HLA-DM and HLA-DO interplay for the peptide editing of HLA class II in healthy tissues and leukemia.

HLA class II antigen presentation is modulated by the activity of the peptide editor HLA-DM and its antagonist HLA-DO, with their interplay controlling the peptide repertoires presented by normal and malignant cells. The role of these molecules in allogeneic hematopoietic cell transplantation (alloHCT) is poorly investigated. Balanced expression of HLA-DM and HLA-DO can influence the presentation of leukemia-associated antigens and peptides targeted by alloreactive T cells, therefore affecting both anti-leukemia immunity and the potential onset of Graft versus Host Disease. We leveraged on a large collection of bulk and single cell RNA sequencing data, available at different repositories, to comprehensively review the level and distribution of HLA-DM and HLA-DO in different cell types and tissues of the human body. The resulting expression atlas will help future investigations aiming to dissect the dual role of HLA class II peptide editing in alloHCT, and their potential impact on its clinical outcome.

Posttranslationally modified self-peptides promote hypertension in mouse models.

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers.

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection. As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Immunogenicity of a peptide-based vaccine for measles: a pilot evaluation in a mouse model.

Although neutralizing antibody is an established correlate of protection for measles, T cell-mediated responses play at least two critical roles in immunity to measles: first, through provision of 'help' enabling robust humoral immune responses; and second, through clearance of measles virus-infected cells. Previously, we identified 13 measles-derived peptides that bound to human leukocyte antigen (HLA) molecules in Priess cells infected with measles virus. In this study, we evaluated the immunogenicity of these peptides in a transgenic mouse model. Our results demonstrated that these peptides induced Th1-biased immune responses at varying levels. Of the 13 peptides, the top four immunogenic peptides were further selected for a viral challenge study in mice. A vaccine based on a combination of these four peptides reduced morbidity and weight loss after viral challenge compared to placebo. Our results emphasize the potential of T cell-mediated, peptide-based vaccines against measles.

Peptide-Loaded HLA Class I Tetramer-Associated Magnetic Bead-Based Enrichment of HBV-Specific CD8+ T Cells.

HBV-specific CD8+ T cells are only present at the low frequency during chronic infection. Thus, they are often undetectable by conventional ex vivo staining methods using peptide-loaded HLA class I tetramers. Detection sensitivity can be increased by magnetic bead-based enrichment strategies following staining with peptide-loaded HLA class I tetramers. Additionally, some downstream applications like e.g., single cell RNA sequencing of virus-specific CD8+ T cells may also require a pre-enrichment step to increase the frequency of the cells of interest. For this, peptide-loaded HLA class I tetramers-associated magnetic bead-based enrichment is also a suitable method.

Nanobody peptide conjugate: a novel CD163 based broad neutralizing strategy against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

B-Cell Epitope Prediction for Antipeptide Paratopes with the HAPTIC2/HEPTAD User Toolkit (HUT).

B-cell epitope prediction is key to developing peptide-based vaccines and immunodiagnostics along with antibodies for prophylactic, therapeutic and/or diagnostic use. This entails estimating paratope binding affinity for variable-length peptidic sequences subject to constraints on both paratope accessibility and antigen conformational flexibility, as described herein for the HAPTIC2/HEPTAD User Toolkit (HUT). HUT comprises the Heuristic Affinity Prediction Tool for Immune Complexes 2 (HAPTIC2), the HAPTIC2-like Epitope Prediction Tool for Antigen with Disulfide (HEPTAD) and the HAPTIC2/HEPTAD Input Preprocessor (HIP). HIP enables tagging of residues (e.g., in hydrophobic blobs, ordered regions and glycosylation motifs) for exclusion from downstream analyses by HAPTIC2 and HEPTAD. HAPTIC2 estimates paratope binding affinity for disulfide-free disordered peptidic antigens (by analogy between flexible-ligand docking and protein folding), from terms attributed to compaction (in view of sequence length, charge and temperature-dependent polyproline-II helical propensity), collapse (disfavored by residue bulkiness) and contact (with glycine and proline regarded as polar residues that hydrogen bond with paratopes). HEPTAD analyzes antigen sequences that each contain two cysteine residues for which the impact of disulfide pairing is estimated as a correction to the free-energy penalty of compaction. All of HUT is freely accessible online ( https://freeshell.de/~badong/hut.htm ).

Identification and Validation of Peptides Specifying SARS-CoV-2 B-Cell Epitopes Eliciting Neutralizing Antibodies.

Vaccination is an effective means of inducing immune protection to prevent transmissible diseases. During the Covid-19 pandemic, immunizations using traditional and novel vaccine platforms such as the inactivated SARSCo-V-2 vaccine, adenoviral-vectored, and nucleic acid-based mRNA vaccines have been relatively successful in controlling the rates of infection and hospitalizations. Nevertheless, the danger posed by the emergence of SARS-CoV-2 variants would set the stage for the design of next generation vaccines. To overcome the lack of efficacy of current vaccines against emerging SARS-CoV-2 variants, new vaccines must be able to overcome the reduced effectiveness of the current vaccines. Since the current Covid-19 vaccines are dependent on the whole S-protein of Wuhan strain as the antigen, mutations have rendered the current Covid-19 vaccines less effective against variants of concern (VoCs). Instead of using the whole S-protein, peptide-based epitopes could be predicted using immunoinformatic approaches, simulation of the 3D structures, overlapping peptides covering the whole length of the S-protein or peptide arrays based on synthetic peptide combinatorial libraries comprising peptides recognizable by monoclonal antibodies. B-cell epitopes were predicted, and immunogenicity of peptides was validated in mice by immunizing mice with peptides conjugated to keyhole limpet hemocyanin (KLH) mixed with Montanide 51 as an adjuvant. The immunogenicity of epitopes that could elicit peptide specific IgGs was determined by peptide-based ELISA. Neutralizing activities were determined by cPass and pseudovirus-based neutralization assays.

Polyclonal Peptide Antisera.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages.The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare.Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.

Structural Characterization of Peptide Antibodies.

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery. To better understand the underlying mechanisms of antibody-antigen interaction, here, we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Cytochemical and Histochemical Staining with Peptide Antibodies.

Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/absorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

Peptide Antibodies: Current Status.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity.

EBV T-cell immunotherapy generated by peptide selection has enhanced effector functionality compared to LCL stimulation.

Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRß) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.

Designing a Conserved Immunogenic Peptide Construct from the Nucleocapsid Protein of Puumala orthohantavirus.

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems.

Newly developed peptide-ELISA successfully detected anti-IgG antibodies against Maedi-Visna virus in sheep.

Maedi Visna Virus (MVV) is a retrovirus that can infect sheep. There is still no effective therapy or vaccine against this virus and timely diagnosis is important to combat the complications of the disease. In this study, we aimed to develop an ELISA using peptides derived from gag protein as antigen. For this purpose, B cell epitopes of gag protein were predicted and a docking analysis with the B cell receptor was performed to select peptides to be used in ELISA. After three soluble epitopes with the highest antigenicity were produced as peptides, the immunogenicity of each peptide was determined by ELISA using sheep serum samples categorized as MVV positive (n=24) and negative (n=13). Subsequently, in house ELISA using above mentioned immunogenic peptides as antigen was used to investigate MVV seroprevalence in sheep (n=88). According to the results, among three peptides, two of them strongly reacted with MVV positive serum samples and the mean absorbance values detected among positive and negative serum samples were statistically significant, indicating that these peptides were immunogenic (P=0.016 and P=0.038). The third peptide also reacted with positive serum samples but the mean absorbance value was not statistically significant and this peptide was considered non-immunogenic (P=0.175). The immunogenic two peptides showed the same high sensitivity and specificity values of 91.60 and 92.80 according to the commercial kit. Moreover, MVV seroprevalence detected by peptide-ELISAs using CKQGSKE and CRPQGKAGHKG peptides as antigen was 3.40 % and 4.5 %, respectively. As a result, it was shown that these peptides can be successfully used for serological diagnosis of MVV.