15-mer Peptides Research Articles

Cellular interferon-gamma and interleukin-2 responses to SARS-CoV-2 structural proteins are broader and higher in those vaccinated after SARS-CoV-2 infection compared to vaccinees without prior SARS-CoV-2 infection.

Class I- and Class II-restricted epitopes have been identified across the SARS-CoV-2 structural proteome. Vaccine-induced and post-infection SARS-CoV-2 T-cell responses are associated with COVID-19 recovery and protection, but the precise role of T-cell responses remains unclear, and how post-infection vaccination (‘hybrid immunity’) further augments this immunity To accomplish these goals, we studied healthy adult healthcare workers who were (a) uninfected and unvaccinated (n = 12), (b) uninfected and vaccinated with Pfizer-BioNTech BNT162b2 vaccine (2 doses n = 177, one dose n = 1) or Moderna mRNA-1273 vaccine (one dose, n = 1), and (c) previously infected with SARS-CoV-2 and vaccinated (BNT162b2, two doses, n = 6, one dose n = 1; mRNA-1273 two doses, n = 1). Infection status was determined by repeated PCR testing of participants. We used FluoroSpot Interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) assays, using subpools of 15-mer peptides covering the S (10 subpools), N (4 subpools) and M (2 subpools) proteins. Responses were expressed as frequencies (percent positive responders) and magnitudes (spot forming cells/106 cytokine-producing peripheral blood mononuclear cells [PBMCs]). Almost all vaccinated participants with no prior infection exhibited IFN-γ, IL-2 and IFN-γ+IL2 responses to S glycoprotein subpools (89%, 93% and 27%, respectively) mainly directed to the S2 subunit and were more robust than responses to the N or M subpools. However, in previously infected and vaccinated participants IFN-γ, IL-2 and IFN-γ+IL2 responses to S subpools (100%, 100%, 88%) were substantially higher than vaccinated participants with no prior infection and were broader and directed against nine of the 10 S glycoprotein subpools spanning the S1 and S2 subunits, and all the N and M subpools. 50% of uninfected and unvaccinated individuals had IFN-γ but not IL2 or IFN-γ+IL2 responses against one S and one M subpools that were not increased after vaccination of uninfected or SARS-CoV-2-infected participants. Summed IFN-γ, IL-2, and IFN-γ+IL2 responses to S correlated with IgG responses to the S glycoprotein. These studies demonstrated that vaccinations with BNT162b2 or mRNA-1273 results in T cell-specific responses primarily against epitopes in the S2 subunit of the S glycoprotein, and that individuals that are vaccinated after SARS-CoV-2 infection develop broader and greater T cell responses to S1 and S2 subunits as well as the N and M proteins.

The lysosomal transporter TAPL has a dual role as peptide translocator and phosphatidylserine floppase

TAPL is a lysosomal ATP-binding cassette transporter that translocates a broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen. Here we report that, in addition to its well-known role as a peptide translocator, TAPL exhibits an ATP-dependent phosphatidylserine floppase activity that is the possible cause of its high basal ATPase activity and of the lack of coupling between ATP hydrolysis and peptide efflux. We also present the cryo-EM structures of mouse TAPL complexed with (i) phospholipid, (ii) cholesteryl hemisuccinate (CHS) and 9-mer peptide, and (iii) ADP·BeF3. The inward-facing structure reveals that F449 protrudes into the cylindrical transport pathway and divides it into a large hydrophilic central cavity and a sizable hydrophobic upper cavity. In the structure, the peptide binds to TAPL in horizontally-stretched fashion within the central cavity, while lipid molecules plug vertically into the upper cavity. Together, our results suggest that TAPL uses different mechanisms to function as a peptide translocase and a phosphatidylserine floppase.

MHC2AffyPred: A machine-learning approach to estimate affinity of MHC class II peptides based on structural interaction fingerprints.

Understanding how MHC class II (MHC-II) binding peptides with differing lengths exhibit specific interaction at the core and extended sites within the large MHC-II pocket is a very important aspect of immunological research for designing peptides. Certain efforts were made to generate peptide conformations amenable for MHC-II binding and calculate the binding energy of such complex formation but not directed toward developing a relationship between the peptide conformation in MHC-II structures and the binding affinity (BA) (IC50 ). We present here a machine-learning approach to calculate the BA of the peptides within the MHC-II pocket for HLA-DRA1, HLA-DRB1, HLA-DP, and HLA-DQ allotypes. Instead of generating ensembles of peptide conformations conventionally, the biased mode of conformations was created by considering the peptides in the crystal structures of pMHC-II complexes as the templates, followed by site-directed peptide docking. The structural interaction fingerprints generated from such docked pMHC-II structures along with the Moran autocorrelation descriptors were trained using a random forest regressor specific to each MHC-II peptide lengths (9-19). The entire workflow is automated using Linux shell and Perl scripts to promote the utilization of MHC2AffyPred program to any characterized MHC-II allotypes and is made for free access at https://github.com/SiddhiJani/MHC2AffyPred. The MHC2AffyPred attained better performance (correlation coefficient [CC] of .612-.898) than MHCII3D (.03-.594) and NetMHCIIpan-3.2 (.289-.692) programs in the HLA-DRA1, HLA-DRB1 types. Similarly, the MHC2AffyPred program achieved CC between .91 and .98 for HLA-DP and HLA-DQ peptides (13-mer to 17-mer). Further, a case study on MHC-II binding 15-mer peptides of severe acute respiratory syndrome coronavirus-2 showed very close competency in computing the IC50 values compared to the sequence-based NetMHCIIpan v3.2 and v4.0 programs with a correlation of .998 and .570, respectively.

A novel telomerase-derived peptide GV1001-mediated inhibition of angiogenesis: Regulation of VEGF/VEGFR-2 signaling pathways

GV1001, a human telomerase reverse transcriptase catalytic subunit-derived 16-mer peptide, has been developed as a novel anticancer vaccine against various cancers including pancreatic cancer. In the current study, we demonstrate the regulatory roles and mechanisms of GV1001 in endothelial cell responses in vitro and microvessel sprouting ex vivo. GV1001 markedly inhibits vascular endothelial growth factor-A (VEGF-A)-stimulated endothelial cell permeability, proliferation, migration, invasion, tube formation as well as microvessel outgrowth from rat aortic rings. These anti-angiogenic effects of GV1001 were associated with the inhibition of VEGF-A/VEGFR-2 signaling pathways, redistribution of vascular endothelial-cadherin to cell–cell contacts, and down-regulation of VEGFR-2 and matrix metalloproteinase-2. Furthermore, GV1001 suppresses the proliferation and invasion of non-small cell lung cancer cells, and the release of VEGF from the cells, suggesting the regulatory role of GV1001 in tumor-derived angiogenesis as well as cancer cell growth and progression. Collectively, our study reports the pharmacological potential of GV1001 in the regulation of angiogenesis, and warrants further evaluation and development of GV1001 as a promising therapeutic agent for a variety of angiogenesis-related diseases including cancer.

Protease-targeting peptide-functionalized porous silicon nanoparticles for cancer fluorescence imaging.

Background: Porous silicon (pSi) nanoparticles (NPs) functionalized with suitable targeting ligands are now established cancer bioimaging agents and drug-delivery platforms. With growing interest in peptides as tumor-targeting ligands, much work has focused on the use of various peptides in combination with pSi NPs for cancer theranostics. Here, the authors investigated the targeting potential of pSi NPs functionalized with two types of peptide, a linear 10-mer peptide and its branched (Y-shaped) equivalent, that respond to legumain activity in tumor cells. Results: In vitro experiments established that the linear peptide-pSi NPconjugate had better aqueous stability under tumor conditions and higher binding efficiency (p<0.001) toward legumain-expressing cells such as RAW 264.7 cells compared with that of its branched equivalent. In vivo studies (analyzed using ex vivo fluorescence) with the linear peptide-pSi NPformulation using a syngeneic mouse model of breast cancer showed a higher accumulation (p>0.05) of linear peptide-conjugated pSi NPs in the tumor site within 4 hcompared with nonconjugated pSi NPs. Theseresults suggest that the linear peptide-pSi NPformulation is a nontoxic, stableand efficient fluorescence bioimaging agent and potential drug-delivery platform.

Inhibition of TREM-2 Markedly Suppresses Joint Inflammation and Damage in Experimental Arthritis.

The triggering receptors expressed on myeloid cells (TREMs) are a family of activating immune receptors that regulate the inflammatory response. TREM-1, which is expressed on monocytes and/or macrophages and neutrophils, functions as an inflammation amplifier and plays a role in the pathogenesis of rheumatoid arthritis (RA). Unlike TREM-1, the role in RA of TREM-2, which is expressed on macrophages, immature monocyte-derived dendritic cells, osteoclasts, and microglia, remains unclear and controversial. TREM-2 ligands are still unknown, adding further uncertainty to our understanding of TREM-2 function. Previously, we demonstrated that TREM-1 blockade, using a ligand-independent TREM-1 inhibitory peptide sequence GF9 rationally designed by our signaling chain homooligomerization (SCHOOL) model of cell signaling, ameliorates collagen-induced arthritis (CIA) severity in mice. Here, we designed a TREM-2 inhibitory peptide sequence IA9 and tested it in the therapeutic CIA model, either as a free 9-mer peptide IA9, or as a part of a 31-mer peptide IA31 incorporated into lipopeptide complexes (IA31-LPC), for targeted delivery. We demonstrated that administration of IA9, but not a control peptide, after induction of arthritis diminished release of proinflammatory cytokines and dramatically suppressed joint inflammation and damage, suggesting that targeting TREM-2 may be a promising approach for the treatment of RA.

Structural insights into engineering a T-cell receptor targeting MAGE-A10 with higher affinity and specificity for cancer immunotherapy

BackgroundT-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being...

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19–198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Longitudinal mapping of hepatitis B vaccine-induced B-cell linear epitopes in healthy individuals.

The elimination of hepatitis B virus (HBV) infection is partially facilitated by the prophylactic HB vaccine. As the loss of seroprotection over time remains a conundrum for long-lasting protection, a comprehensive dynamic analysis of immunogenic targets of the HB vaccine will provide novel insights into the improvement and design of potential targets. In this study, 36 healthy subjects without prior history of hepatitis B infection and negative for hepatitis B surface antibody (anti-HBs) were enrolled. Participants were given a series of three doses of HB vaccine on a 0-, 1-, and 6-month schedule and longitudinally followed up. We systematically mapped 55 overlapping 15-mer peptides covering the small S protein of hepatitis B virus (SHBs) of vaccinees' serum samples at seven time points by performing an ELISA assay. Additionally, the frequencies and function dynamics of adaptive immune response were assessed by flow cytometry. We found that the SHBs peptide coverage presented an overall upward trend along with the vaccination progress, and the individual subpartition recognition was strongly correlated with the anti-HBs titers. Moreover, we identified one dominant epitope (S29) located on "a determinant region" associated with effective vaccine response. Besides, significant correlations between the proportion of plasmablasts and proliferating B cells and levels of anti-HBs were ascertained. Taken together, our data characterized the dynamics of HB vaccine-induced neutralizing antibodies against B-cell linear epitopes on SHBs and adaptive immune response, which will be constructive to develop the next-generation vaccine.

Functional analysis of mutations endowing rAAV2-retro with retrograde tracing capacity

Recombinant adeno-associated viruses (rAAVs) are widespread vectors in neuroscience research. However, the nearly absent retrograde access to projection neurons hampers their application in functional dissection of neural circuits and in therapeutic intervention. Recently, engineering of the AAV2 capsid has generated an AAV variant, called rAAV2-retro, with exceptional retrograde functionality. This variant comprises a 10-mer peptide insertion at residue 587 and two point mutations (LADQDYTKTA + V708I + N382D). Here, we evaluated the contribution of each mutation to retrograde transport in prefrontal cortex -striatum and amygdala-striatum pathways, respectively. Results showed that disruption of the inserted decapeptide almost completely abolishes the retrograde access to neurons projecting to striatum. Eliminating N382D has little effect on the retrograde functionality. Restoring another mutation V708I, however, even improves its performance in amygdala-striatum pathway. Parallel comparison within same animal further confirms this conflicting effect of V708I. These results demonstrate a pivotal role of decapeptide insertion in gaining the capacity of retrograde transport and highlight a neural circuit-dependent contribution of V708I. It suggests constant and custom engineering of rAAV2-retro might be required to tackle the challenge of tremendous neuronal heterogeneity.

Abstract 2719: Experimental comparison of two pipelines to identify neo-epitopes in head and neck cancer

Abstract Through somatic mutations, cancer cells accumulate cancer-specific mutated peptides, commonly referred to as neoepitopes or neoantigens, that can be recognized by T cells. Due to their cancer-specific nature, neoantigens are a highly attractive area of study for developing cancer immunotherapies. By comparing whole-exome sequencing of patient tumor tissue and matched normal tissue, researchers have been able to identify cancer-specific neoepitopes. However, the filters that can accurately delineate immunogenic neoepitopes from non-immunogenic remain elusive. Current methods, such as the pipeline developed by the Tumor Neoantigen Selection Alliance (TESLA), typically select short peptides based on hypothesized rules of immunogenicity. Importantly, only a small percentage of these epitopes have been reported to be recognized by T cells through experimental validation assays. In this study, we systematically tested the neoantigen prediction capabilities of our in-house pipeline, Identify-Prioritize-Validate (IPV), and the TESLA pipeline. Whole-exome sequencing from 11 head and neck squamous cell carcinoma patients was used to identify somatic variants. With IPV, variants were filtered and ranked primarily based on variant allele frequencies in RNA and DNA and 20mer peptides were generated from each mutation. In parallel, the TESLA pipeline was used to rank 8- 12-mer peptides based on a combination of HLA binding and stability predictions as well as expression levels of source antigens. Pools of the top 10 peptides from both methods were generated. To enable the direct comparison of TESLA and IPV while accounting for the disparate peptide lengths produced by the pipelines, two additional pools were generated: one pool of 20mers from the TESLA pipeline and one pool of 8-12mers from IPV. The four peptide pools were individually cultured in vitro with patient-matched PBMCs and immune recognition was measured through IFNg and IL5 using fluorospot assays. The IPV pipeline consistently outperformed the TESLA pipeline in predicting neoantigens that elicited an immune response. 5 of the 11 patients showed significant immune recognition of the IPV 20mer peptide pool, only 2 patients responded to the 20mer TESLA pool, while no patients responded to the TESLA and IPV 8-12mer pools. Through this data, we prove the efficacy of IPV, and its filters, and highlight the importance of utilizing 20mer peptides over shorter peptides. Finally, we deconvoluted the positive IPV responses and were able to provide the sequences of novel immunogenic neoepitopes. Our work underscores the improvement in the predictive ability of IPV in comparison to typical pipelines that output short peptides based on parameters derived from a priori knowledge and demonstrates the ability of IPV to be utilized as a tool to identify novel neoantigens. Citation Format: Leila Yasmine Chihab, Zeynep Kosaloglu-Yalcin, Jason Greenbaum, Aaron Miller, Stephen Schoenberger, Bjoern Peters. Experimental comparison of two pipelines to identify neo-epitopes in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2719.

Abstract LB099: Direct identification of MHC class I and class II epitopes for TCR-based T cell therapy for pancreatic cancer

Abstract Background: Tumor-associated antigens and neoantigens serve as primary targets for cancer immunotherapies such as vaccines and T-cell based therapy. However, identifying pancreatic ductal adenocarcinoma (PDAC) associated T-cell epitopes have been challenging due to its low genomic mutational burden. In this study, we attempted to directly identify PDAC T-cell epitopes by using mass spectrometry. Methods: The protein lysate from PDAC specimens and cell lines were subjected to the antibody affinity purification of human major histocompatibility complexes (MHC) including both HLA Class I and Class II complexes. Peptides bound to the MHC were eluted and identified through LC-MS/MS. Peptide sequences were analyzed with MAXQUANT and Novor Denovo. HLA-binding affinity of peptides were predicted using NetMHC4.0 and validated by in vitro T2 binding assays. Their ability to induce T cell response were measured in a cytokine-Flurospot assay. TCRs specific for selected peptides were cloned by single-cell TCR sequencing and their anti-PDAC activity were tested in vivo on the patient-derived xenograft(PDX) models. Results: We identified 6553 unique HLA-I bound 9-mer peptides from eight PDAC specimens and two PDAC cell lines (Panc10.05 and Pan06.03). Among them, 1163 peptides and 1354 proteins were found in two or more PDAC specimens. We identified 8 potentially immunogenic peptides that bind strongly to matched and nonmatched HLA molecules and induced T cell response in peripheral T cells from both HLA-type matched and non-matched patients. We also identified HLA-II bound peptides in six PDAC tissues and found that the HLA-I and HLA-II peptides isolated from the same patient are highly overlapped. These overlapped peptides were able to induce polyfunctional cytokine response (IFN-γ, TNF-α, and IL-2) in peripheral T cells from patient PBMC. We further investigated the anti-tumor capability of T cell receptors (TCR) for an LAMC-2 derived HLA-class I epitope and a TMSB10 derived peptide eluted from both HLA-Class I and Class II affinity purification. Immunohistochemistry revealed both proteins to be more highly expressed in PDAC tissue compared to paranormal normal tissue. We stimulated HLA-type matched patient’s PBMC with LAMC-2 and TMSB10 peptides to induce clonal expansion of epitope-specific CD8+ and CD4+ T-cells, respectively. We subsequently performed single-cell TCR sequencing of these expanded T cells and infected Jurkat cells with the lentivirus expressing TCR of interest. Mice with orthotopically implanted PDAC tumors that were treated with Jurkat cells expressing LAMC-2 specific cells showed significant slowdown tumor growth. We are currently in the processing of testing TMSB10 peptide targeting TCRs. Conclusion: This study provides a novel venue for identifying T cell epitopes in a nonimmunogenic tumor such as PDAC for the design and development of cancer vaccine and T cell therapy. Citation Format: Tengyi Zhang, Jianxin Wang, Yingkuan Shao, Pan Li, Nan Niu, Brian Herbst, Jessica Gai, Juan Fu, Pingbo Zhang, Jun Yu, Kenji Fujiwara, Lei Zheng. Direct identification of MHC class I and class II epitopes for TCR-based T cell therapy for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB099.

Abstract 1743: Identification of mesothelin binding peptide for targeted therapy against pancreatic cancer

Abstract Pancreatic cancer is the main cause of cancer-related deaths worldwide and is difficult to diagnose before the extensive local invasion and distant organ metastasis. Mesothelin is abnormally overexpressed in tumors such as ovary cancer and pancreatic cancer. Studies show a significant link between mesothelin overexpression and short survival in patients with pancreatic cancer. In this study, we screened a phage displayed-peptide library for peptides that selectively bind to mesothelin using mesothelin-overexpressing cells. After five rounds of screening, we selected a 9-mer peptide (named MSLN-Pep) that preferentially bound to mesothelin-high pancreatic cancer cells such as ASPC-1 and Panc-1 cells over mesothelin-low cells such as HEK 293 cells. MSLN-Pep was efficiently internalized into ASPC-1 cells and inhibited the cell migration and invasion while little affected the phosphorylation of Akt. Moreover, a MSLN-Pep-guided proapoptotic peptide (MSLN-Pep-KLA) exerted selective cytotoxicity against pancreatic cancer cells over mesothelin-low cells. MSLN-Pep-KLA when combined with gemcitabine, a chemotherapeutic agent against pancreatic cancer, sensitized ASPC-1 cells to the gemcitabine treatment. These results suggest that MSLN-Pep is a promising tool for a targeted therapy against pancreatic cancer expressing mesothelin at high levels. Citation Format: Min-Sung Park, Byungheon Lee. Identification of mesothelin binding peptide for targeted therapy against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1743.

Abstract 5562: Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice

Abstract Telomerase plays a vital role in cell senescence and cellular replication and has been described as a leading regulator in several human cancers, including prostate, thyroid, breast, colon, and bladder cancer. Telomerase is responsible for elongating telomere length and prolonging cellular lifespan as well as acting as a transcriptional modulator in cancer signaling pathways. Because of its enhanced expression in primary tumor cells as well as incipient tumor-initiating stem cells and with limited distribution in normal somatic cells, the catalytic component of telomerase, TERT (telomerase reverse transcriptase), is regarded as a high-impact target for immunotherapeutic agents and vaccines. In order to derive a mouse TERT (mTERT)-specific vaccine which could be useful in a preventive approach, we implemented an epitope-mapping stratagem by which pools of 30-mer peptides overlapping by 5 spanning the entire mTERT sequence were used to immunize C57BL/6 mice. Several immunogenic peptides were identified by matrixed peptide pool IFN-ɣ ELISPOT screening in a 2-part process that first identified positive pools, then identified individual peptides. 15-mer and 31-mer versions of the top 7 immunogenic mTERT peptides were synthesized and used as a peptide pool combined separately with several adjuvants to optimize immunogenicity. Results suggested a peptide sequence-specific preference for TLR3-mediated Hiltonol versus TLR9-mediated CpG-based adjuvants. In addition, the majority of T cell responses specific to the TERT peptides was identified through flow cytometry as belonging to the CD4 compartment, although one class I-restricted epitope was also identified. Multifunctional CD4+ T cells expressing IFN-ɣ, TNF-α, IL-2, and CD107a were identified specific to several mTERT peptides. In vivo cytotoxic effects exerted on TERT peptide pool-loaded target cells were also uncovered that were specific to a CpG-adjuvanted version of the vaccine. Finally, multiple versions of the mTERT peptide/adjuvant vaccine with highest immunogenicity scores are being tested in a syngeneic graft mouse model using subcutaneous implantation of lung cancer-derived SPON10 cells expressing mTERT. If proven efficacious, these results will support the feasibility of a TERT peptide/adjuvant approach for prophylactically engendering broad antitumor immunity to TERT-expressing cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Jason D. Marshall, Yurong Song, Hamid Zarkesh, Rebecca L. Matthews, Chelsea Sanders, Simone Difilippantonio, Ligia A. Pinto, Shizuko Sei, Robert H. Shoemaker. Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5562.

1473-P: Combination Therapy with Dual-Function Peptide S1K and Insulin Reduces Blood Glucose Levels in Mouse Models of Type 1 Diabetes through Beta-Cell Regeneration and Immunosuppression

β cell regeneration is necessary to cure patients with type 1 diabetes (T1D) whose β cells are destroyed by immune cells. Although insulin therapy can control blood glucose levels, it must be administered throughout life and occurs several side effects such as hypoglycemia. New β cell can be generated through neogenesis, β cell replication or transdifferentiation from other cells in diabetic animals. In addition to β cell regeneration, immunosuppression is another important mechanism to prevent β cell destruction, but currently any drugs haven’t been approved as immunosuppressants for T1D treatments yet. A novel 9-mer peptide, Shiloah 1000 (S1K) , has dual functions of β cell regeneration and immunosuppression. In streptozotocin-induced hyperglycemic mouse model, the number of mice recovered to normoglycemia was increased by the combined treatment of S1K and insulin compared to S1K or insulin alone. In addition, the combined treatment more increased the number of β cells than S1K or insulin alone. Interestingly, insulin/glucagon double-positive cells were increased by both combined and S1K alone treatments. The increase in the number of mice recovered to normoglycemia is considered to be due to β cell regeneration through α- to β-cell transdifferentiation by S1K. In the NOD mouse model, the combined treatment of S1K and insulin suppressed hyperglycemia more than insulin alone. In addition, the serum C-peptide levels were increased by both combined and S1K treatments, but not by insulin treatment. The increase in the serum C-peptide level suggest that β cells remain undestroyed by immunosuppression. Taken together, our data show that S1K, as a drug candidate for T1D treatment in combination with insulin reduces blood glucose levels in the T1D mouse models through β cell regeneration and immunosuppression, suggesting a potential role in preventing and curing T1D. Disclosure Y. Lee: Employee; Ensolbiosciences. H. Kim: Employee; Ensolbiosciences. J. Kim: Employee; Ensol Bioscience. Y. Jang: Employee; Ensolbiosciences.

Cancer immunotherapy: Identifying cancer testis antigen peptides to enhance antitumor response.

e20022 Background: Cancer testis antigens (CTAs) are highly immunogenic, tissue-restricted proteins that may be over expressed in hematological malignancies. In this study, an in silico analysis of CTA-derived peptide presentation on HLA class I molecules is reported with an aim to identify putative CTA peptides with a high estimated binding affinity to known HLA class I molecules. Methods: Eighty unique HLA Class I alleles (HLA-A, B, and C), across 78 hematopoietic cell transplant recipients were computationally studied for their ability to bind peptides derived from three CTAs. The amino acid sequences of all three CTA proteins were obtained from UniProtKB. NetMHCPan-4.1 was utilized for in silico prediction of HLA class I binding affinities of the 9-mer peptides generated from these CTAs. Results: The CTA MAGE-A1 had 1202 unique peptides predicted to bind at least one HLA class I allele and 176 of these peptides were classified as strong binders (Rank &lt;0.5 and IC50&lt;500nM). Conversely, 71 unique HLA class 1 alleles were predicted to bind strongly to at least one MAGE-A1 derived peptide. The ability of different MAGE-A1 peptides to bind HLA varied considerably, e.g., peptide TSYVKVLEY, was predicted to bind strongly to 22 different HLA class I alleles. Similarly, 41 CTAG-1B-derived and 164 MAGE-A2-derived peptides were predicted to generate strong binders with at least one HLA class 1 allele. Conversely, 37 and 71 unique HLA class I alleles were predicted to bind strongly to at least one CTAG1B peptide and one MAGE-A1 peptide, respectively. CTAG1-B derived peptide FATPMEAEL and MAGE-A2-derived peptide FAHPRKLLM were predicted to bind strongly to 17 and 22 HLA class 1 alleles, respectively. In aggregate most patients presented multiple peptides across the 6 HLA class I molecules studied (Table). The number of strongly binding MAGE-A1 and CTAG-1B peptides from unique patients. Strong binding defined by Rank &lt;0.5 and IC50&lt;500nM. Conclusions: Most patients presented several CTA derived peptides, suggesting that the aggregate HLA presentation across the 6 classical HLA class I molecules will yield high odds of presentation of CTA expressed by target tissues. These HLA genotype-informed quantitative analyses raise the possibility that agents such as checkpoint inhibitors and immunomodulatory drugs may synergize with hypomethylating agents and augment CTA-directed T-cell responses.[Table: see text]

Picturins and Pictuseptins, two novel antimicrobial peptide families from the skin secretions of the Chachi treefrog, Boana picturata

The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named “Picturins” (PTR), “Pictuseptins” (PTS), and “Boanins” (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, −2 and − 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, −2 and − 4 have 22 amino acid residues in length, while PTS -3, −5 and − 6 are composed of 26 residues. Whereas BNS are four 28–37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to −3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 μM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 μM) followed by PTS-3 (11.7 μM) and PTS-2 (14.24 μM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. Biological significanceNature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Signaling via dopamine and adenosine receptors modulate viral peptide-specific and T-cell IL-8 response in COVID-19

B-cell but not T-cell responses have been extensively studied using peripheral blood mononuclear cells (PBMCs) obtained from patients with coronavirus disease 2019 (COVID-19). Our recent study showed that not only T-helper (Th) 17 but also Th1 cells directly produce interleukin (IL)-8, a major source of neutrophilic inflammation, which is also known to induce disseminated intravascular coagulation (DIC) in COVID-19 patients. Neutrophilic inflammation caused by IL-17A or IL-8 can be fatal; thus, therapeutic intervention is highly expected. The present study aimed to investigate the T-cell responses in the Japanese patients. We synthesized spike protein-derived 15-mer peptides that are expected to bind to HLA class II allelic products frequently observed in the Japanese population, and checked the T-cell responses in Japanese patients with COVID-19. We have found that (i) patients show marked IL-8 but not IL-17A responses; (ii) these responses are restricted by HLA-DR; and (iii) IL-8 responses are abrogated by a dopamine D2 like receptor (D2R) agonist, ropinirole, and an adenosine A2a receptor (A2aR) antagonist, istradefylline. Compounds used for the treatment of Parkinson’s disease may ease DIC in COVID-19. (183 words)

Caloxin-derived peptides for the inhibition of plasma membrane calcium ATPases

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.