Peptide Binding Specificity Research Articles

Functional Living Materials for Virus Disinfection Based on Engineered Bacillus subtilis Biofilm by Displaying Viral Affinity Peptides and Expressing Protease

AbstractGlobal pandemics caused by pathogenic viruses have highlighted the need to develop effective and sustainable materials to defend against these viruses. However, most commercial viral disinfection materials rely on precious metals, which are toxic. In this study, an engineered living material based on Bacillus subtilis biofilms is presented that can effectively remove Influenza A virus and SARS‐CoV‐2 from water. First, B. subtilis biofilm is used to display the H1N1 and H5N1 specific binding peptide C40 and SARS‐CoV‐2 specific binding peptide SBP1; thus, the biofilm materials has outstanding efficacy against H1N1, H5N1, and SARS‐CoV‐2 pseudovirus from water. In addition to capturing the virus, these biofilm materials can also inactivate the virus by secreting alkaline protease AprE, which effectively degrades the viral proteins at temperatures of 4, 16, 25, and 37 °C. Furthermore, to constrain bacterial growth and prevent the leakage of genetically modified organisms, a suicide genetic circuit using the quorum‐sensing system ComQXPA to control Lytc expression is constructed, leading to bacterial lysis. Overall, this study demonstrates a novel, effective, and sustainably engineered living material for preventing viral transmission. This material provides a safeguard for public health and promotes environmentally friendly governance.

Electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta

This paper reports the development of a highly sensitive and selective electrochemical peptide-based biosensor for the detection of the inflammatory disease biomarker, interleukin-1beta (IL-1β). To this end, flower-like Au–Ag@MoS2-rGO nanocomposites were used as the signal amplification platform to achieve a label-free biosensor with a high sensitivity and selectivity. First, a high-affinity peptide for IL-1β was identified through biopanning with M13 random peptide libraries, and was newly designed by incorporating cysteine at the C-terminus. An IL-1β specific binding peptide was used as the bio-receptor, and the interaction between the IL-1β binding peptide and IL-1β was confirmed via enzyme-linked immunosorbent assay and various physicochemical and electrochemical analyses. Under optimal conditions, the biosensor achieved an ultrasensitive and specific IL-1β detection in a wide linear concentration range of 0–250 ng/mL with a picomolar-level detection limit (∼2.4 pM), low binding constant (∼0.62 pM), and a low coefficient of variation (<1.65 %). The biosensor was successfully utilized for IL-1β determination in the serum of Crohn's disease patients with a good correlation coefficient. In addition, the detection performance was comparable to that of commercially available IL-1β ELISA kit. This indicates that the electrochemical peptide-based biosensor may offer a potentially valuable platform for the clinical diagnosis of various inflammatory disease biomarkers.

Development of fluorometric detection for saxitoxin with its specific binding peptide

A fluorometric sensor for marine saxitoxin was developed using its specific binding peptide based on the graphene oxide-mediated quenching phenomenon.

HLA-DRB1 Hed Is Associated with Improved Survival and Decreased Relapse in Patients with Hematologic Malignancies Following Allogeneic Hematopoietic Stem Cell Transplant

Introduction: Despite advances in disease monitoring and maintenance therapies, the risk of relapse following allogeneic hematopoietic cell transplant (HCT) remains a major concern. Attention has turned to understanding how diversity across the immune system can influence tumor surveillance. Due to their protein's integral role in binding and presenting antigens to immune cells, HLA genes have been a major focus because a high number of polymorphisms alter their peptide-binding grooves and impact their repertoire of antigens. Based on the heterozygote advantage hypothesis, heterozygous HLA genotypes were thought to better elicit immune responses due to a higher probability of binding antigens. Further refinement of HLA diversity centers on the immunopeptidome and variations in peptide-binding specificities. HLA evolutionary divergence (HED) represents a quantitative measure of physiochemical sequence divergence between peptide-binding grooves encoded by HLA alleles. Greater sequence divergence across HLA alleles leads to a more expansive repertoire of antigens and helps to explain that high HED was associated with improved overall survival (OS) in patients with solid tumors treated with immune checkpoint inhibitors (Chowell et al. Nat Med, 2019). With a more diversified immune response, we hypothesize that high HED will improve tumor surveillance and impact survival and relapse following allogeneic HCT. Methods: Patients who underwent 8/8 HLA-A, -B, -C and -DRB1 matched first allogeneic HCT for AML, MDS, ALL, CML, or lymphoma between 2008-2018 were identified from the CIBMTR database. All patients received myeloablative conditioning, except for those patients with lymphoma. Using donor HLA typing to the allele level and reference amino acid sequences for peptide binding domains, HLA HED was calculated for each HLA class I allele (-A,-B,-C) and HLA class II allele (-DRB1) using the Grantham Distance metric (Pierini, et al. Mol Biol Evol, 2018). An overall mean HED score was calculated for HLA class I and HLA-DRB1 alleles and was initially divided at the median to classify high versus low HED. HED was then evaluated based on quartiles with high HED defined as overall mean HED greater or equal to the upper quartile. The primary outcomes were OS and relapse. Secondary outcomes included transplant-related mortality (TRM), disease-free survival (DFS), and graft-versus-host disease (GVHD). Multivariate analysis using Cox proportional hazards model was performed with factors violating the proportional hazards assumptions adjusted through stratification. P-value ≤ 0.01 was significant for this study. Results: A total of 9,231 patients received an 8/8 HLA-matched allogeneic HCT from 2008-2018 according to selection criteria. With high HED defined by the median, there was no association with OS and HLA class I HED (HR 1.00; 95% CI 0.93 - 1.07, P=0.935) or with HLA-DRB1 HED (HR 0.96; 95% CI 0.91 -1.02, P=0.225). Relapse was not associated with HLA class I HED (HR 1.04, 95% CI 0.96-1.12, P=0.354) or with HLA-DRB1 HED (HR 0.99, 95% CI 0.93 - 1.06, P=0.857). There were no significant associations with HED and the secondary outcomes of TRM, DFS, or GVHD. HED was then characterized by quartiles. High HLA-DRB1 HED (upper quartile) was associated with improved OS (HR 0.91; 95% CI 0.85 - 0.96, P=0.0019) and decreased relapse (HR 0.87; 95% CI 0.80 - 0.95, P=0.0014) when compared to low HED (Q1-Q3). Improved DFS was observed with high HLA-DRB1 HED but failed to achieve statistical significance (HR 0.92; 95% CI 0.86 - 0.98, P=0.011). There was no association between HLA class I HED and OS, relapse, or DFS. Conclusions: High HLA-DRB1 HED, defined as upper quartile, was associated with improved OS and decreased relapse in patients with hematologic malignancies following allogeneic HCT. High HED promotes a diverse repertoire of antigens, which may heighten tumor surveillance and enhance graft versus leukemic effect. Since similar associations were not replicated when high HED was defined by the median, the effects of HLA-DRB1 HED are most likely observed in the most divergent donor-recipient HLA pairings. Previous work has implicated the role of HLA class II alleles in affecting immune function and contributing to relapse following HCT. Therefore, HLA-DRB1 may play a more integral role in tumor surveillance post-HCT, which can have important implications in donor selection, especially in HLA mismatched settings.

Mass Spectrometric Profiling of HLA-B44 Peptidomes Provides Evidence for Tapasin-Mediated Tryptophan Editing.

The extreme polymorphisms of HLA class I proteins result in structural variations in their peptide binding sites to achieve diversity in Ag presentation. External factors could independently constrict or alter HLA class I peptide repertoires. Such effects of the assembly factor tapasin were assessed for HLA-B*44:05 (Y116) and a close variant, HLA-B*44:02 (D116), which have low and high tapasin dependence, respectively, for their cell surface expression. Analyses of the HLA-B*44:05 peptidomes in the presence and absence of tapasin reveal that peptides with C-terminal tryptophans and higher predicted affinities are preferentially selected by tapasin, coincident with reduced frequencies of peptides with other C-terminal amino acids, including leucine. Comparisons of the HLA-B*44:05 and HLA-B*44:02 peptidomes indicate the expected structure-based alterations near the peptide C termini, but also C-terminal amino acid frequency and predicted affinity changes among the unique and shared peptide groups for B*44:02 and B*44:05. Overall, these findings indicate that the presence of tapasin and the tapasin dependence of assembly alter HLA class I peptide-binding preferences at the peptide C terminus. The particular C-terminal amino acid preferences that are altered by tapasin are expected to be determined by the intrinsic peptide-binding specificities of HLA class I allotypes. Additionally, the findings suggest that tapasin deficiency and reduced tapasin dependence expand the permissive affinities of HLA class I-bound peptides, consistent with prior findings that HLA class I allotypes with low tapasin dependence have increased breadth of CD8+ T cell epitope presentation and are more protective in HIV infections.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses.

Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

HLA-Clus: HLA class I clustering based on 3D structure

BackgroundIn a previous paper, we classified populated HLA class I alleles into supertypes and subtypes based on the similarity of 3D landscape of peptide binding grooves, using newly defined structure distance metric and hierarchical clustering approach. Compared to other approaches, our method achieves higher correlation with peptide binding specificity, intra-cluster similarity (cohesion), and robustness. Here we introduce HLA-Clus, a Python package for clustering HLA Class I alleles using the method we developed recently and describe additional features including a new nearest neighbor clustering method that facilitates clustering based on user-defined criteria.ResultsThe HLA-Clus pipeline includes three stages: First, HLA Class I structural models are coarse grained and transformed into clouds of labeled points. Second, similarities between alleles are determined using a newly defined structure distance metric that accounts for spatial and physicochemical similarities. Finally, alleles are clustered via hierarchical or nearest-neighbor approaches. We also interfaced HLA-Clus with the peptide:HLA affinity predictor MHCnuggets. By using the nearest neighbor clustering method to select optimal allele-specific deep learning models in MHCnuggets, the average accuracy of peptide binding prediction of rare alleles was improved.ConclusionsThe HLA-Clus package offers a solution for characterizing the peptide binding specificities of a large number of HLA alleles. This method can be applied in HLA functional studies, such as the development of peptide affinity predictors, disease association studies, and HLA matching for grafting. HLA-Clus is freely available at our GitHub repository (https://github.com/yshen25/HLA-Clus).

Novel peptide identified from viable-cell based phage display technique regulates growth cycle of Daphnia magna

Phage display is a widely used technique for selecting specific binding peptides, but presenting antigens in their natural form can be challenging, as protein coating may induce structural changes. In this study, we employed a whole cell-based phage display technique without a coating step to select peptides that bind specifically to Daphnia magna eggs. Boiled eggs were used as a control to ensure that antigens were presented in their natural forms. We identified a peptide, DEP1 (LYALPLSHLKSHGGG), with the highest binding affinity to D. magna eggs. DEP1 did not affect zebrafish eggs, but it inhibited normal hatching and reproductive ability in D. magna eggs, and hindered growth in neonates before their first ecdysis. Morphological analysis revealed that DEP1 caused intestinal damage and tissue abnormalities. Our findings demonstrate that the whole cell-based phage display technique is successful in presenting antigens in their natural form, and that the DEP1 peptide can be applied to regulate the growth cycle of D. magna. These results have implications for the use of phage display in environmental research and the potential use of DEP1 for hazardous organisms in aquatic systems.

Abstract 5556: Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of ovarian cancer

Abstract The objective of these in vitro and in vivo studies was to validate Lemur Tyrosine Kinase 3 (LMTK3) as a specific target and predictor of clinical outcome in ovarian cancer. LMTK3 belongs to a family of regulated tyrosine kinases with three structurally related isoforms, LMTK1, LMTK2, and LMTK3. Both nuclear and cytoplasmic LMTK3 expressions correlated with tumor grade and patient survival in cancers such as breast and colorectal cancer. We tested the clinical significance of the LMTK3 gene by immunohistochemistry (IHC) using LMTK3 monoclonal antibody on formalin-fixed paraffin-embedded sections (FFPE) collected from 204 early-stage (stage I-II) ovarian cancer patients. Results from this IHC LMTK3 study revealed a higher cytoplasmic to nuclear localization of LMTK3 correlated with worse overall survival (P&amp;lt;0.01). Further investigation of LMTK3’s prognostic value by screening LMTK3 signaling in 270 stage III-IV ovarian cancer patients is ongoing. We utilized the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for testing the killing efficacy of targeting LMTK3 by a monoclonal antibody, siRNA, and specific LMTK3 binding peptides (LMTK3BP) in ovarian cancer cell lines SKOV3, MDAH-2774, A2780, and TOV-21G. Treatment with LMTK3 specific monoclonal antibody, siRNA, and LMTK3BP significantly induced killing of both chemosensitive and chemoresistant ovarian cancer cells without affecting normal cells in vitro. Moreover, we observed this killing as synergistic with both Cisplatin and Taxotere treatment in vitro. Lastly, we used an A2780 cell line derived orthotopic xenograft mouse model of ovarian cancer to test the efficacy of specific LMTK3BP in vivo. Strikingly, LMTK3BP 20 mg/kg IV dose given three times a week for three weeks showed a 35% tumor reduction. Furthermore, in vivo safety studies showed no signs of toxicity of the LMTK3BP, even at a very high dose of 40 mg/kg. Thus, with dose optimization in ongoing experiments, we could expect a higher efficacy. In conclusion, this study highlighted the importance of LMTK3 as a predictor of patient clinical outcome and potential target for treatment in ovarian cancer. Citation Format: Thea K. Kirsch-Mangu, Axel S. Tullberg, Anna Portela, Khalil Helou, Ghassan M. Saed. Lemur tyrosine kinase 3 serves as a predictor of patient outcome and a target for the treatment of 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 5556.

Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

Peptide-binding specificity prediction using fine-tuned protein structure prediction networks

Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.

Correlating HLA variants and the emergence of immune-related adverse events (irAEs) from ipilimumab/nivolumab (I/N) in patients (pts) treated on CheckMate 214.

659 Background: Combination therapy with I/N can achieve long-term benefit in a sizable proportion of RCC pts, but broad application has been challenged by a high incidence of irAEs. Host features affecting antigen presentation are candidate determinants of irAE pathogenesis, and isolated reports have linked Human Leukocyte Antigen (HLA) variations to organ- specific irAEs. We analyzed data for pts treated on a phase 3 trial to investigate association of class 1 HLA variants with development of irAEs from I/N and develop a tool to predict risk for toxicity. Methods: We analyzed germline HLA + clinical data for 472 pts receiving I/N vs sunitinib (SUT). Based on allelic variants for HLA-A and B pts were categorized into 12 established HLA "Super-Types" (ST), sets of HLA variants with largely overlapping peptide binding specificity. We conducted uni- and multivariate (UV; MV) Cox proportional hazards regression to correlate HLA-ST variables with time to treatment-related AEs (TTrAE, grade 2+) in I/N pts using the Kaplan Meier method. Several MV HLA-ST models were developed on a discovery set (DISC; 2/3 random sample of I/N pts without replacement ) and compared using model concordance (c-index) on a validation set (VAL; remaining 1/3). Model coefficients were used to create a “HLA-ST score” that could be computed for individual pts. Results: 235 pts and 237 pts received I/N vs SUT and had available HLA data, respectively. On UV analysis for I/N, HLA-B07 ST had protective association TTrAE (HR= 0.65, 95% CI: 0.46,0.90; p= 0.010), while B62 associated adversely (HR=1.64, 95% CI: 1.12, 2.40; p=0.014). Relevant to the development of our model we identified interactions between several pairs of HLA ST. The HLA ST model with best performance in DISC (c-index= 0.606) and VAL (c-index=0.595) integrates B07, B62, A01, B08, and two interactions: B07-B08 and B07-A01. The model-generated HLA-ST score was significantly associated with TTrAE after adjusting for race, BMI, region, PDL1 status, and MSKCC risk score (DISC p&lt;0.001; VAL p=0.028). No association with TTrAE was seen in SUT treated pts (p=0.655), neither in UV nor MV model. I/N-treated patients with HLA-ST score dichotomized &gt;= vs &lt; median had significantly different TTrAE both in DISC (p=0.004) and VAL (p=0.009); again, no difference was observed in the SUT arm (p=0.5). The weighted HLA-ST score had no association with PFS or OS for I/N pts. Conclusions: In this large sample of I/N-treated patients, class I HLA variants were associated with the risk of developing irAEs. We developed a HLA ST based score that correlated with treatment toxicity independent of relevant clinical and demographic features. No association with TTrAE was seen in SUT-treated pts. These results highlight the potential of characterizing germline features to predict immune related toxicity upfront and deserves further study. Clinical trial information: NCT02231749 .

HLA Class I Supertype Classification Based on Structural Similarity.

HLA class I proteins, a critical component in adaptive immunity, bind and present intracellular Ags to CD8+ T cells. The extreme polymorphism of HLA genes and associated peptide binding specificities leads to challenges in various endeavors, including neoantigen vaccine development, disease association studies, and HLA typing. Supertype classification, defined by clustering functionally similar HLA alleles, has proven helpful in reducing the complexity of distinguishing alleles. However, determining supertypes via experiments is impractical, and current in silico classification methods exhibit limitations in stability and functional relevance. In this study, by incorporating three-dimensional structures we present a method for classifying HLA class I molecules with improved breadth, accuracy, stability, and flexibility. Critical for these advances is our finding that structural similarity highly correlates with peptide binding specificity. The new classification should be broadly useful in peptide-based vaccine development and HLA-disease association studies.

The antigen recognition portion of African buffalo class I MHC is highly polymorphic, consistent with a complex pathogen challenge environment, and the 3’ region suggests distinct haplotype configurations

African buffalo (Syncerus caffer) have been distinct from the Auroch lineage leading to domestic cattle for 5 million years, and are reservoirs of multiple pathogens, that affect introduced domestic cattle. To date, there has been no analysis of the class I MHC locus in African buffalo. We present the first data on African buffalo class I MHC, which demonstrates that gene and predicted protein coding sequences are approximately 86–87% similar to that of African domestic cattle in the peptide binding region. The study also shows concordance in the distribution of codons with elevated posterior probabilities of positive selection in the buffalo class I MHC and known antigen binding sites in cattle. Overall, the diversity in buffalo class I sequences appears greater than that in cattle, perhaps related to a more complex pathogen challenge environment in Africa. However, application of NetMHCpan suggested broad clustering of peptide binding specificities between buffalo and cattle. Furthermore, in the case of at least 20 alleles, critical peptide-binding residues appear to be conserved with those of cattle, including at secondary anchor residues. Alleles with six different length transmembrane regions were detected. This preliminary analysis suggests that like cattle, but unlike most other mammals, African buffalo appears to exhibit configuration (haplotype) variation in which the loci are expressed in distinct combinations.

Self-assembled peptide-paclitaxel nanoparticles for enhancing therapeutic efficacy in colorectal cancer.

Chemotherapy is one of the main treatments for colorectal cancer, but systemic toxicity severely limits its clinical use. Packaging hydrophobic chemotherapeutic drugs in targeted nanoparticles greatly improve their efficacy and reduce side effects. We previously identified a novel colorectal cancer specific binding peptide P-LPK (LPKTVSSDMSLN) from phage display peptide library. Here we designed a self-assembled paclitaxel (PTX)-loaded nanoparticle (LPK-PTX NPs). LPK-PTX NPs displayed a superior intracellular internalization and improved tumor cytotoxicity in vitro. Cy5.5-labeled LPK-PTX NPs showed much higher tumor accumulation in colorectal cancer-bearing mice. Furthermore, LPK-PTX NPs exhibit enhanced antitumor activity and decreased systemic toxicity in colorectal cancer patient-derived xenografts (PDX) model. The excellent in vitro and in vivo antitumor efficacy proves the improved targeting drug delivery, suggesting that peptide P-LPK has potential to provide a novel approach for enhanced drug delivery with negligible systemic toxicity.

Diversity and distribution of MHC class I and II alleles in chicken populations worldwide

Almost everything known about antigen processing and presentation outside of mammals is from work on chickens. In contrast to mammals, unusually strong genetic associations exist between MHC haplotype and responses to infection in chickens. These associations, at least in part, seem to be due to the expression of a single classical class I and II molecule in most tissues. Unlike in mammals, chicken TAP and tapasin genes are highly polymorphic. Furthermore, peptide translocation specificity is very similar to the peptide binding specificity of the dominantly expressed MHCI molecule in each haplotype. These genes appear to be co-evolving – a process made possible by strong linkage disequilibrium across the ‘minimal essential MHC’. Similar co-evolution with the chaperone DM has been proposed for the single dominantly expressed class II gene. This study aimed to begin the most complete and exhaustive analysis of global MHC population genetics outside of humans. Once haplotypes have been defined by their class I and II alleles, interesting haplotypes would be selected for more detailed studies. Illumina MiSeq was used with a novel double-barcoding system to perform multiplexed amplicon se- quencing of exons 2 and 3 from all classical class I and class II loci. Samples were obtained from all major commercial breeders, ‘fancy’ breeds and free-range local chickens from Ethiopia, Nigeria, Tan- zania and Ghana. Our results show that, globally, there is huge diversity in chicken MHC alleles, most of which has never previously been reported from studies on experimental lines. Commercial flocks, however, have extremely low diversity, suggesting that artificial selection and population bottlenecks have had a drastic effect on the immune phenotypes of commercial flocks. These populations of commercial birds are also enriched in haplotypes with apparently promiscuous class I alleles that bind a wide variety of peptides and confer resistance to many common infectious diseases.

Presentation of HIV and Mtb derived peptides by HLA-E

Unconventional, Mamu-E-restricted CD8+ T cell responses directed against a diverse range of epitopes have been implicated as immune correlates of sterile immune protection in rhesus CMV-vectored SIV vaccination. In light of these findings, we have re-explored the peptide binding specificity of the human ortholog, HLA-E, from the view of developing a parallel HIV vaccination strategy in humans. Given the extraordinary breadth of vaccine-elicited, Mamu-E-restricted responses, we first optimised and developed two highly sensitive peptide binding affinity assays to determine whether broad epitope sampling also extends to HLA-E. We found extensive overlap in the binding repertoires of the human and rhesus orthologs, and the majority of SIV-derived Mamu-E-restricted epitopes tested also bound HLA-E. We also demonstrated binding for a number of HIV and Mtb derived epitopes identified from rhesus macaques vaccinated with rhCMV-HIV Gag immunogens or from active tuberculosis infection in humans, respectively. As previous crystal structures of HLA-E were exclusively solved in complex with canonical MHC-I signal peptides, we next investigated the structural basis underlying pathogen-derived peptide binding. We obtained a panel of crystal structures of HLA-E in complex with different epitopes including a number from Mtb in addition to the HIV counterpart to the vaccine-identified SIV Gag ‘supertope’69. Strikingly, despite the presence of canonical primary anchor residues, the HIV pep- tide adopted an unusual motif within the peptide binding groove, generating a solvent exposure profile that deviated from canonical signal peptide motifs, with potential implications for T cell receptor recognition and immunogenicity. Further, structural and mutagenesis analyses illustrated a greater tolerance for hydrophobic and polar residues in the primary pockets than previously anticipated. In conclusion, our combined structural and peptide binding data suggest a previously underestimated breadth of the HLA-E peptidome and support the potential for broadly targeted HLA-E-restricted HIV vaccine development.

Affinity Peptide-based Electrochemical Biosensor for the Highly Sensitive Detection of Bovine Rotavirus

Bovine diarrhea is a major concern in the global bovine industry because it can cause significant financial damage. Of the many potential infectious agents that can lead to bovine diarrhea, bovine rotavirus (BRV) is a particular problem due to its high transmissibility and infectivity. Therefore, it is important to prevent the proliferation of BRV using an early detection system. This study developed an affinity peptide-based electrochemical method for use as a rapid detection system for BRV. A BRV-specific peptide was identified via the phage display technique and chemically synthesized. The synthetic peptide was immobilized on a gold electrode through thiol-gold interactions. The performance of the BRV specific binding peptides was evaluated using square wave voltammetry. The developed detection system exhibited a low detection limit (5 copies/mL) and limit of quantitation (2.14 × 102 copies/mL), indicating that it is a promising sensor platform for the monitoring of BRV.

Abstract 1992: Evaluating in vivo efficacy of peptide vaccines in humanized B-HLA-A2.1 mice

Abstract HLA-A molecules are αβ heterodimers that belong to the human leukocyte antigen (HLA) class I major histocompatibility complex (MHC) in humans. Class I molecules play a central role in the immune system by presenting intracellular peptides derived from the endoplasmic reticulum lumen, enabling recognition by cytotoxic T cells. The heavy α chain structure of HLA-A, encoded by the HLA-A gene, contains the leader peptide, the α1 and α2 domains that both bind the peptide, and the α3 domain that binds CD8 molecules, the transmembrane region, and the cytoplasmic tail. Polymorphisms within α1 and α2 domains of the heavy chain are responsible for the peptide binding specificity of each class I molecule, while the light β2-microglobulin (B2M) chain encoded by the B2M gene, is not known to be polymorphic. Among more than 6,000 HLA-A alleles, HLA-A2.1 is the most commonly expressed, and its peptide-binding motif is well understood. We developed MHC I humanized mice (B-HLA-A2.1 mice) expressing human HLA molecules to establish an in vivo experimental model for evaluating human HLA restricted T cell-mediated vaccine efficacy in oncology applications. Our strategy was to replace the B2m gene of the mouse with the sequences encompassing the human B2M CDS and HLA-A2.1 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the mouse H-2Db gene encoding the α3, transmembrane and cytoplasmic domains. Using flow cytometry analysis, human B2M and HLA-A2.1 were detected in splenocytes of homozygous B-HLA-A2.1 mice but not in wild type mice. Mouse B2M was detectable in splenocytes of wild type mice but not in homozygous B-HLA-A2.1 mice. The percentage of T cells, B cells, CD4+ T cells, and CD8+ T cells within the spleens of homozygous B-HLA-A2.1 mice were similar to those in the wild type mice, demonstrating that introduction of human B2M and HLA-A2.1 does not change the overall development, differentiation, or distribution of these cell types. Next, in vivo efficacy evaluation of two peptide vaccines (V1 and V2) under development was performed with the humanized mice. Mice were inoculated with vaccines by intramuscular injections into the inner thigh (L and R) muscles and sacrificed three weeks after the last immunization. The splenocytes were extracted, stimulated with individual peptides, and measured for IFN-γ secretion. HLA-A2.1-restricted CTL responses of the vaccines were successfully detected in B-HLA-A2.1 mice, and no significant difference in body weight was observed among groups. Our results show that B-HLA-2.1 mice provide a powerful preclinical mouse model for in vivo evaluation of peptide vaccines. Citation Format: Jiawei Yao, Chengzhang Shang, Shivam Madaan, Yanhui Nie, Yi Yang. Evaluating in vivo efficacy of peptide vaccines in humanized B-HLA-A2.1 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 1992.