much more hydrophobic than expected. This paper expands that study by also focusing on HLA-B*2705 and HLA-B*0801, which prefer binding hydrophilic and balanced peptides respectively. The correlation of hydrophobicity of 9-mer peptides with their predicted binding strengths to these various HLAs was investigated. Two studies were performed, one using the data that the two neural networks were trained on, and the other using a sample of the human proteome. NetMHC-4.0 was found to have a statistically significant bias towards predicting highly hydrophobic peptides as strong binders to HLA-A*0201 and HLA-B*2705 in both studies. Machine Learning metrics were used to identify the causes for this bias: hydrophobic false positives and hydrophilic false negatives. These results suggest that the retraining the neural networks with biochemical attributes such as hydrophobicity and better training data could increase the accuracy of their predictions. This would increase their impact in applications such as vaccine design and neoantigen identification.

Identification of Cancer Cells in the Human Body by Anti-Telomerase Peptide Antibody: Towards the Isolation of Circulating Tumor Cells.

Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (α-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611-626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab')2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs.