Minimal Epitope Research Articles

Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridiumthermocellum.

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridiumthermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation-selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. DATABASE: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

ER‐aminopeptidase 1 determines the processing and presentation of an immunotherapy‐relevant melanoma epitope

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

Characterization of Antigenic MHC-Class-I-Restricted T Cell Epitopes in the Glycoprotein of Ebolavirus

SummaryEbolavirus causes highly lethal hemorrhagic fever in humans. The envelope-displayed viral glycoprotein (GP) is the primary target of humoral immunity induced by natural exposure and vaccination. No T cell epitopes in the GP have been characterized in humans. A phase I clinical trial of a heterologous prime-boost vaccination regime with viral vectors encoding filovirus antigens elicits humoral and T cell responses in vaccinees. The most frequently recognized peptide pools are deconvoluted to identify the minimal epitopes recognized by antigen-specific T cells. We characterize nine immunogenic epitopes on the Ebolavirus GP. Histocompatibility leukocyte antigen (HLA) typing with in silico epitope analysis determines the likely MHC class I restriction elements. Thirteen HLA-A and -B alleles are predicted to present the identified CD8+ T cell epitopes, suggesting promiscuous recognition and a broad immune response. Delivery of the Ebolavirus GP antigen by using a heterologous prime-boost approach is immunogenic in genetically diverse human populations, with responses against multiple epitopes.

Evaluation of Immune Responses to Group B Streptococcus Type III Oligosaccharides Containing a Minimal Protective Epitope.

Recent structural studies demonstrated that the epitope recognized by a monoclonal antibody representative of the protective response against the type III group B Streptococcus polysaccharide was comprised within 2 of the repeating units that constitute the full-length native structure. In the current study, we took advantage of this discovery to design a novel vaccine based on multivalent presentation of the identified minimal epitope on a carrier protein. We show that highly glycosylated short oligosaccharide conjugates elicit functional immune responses comparable to those of the full-length native polysaccharide. The obtained results pave the way to the design of well-defined glycoconjugate vaccines based on short synthetic oligosaccharides.

Identification of antigenic epitopes in the haemagglutinin protein of H7 avian influenza virus

ABSTRACT The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The haemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs, 2F8, 3F6, 5C11, 5E2 and 5C12) against the HA protein of H7 virus were produced and characterized. Epitope mapping indicated that 103RESGSS107 was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and mAbs 5E2/5C12 recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and none of the five mAbs cross-reacted with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope 103RESGSS107 was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations prevented the mutated epitope from being recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that 103RESGSS107 may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus. RESEARCH HIGHLIGHTS Five mAbs against HA protein of H7 AIV were generated and characterized. Two novel epitopes 103RESGSS107 and 103-145aa were identified. The epitope 103RESGSS107 differs between Eurasian and North-American lineages. The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.

Characterization of Monoclonal Antibodies against σA Protein and Cross-Reactive Epitope Identification and Application for Detection of Duck and Chicken Reovirus Infections.

Although σA is an important major core protein of duck reovirus (DRV), the B-cell epitopes of this protein remain unknown to reseacrhers. Six monoclonal antibodies (MAbs) (1A7, 3F4, 5D2, 4E2, 3C7, and 2B7) were developed by using prokaryotic-expressed recombinant His-σA protein. Five of six MAbs (1A7, 3F4, 4E2, 3C7, and 2B7) reacted with His-σA protein in a conformation-independent manner, while 5D2 reacted with σA in a conformation-dependent manner. Immunofluorescence assays showed that the MAbs could specifically bind to DRV infected BHK-21 cells. The MAbs were delineated as three groups by a competitive binding assay. By using 12-mer peptide phage display and mutagenesis, MAb 4E2 was identified to recognize minimal epitope 56EAPYPG61 and MAb 1A7 recognize 341WVV/MAGLI/V347, residues 341V/M and 347I/V are replaceable. Dot blotting and sequence analysis confirmed that EAPYPG and WVV/MAGLI/V are cross-reactive epitopes in both DRV and avian reovirus (ARV). An enzyme-linked immunosorbent assay (ELISA) based on two expressed EAPYPG and WVVAGLI as antigen demonstrated its diagnostic potential by specific reacting with serum samples from DRV- or ARV-infected birds. Based on these observations, an epitope-based ELISA could be potentially used for DRV or ARV surveillance. These findings provide insights into the organization of epitopes on σA protein that might be valuable for the development of epitope-based serological diagnostic tests for DRV and ARV infection.

Identification of three linear B cell epitopes against non-structural protein 3ABC of FMDV using monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "92EYIEKA97" "23EGPYAGPLE31" and "209EPHH212", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "92EYIEKA97" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "23EGPYAGPLE31" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "23EGPYAGPLE31" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.

Abstract 4059: Identification of pre-existing neoantigen-specific T cells in patients receiving checkpoint inhibitor therapy using a deep learning antigen prediction model

Abstract Immune Checkpoint Blockade (ICB) has revolutionized the treatment of patients with cancer but provides durable clinical benefit to only a minority of patients. Neoantigens, antigens derived from tumor mutations, are emerging as key targets of T cells in patients with clinical responses to ICB whose tumors exhibit high mutational burden. T cells specific to some neoantigens appear to be naturally generated in these patients and can be further expanded upon ICB. Accurate neoantigen identification may fuel therapeutic advances by driving potent anti-neoantigen immune responses through therapeutic immunization or adoptive cell therapy. However, identification of neoantigens and neoantigen-reactive T cells remains a significant challenge. Current methods are time consuming and labor intensive and require non-routine clinical specimen collections. Here, we apply deep learning to a large HLA peptide and genomic dataset from NSCLC patients to create a computational model of minimal epitope presentation for neoantigen prediction. We demonstrate that our model allows rapid identification of neoantigens and neoantigen reactive T cells using routine clinical biopsies and blood specimens. This methodology does not require HLA-specific reagents and enables identification of T cell responses for every patient. To identify neoantigen-specific T cells, selected peptides, prioritized by our prediction model, were synthesized as minimal epitopes and added to short-term in vitro stimulation (IVS) cultures to expand neoantigen-reactive T cells. The presence of antigen-specific T cells was assessed two weeks later using IFN-gamma (IFN-g) ELISpot against pools of the prioritized neoantigens. Patient-specific peptide pools and IFN-g ELISpot enabled identification of neoantigen-reactive T cells in 5/9 (56%) patients on ICB. Deconvolution yielded an average of 2 recognized neoantigens per patient, with increased responses over time observed in one patient where multiple time points were available. Neoantigen responses were confirmed in repeat IVS cultures where additional samples were available. Importantly, expansion of HLA-matched healthy controls in the presence of patient peptide pools did not result in any responses by IFN-g ELISpot. In addition, antigen-reactive T cells were sorted for single cell sequencing on the 10x platform using CD137 as an HLA-independent T-cell activation marker. In summary, pre-existing neoantigen specific T cells and TCR clones can be rapidly identified utilizing routine clinical specimens, through (i) a powerful neoantigen prediction model, (ii) short-term in vitro T-cell expansion followed by IFN-gamma ELISpot, and (iii) HLA-independent selection and single cell sequencing. This methodology may allow rapid design of neoantigen-targeting vaccines or selection of TCRs for cell therapeutic approaches. Citation Format: Christine D. Palmer, Brendan Bulik, Aaron Yang, Meghan Hart, Matthew Davis, Joshua Francis, Fujiko Duke, Rita Zhou, Jennifer Busby, Tyler Murphy, Andrew Clark, Lauren Young, Mike Zhong, Kamilah Caldwell, Jesse Abhyankar, Alison Cook, Tommy Boucher, Raphaël Rousseau, Cynthia Voong, Naiyer Rizvi, Mark Frattini, Roman Yelensky, Karin Jooss, Mojca Skoberne. Identification of pre-existing neoantigen-specific T cells in patients receiving checkpoint inhibitor therapy using a deep learning antigen prediction model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4059.

Abstract 1204: Design and development of anti-linker antibodies for the detection and characterization of CAR T cells

Abstract Introduction: Chimeric antigen receptor (CAR) T cell therapy is a transformative treatment modality in B cell malignancies. As next generation CARs are developed to improve durable response rates and expand into other indications of unmet need, the proper tools to identify, characterize, and modulate CAR activity will play a pivotal role in the optimization of this therapeutic option. This study developed and evaluated one approach, that of using monoclonal antibodies (mAbs) against common elements utilized in approved and investigational CARs. Methods: The mAbs KIP-1 and KIP-4 were raised in rabbits against the linkers of 2 single-chain variable fragments, the Whitlow linker (Whitlow, et al. Protein Eng. 1993) and the G4S linker (Huston JS, et al. PNAS. 1988), respectively, to generate universal detection reagents without adding an exogenous peptide recognition motif. Specificity and sensitivity were assessed by flow cytometric analysis of CAR T cells vs nontransduced T cells, immunohistochemistry (IHC) staining of embedded cell pellets with or without a CAR carrying the relevant linker, and epitope mapping by ELISA. Activation was assessed in a competitive stimulation assay in which nontransduced T cells and CAR T cells were mixed at defined ratios and stimulated broadly with OKT3 or specifically with KIP-1 or KIP-4. Results: Flow cytometric analysis demonstrated that CAR T cells with the Whitlow linker could be detected by phycoerythrin-conjugated KIP-1 at ≤ 10 ng per million cells with no detectable staining on nontransduced T cells above background levels. To further demonstrate specificity, IHC data revealed that KIP-1 bound to T cell pellets expressing CARs containing the Whitlow linker, but not the G4S linker. Epitope mapping revealed that the minimal KIP-1 epitope was contained within the amino acid sequence SGKPGSGE. Furthermore, KIP-1 was able to effectively identify CAR T cells in patient samples by both flow cytometry and IHC. Having established that KIP-1 was specific and sensitive, activation assays were performed to determine its CAR T cell-specific activating capacity. KIP-1 specifically activated CAR T cells, but not nontransduced T cells, as demonstrated by CAR T cell expansion and upregulation of cell surface activation markers, including CD69 and 4-1BB. KIP-4 was also assessed for specificity, sensitivity, and activation capacity with similar results. Conclusions: Taking advantage of the linear epitopes within the commonly-used Whitlow and G4S linkers, KIP-1 and KIP-4, respectively, can be used in a wide variety of phenotypic and functional assays with CARs of various specificities that share these linkers. These tools provide the means to realize the full potential of investigational CAR T cell products by supporting research, clinical, and manufacturing efforts. Citation Format: Stuart A. Sievers, Keith A. Kelley, Stephanie H. Astrow, Adrian Bot, Jed J. Wiltzius. Design and development of anti-linker antibodies for the detection and characterization of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1204.

Identification of three linear B cell epitopes using monoclonal antibodies against bovine enterovirus VP2 protein.

Bovine enterovirus (BEV) VP2 protein is a structural protein that plays an important role in inducing protective immunity in the host. The function of VP2 has been characterized, but there is little information on its B cell epitopes. Three monoclonal antibodies (mAbs) directed against BEV VP2 were generated and characterized from mice immunized with the recombinant VP2 protein. Three minimal linear epitopes 152FQEAFWLEDG161, 168LIYPHQ173, and 46DATSVD51 reactive to the three mAbs were identified using western blotting analysis. Three-dimensional model of the BEV-E virion and the VP2 monomer showed that epitope 152FQEAFWLEDG161 is exposed on surface of the virion and epitopes 46DATSVD51 and 168LIYPHQ173 are located inside the virion. Alignment of the amino acid sequences corresponding to the regions containing the three minimal linear epitopes in the VP2 proteins and their cross-reactivity with the three mAbs showed that epitope 168LIYPHQ173 is completely conserved in all BEV strains. Epitope 46DATSVD51 is highly conserved among BEV-E strains and partly conserved among BEV-F strains. However, epitope 152FQEAFWLEDG161 is not conserved among BEV-F strains. Using the mAbs of 3H4 and 1E10, we found that VP2 localized in the cytoplasm during viral replication and could be used to monitor the viral antigen in infected tissues using immunohistochemistry. A preliminary 3H4-epitope-based indirect ELISA allowed us to detect anti-BEV-strain-HY12 antibodies in mice. This study indicates that the three mAbs could be useful tools for investigating the structure and function of the viral VP2 protein and the development of serological diagnostic techniques for BEV infection.

Antimicrobial Peptides and Vaccine Development to Control Multi-drug Resistant Bacteria.

Antimicrobial resistance (AMR) reported to increase globally at alarming levels in the recent past. A number of potential alternative solutions discussed and implemented to control AMR in bacterial pathogens. Stringent control over the clinical application of antibiotics for a reduction in uses is a special consideration along with alternative solutions to fight against AMR. Although alternatives to conventional antibiotics like antimicrobial peptides (AMP) might warrant serious consideration to fight against AMR, there is a thriving recognition for vaccines in encountering the problem of AMR. Vaccines can reduce the prevalence of AMR by reducing the number of specific pathogens, which result in cutting down the antimicrobial need and uses. However, conventional vaccines produced using live or attenuated microorganisms while the presence of immunologically redundant biological components or impurities might cause major side effects and health related problems. Here we discussed AMPs based vaccination strategies as an emerging concept to overcome the disadvantages of traditional vaccines while boosting the AMPs to control multidrug resistant bacteria or AMR. Nevertheless, the poor immune response is a major challenge in the case of peptide vaccines as minimal antigenic epitopes used for immunization in peptide vaccines.

Identification of Two Distinct Linear B Cell Epitopes of the Matrix Protein of the Newcastle Disease Virus Vaccine Strain LaSota.

Matrix (M) protein of Newcastle disease virus (NDV) is an abundant protein that can induce a robust humoral immune response. However, its antigenic epitopes remain unknown. In this study, we used a pepscan approach to map linear B cell immunodominant epitopes (IDEs) of M protein with NDV-specific chicken antisera. The six epitopes with the highest reactivity by peptide scanning were obtained as IDE candidates. Among them, aa71-85 and aa349-363 were identified by immunological assays with NDV-specific or IDE-specific antisera. The minimal antigenic epitopes of the two IDEs were further characterized as 77MIDDKP82 and 354HTLAKYNPFK363. Moreover, an amino acid sequence alignment and immunoblot analysis revealed the conservation of the two IDEs in the M protein of strains of different genotypes. These two IDEs of M protein could be genetically eliminated as negative markers in recombinant NDV for serologically differential diagnosis in the development of marker vaccines.

Performance evaluation of 3 serodiagnostic peptide epitopes and the derived multi-epitope peptide OvNMP-48 for detection of Onchocerca volvulus infection

Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite’s proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA’s and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox.

Substitution of alpha aminobutyric acid for cysteine at class I MHC anchor positions can enhance recognition of cancer epitopes

Abstract We curated a list of 11 known and validated class I-restricted cancer neoepitopes and tumor antigens that contain cysteine, six of which were restricted by HLA-A*02:01. We synthesized native (Cys-peptide) and alpha aminobutyric acid substituted (AABA-peptide) minimal epitopes and carried out co-culture assays in which responses of relevant T cell populations to titered doses of the native and substituted peptides were compared. The results indicated that T cells reactive with 6 of the 6 HLA-A*02:01 epitopes recognized peptides containing AABA substitutions at putative MHC anchor positions-at significantly lower concentrations than their Cys-pep counterpart. For 4 of the 5 non-HLA-A*02:01 restricted epitopes, T cells recognized AABA-peptides at lower concentrations than their native counterparts, while for the remaining epitope, the Cys-peptide was recognized at lower concentration than its AABA-peptide counterpart. Three of the HLA-A*02:01 restricted peptides, PHLPP1, HIVEP2, and gp100 contained two cysteine residues, one a putative MHC anchor and the second a putative T cell contact position. Substitution of ABBA for the putative T cell contact residue in these peptides appeared to reduce their ability to stimulate relevant cells. Overall, for 9 of the 11 (82%) cysteine-containing peptides evaluated, substitution of AABA for cysteine resulted in 10-fold or greater decrease in the minimum concentration of peptide recognized by T cells. We conclude that substitution of cysteine with AABA at MHC anchor positions can significantly improve the sensitivity of neoantigen screening assays by offsetting impairment that is likely to be due to cysteine oxidation, an effect that was observed for five HLA class I restriction elements.

Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma

Abstract The comparative resistance of some cancers including head and neck squamous cell carcinoma (HNSCC) to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens (NeoAg). SCC VII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity in syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation containing overlapping minimal epitopes during in vitro recall assays. NeoAgs eliciting CD4+ T cell responses alone were not protective in vivo. However, the more immunogenic Cltc NeoAg prophylactically protected hosts from tumor challenge and caused regression of established tumors as an immunotherapy. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection by boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against malaria

Abstract Liver resident-memory CD8+ T cells (TRM cells) provide important protection against liver-stage Plasmodium infections. Here, we show that a glycolipid-peptide conjugate vaccine designed to be cleaved in vivo to release the NKT cell agonist a-galactosylceramide (a-GalCer), together with a CD8+ T cell malarial epitope, efficiently induced intrahepatic malaria-specific T cells expressing canonical markers of liver TRM (CD69, CXCR6 and CD101). While sterile protection was achieved in a proportion of mice using a single vaccine dose, a combined prime-boost regimen induced higher numbers of liver TRM cells and more robust protection. Improved liver TRM cell generation and sterile immunity using a single dose of the vaccine were obtained in all mice by extending the C-and N-terminal residues of the minimal epitope, and by modifying the linker to enhance a-GalCer release. Our findings describe an ideal synthetic vaccine platform to generate large numbers of liver TRM cells that is potentially valuable for controlling a variety of hepatotropic infections.

Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus.

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

Identification of a conserved linear epitope using monoclonal antibody against non-structural protein 3A of foot-and-mouth disease virus with potential for differentiation between infected and vaccinated animals

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.

GlcNAc6ST3 is a keratan sulfate sulfotransferase for the protein-tyrosine phosphatase PTPRZ in the adult brain

Keratan sulfate (KS) is a carbohydrate side chain covalently attached to extracellular proteoglycans. KS is composed of disaccharide units of 6-sulfated N-acetylglucosamine (GlcNAc) and galactose. We have previously shown that GlcNAc-6-O-sulfotransferase (GlcNAc6ST) 1 encoded by Chst2 is an enzyme necessary for the synthesis of GlcNAc-6-sulfated KS chains that are required for neuronal plasticity in the visual cortex of the mouse brain during the critical period, but not in adulthood. Here, we show that GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody, of which the minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S), distributes diffusely in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net (PNN)-positive neurons in the adult visual cortex. Surprisingly, GlcNAc6ST3, which was discovered as an intestinal GlcNAc6ST encoded by Chst5, is a major brain KS sulfotransferase expressed in oligodendrocytes in adulthood. Moreover, we identified an isoform of the protein-tyrosine phosphatase PTPRZ as a R-10G-reactive KS proteoglycan. These results indicate that GlcNAc6ST3 may play a role in synthesis of a component of PNN in the adult brain, and that the KS-modified isoform of PTPRZ encoded by Ptprz1 could be an extracellular molecule associated with PNNs.

Mapping a highly conserved linear neutralizing epitope on gD glycoprotein of bovine herpesvirus type I using a monoclonal antibody.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae, causes a variety of diseases, which result in significant economic losses worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an important role in viral entry into the permissive cells, and protective immune response. The fine mapping epitope on the gD will contribute to the understanding of viral pathogenesis and development of alternative vaccines against various diseases associated with BoHV-1. We previously reported the preparation of a monoclonal antibody (MAb) 2B6, which was raised by a truncated recombinant gD protein, demonstrating a neutralizing activity against BoHV-1 infection in Madin–Darby bovine kidney cells. This study described the identification of a linear B-cell epitope on gD using MAb 2B6. A series of partially overlapping gD proteins with glutathione S-transferase tag were generated to define the epitope recognized by MAb 2B6. The amino acid (aa) sequence 323GEPKPGPSPDADRPE337 was recognized by MAb 2B6 using Western blot with the variedly truncated recombinant proteins. Importantly, this epitope was highly conserved among the typical members of BoHV-1, indicating that the epitope may be utilized in diagnosis of diseases due to BoHV-1 infection. Furthermore, the minimal linear epitope sequence 323GEPKPGP329 on gD recognized by MAb 2B6 was confirmed using single-aa residue deletion mutation in carboxyl terminal. This finding not only contributes to our understanding of gD of BoHV-1 virion but also shows a potential for the development of vaccine candidates and diagnostic techniques.