Immunogenic Epitopes Research Articles

Novel RNA-based Cancer Vaccine for AML immunotherapy

Abstract Acute Myeloid Leukemia is a rapidly progressing malignancy relying primarily on chemotherapy as the frontline treatment option. Success of personalized neoantigen vaccines in melanoma patients has garnered great attention in the field of immunotherapy. We hypothesize that personalized cancer vaccine, in the form of mRNA, can be a viable treatment strategy for AML patients. Next generation sequencing was performed on both C57BL6Jinv mouse’s and C1498 cell line’s nucleic acid materials. Three different neoepitope prediction pipelines were utilized to identify potential immunogenic neoepitopes. Candidate neoepitopes within intron polyadenlyation sites (iPAS), retained intron (RI) sites and somatic mutation (SM) sites were found. A total of 285 SM-derived neoepitopes were identified. Top 38 highly expressed genes were selected for further analysis. Using Sanger sequencing, we confirmed 37 out of the 38 mutations. A total of 5 iPAS-derived neoepitopes were identified. A total of 26 RI-derived neoepitopes were identified in our first RNAseq analysis and a separate 476 RI-derived neoepitopes were identified in our second RNAseq analysis. With these three categories of candidates, we narrowed down our selection and did a preliminary screen for immunogenic epitopes using a total of 71 synthetic peptides in an ELISpot assay. Conventional ELISpot assay’s results were however non-reproducible, thereby propelling us to adopt Culture ELISpot assay. Subsequently, 10 neoepitope candidates were short-listed for further in-vivo evaluation. mRNA vaccine was synthesized and evaluated in a murine AML model. T cell responses against the 10 neoepitopes were analyzed to provide pre-clinical evidence for patient studies. Supported by NMRC CS-IRG grant

Novel gene immunotherapy prevents Aquaporin-4 (AQP4) mediated neuroinflammation and demyelination in a mouse model of multiple sclerosis

Abstract Neuromyelitis optica (NMO) is an auto-inflammatory demyelinating disease that typically affects optic nerves and spinal cord that is characterized by the presence of serum aquaporin-4 immunoglobulin G antibodies (AQP4-IgG). NMO accounts for >45% of the demyelinating disease in Asians and warrants the development of a suitable therapy other than generalized immunosuppressants. It is thought that autoimmune activated AQP4-specific T cells disrupt the BBB and allow increased entry of AQP4-IgG and other immune effectors into tissues containing astrocytes expressing AQP4 in their membranes. Recently we have established an adeno-associated viral (AAV) gene immunotherapy that effectively prevents and reverses experimental autoimmune encephalomyelitis (EAE) via generation of suppressive antigen-specific regulatory T cells (Tregs). In this report, using a similar antigen-specific approach, we have demonstrated that AAV.AQP4 gene immunotherapy prevented the development of AQP4-mediated neuroinflammation and clinical neurological disability in almost 100% of C57BL/6J mice when AAV.AQP4 vector was administered to mice 2 weeks prior to immunization with an immunogenic epitope of AQP4. Moreover, in contrast to AAV.AQP4 treated mice which remained unremarkable, histological analysis of the spinal cord sections from untreated vehicle only mice showed multiple areas of significant focal inflammation within the spinal cord. Based on our clinical and pathological data, we’ve successfully demonstrated that our novel AAV.AQP4 gene immunotherapy can indeed suppress the induction of AQP4 mediated autoimmune disease. Further evaluation will determine if it can also treat preexisting disease.

In Silico Analysis Prediction of B-Cell Epitope as a Vaccine Candidate for SARS-CoV-2 B.1.617.2 (Delta) Variant

Background: The COVID-19 pandemic by SARS-CoV-2 has caused many losses. One way to prevent the spread of this virus is to get vaccinated. However, the latest SARS-CoV-2 variants, including variant B.1.617.2 (Delta) are doubtful to be inhibited by existing vaccines because of mutations. Therefore, we need a new vaccine candidate that is effective against this SARS-CoV-2 variant. Through an immunoinformatics approach with various software and analysis websites, vaccine candidates can be predicted in a short time.Objective: Identity, analyze, obtain, and confirm the selected B-cell epitope sequence that can be used as a vaccine candidate for the SARS-CoV-2 B.1.617.2 (Delta) variant.Methods: This research was conducted by isolating the amino acid peptide sequence in the SARS-CoV-2 B.1.617.2 (Delta) variant protein spike from the Protein Data Bank which is suspected to be an immunogenic epitope and can be used as a vaccine candidate. A Series of tests were carried out such as antigenicity, toxicity, allergenicity, and BLAST® protein to ensure that this vaccine candidate is safe for later application into the human body. The next stage is a conservation analysis to see its potential by comparing it with the SARS-CoV-2 Delta (B.1.617.2) variant spike protein sequence in Indonesia. The study ended by mapping amino acid peptides to the SARS-CoV-2 Delta (B.1.617.2) variant spike protein using the Biovia Discovery Studio Visualizer v21.1.0.20298 2020 software to ensure that the selected sequences were epitope.Results: From the five amino acid peptides that have been isolated, the FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT epitope sequence has good results than the others. It is probable an antigen, non-toxic, non-allergen, and non-homolog to the human body protein.Conclusion: Based on this in silico study, it was found that the FTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT epitope sequence was the best to be used as a vaccine candidate of SARS-CoV-2 B.1.617.2 (Delta) variant.Keywords: SARS-CoV-2 B.1.617.2 (Delta) variant, B-cell epitope, vaccine, in silico, immunoinformatics.

Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation.

Neutrophil extracellular traps (NETs) are web-like chromatin structures composed by dsDNA and histones, decorated with antimicrobial proteins. Their interaction with dendritic cells (DCs) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and the induction of a CD8+ T-cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow (BM) chimeras in a competitive BM transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an antileukemia CD8 T-cell memory response.

A multi-epitope vaccine candidate developed from unique immunogenic epitopes against Cryptosporidium hominis by utilizing an immunoinformatics-driven approach

An immunoinformatics-based strategy is being investigated to identify prospective multi-subunit vaccine candidates against Cryptosporidium hominis (C. hominis). We used a systematic technique based on protein structure to create a competent multi-subunit vaccine candidate against C. hominis, with the likelihood of antigenicity, allergenicity, and transmembrane helices as the screening criteria. Using the suitable linkers, the best-screened epitopes such as B-cell epitopes (BCL), Helper T-lymphocytes (HTL), and cytotoxic T-lymphocytes (CTL) were linked together to intensify and develop the presentation and processing of the antigenic molecules. The greatest 3 D model of the component protein was created with the help of modeling software called Raptorax. The validation of the modeled protein was accomplished via the use of PROCHECK. Furthermore, using the ClusPro web server, the projected modeled structure was docked with known receptor TLR-4 to determine their interactions. A molecular dynamics (MD) simulation was used to investigate the stability of the multi-subunit vaccine bound with TLR-4 based on the docking score. Aside from that, the codon optimization and in silico expression demonstrate the possibility of high expression and simple purification of the vaccine product resulting from codon optimization. The overall findings indicated that the multi-subunit vaccine might be a viable vaccination candidate against C. hominis. Communicated by Ramaswamy H. Sarma

Determination of immunogenic epitopes in major house dust mite allergen, Der p 2, via nanoallergens

BackgroundDespite the high prevalence of allergic asthma, currently, avoidance of the responsible allergens, which is nearly impossible for allergens such as house dust mite (HDM), remains among the most effective treatment. Consequently, determination of the immunogenic epitopes of allergens will aid in developing a better understanding of the condition for diagnostic and therapeutic purposes. Current methods of epitope identification, however, only evaluate immunoglobulin E-epitope binding interactions, which is not directly related to epitope immunogenicity. ObjectiveTo determine and rank the immunogenicity of the epitopes of major HDM allergen, Der p 2. MethodsWe performed degranulation assays with RBL-SX38 cells primed using patient plasma and challenged with nanoallergens which multivalently displayed epitopes to study the relative immunogenicity of various epitopes of Der p 2. Nanoallergens were used to evaluate epitopes individually or in combination. ResultsWhen evaluated using 3 patient samples, 3 epitopes in 2 distal regions of Der p 2 were identified as highly immunogenic when presented in combination, whereas no individual epitope triggered relevant degranulation. One of the epitopes (69-DPNACHYMKCPLVKGQQY-86) was identified to be cooperatively immunogenic when combined with other epitopes. ConclusionOur study highlights the importance of conformational epitopes in HDM-related allergies. This study also provides further evidence of the versatility of nanoallergens and their value for functional characterization of allergy epitopes, by ranking the Der p 2 epitopes according to immunogenicity. We believe that nanoallergens, by aiding in identification and understanding of immunogenic epitopes, will provide a better understanding of the manifestation of the allergic condition and potentially aid in developing new treatments.

Determination of B and T Cell Epitopes in Neospora caninum Immune Mapped Protein-1 (IMP-1): Implications in Vaccine Design against Neosporosis.

Prevention of neosporosis is advantageous for cattle health and productivity. Previously, several vaccine candidates were nominated for vaccination against Neospora caninum. This study was premised on in silico evaluation of N. caninum IMP-1 in order to determine its physicochemical features and immunogenic epitopes. We employed a wide array of network-based tools for the prediction of antigenicity, allergenicity, solubility, posttranslational modification (PTM) sites, physicochemical properties, transmembrane domains and signal peptide, secondary and tertiary structures, and intrinsically disordered regions. Also, prediction and screening of potential continuous B cell peptides and those epitopes having stringent affinity to couple with mouse major histocompatibility complex (MHC) and cytotoxic T lymphocyte (CTL) receptors were accomplished. The protein had 393 residues with a molecular weight of 42.71 kDa, representing aliphatic index of 85.83 (thermotolerant) and GRAVY score of -0.447 (hydrophilic). There were 47 PTM sites without a signal peptide in the sequence. Secondary structure comprised mostly of extended strand and helices, followed by coils. The Ramachandran plot of the refined model showed 90.1%, 9.9%, 0.0%, and 0.0% residues in the favored, additional allowed, generously allowed, and disallowed regions, correspondingly. Additionally, various potential B cell (linear and conformational), CTL, and MHC binding epitopes were predicted for N. caninum IMP-1. The findings of the present study could be further directed for next-generation vaccine design against neosporosis.

Antibody Responses Against Plasmodium falciparum MSP3 Protein During Natural Malaria Infection in Individuals Living in Malaria-Endemic Regions of India.

The humoral immune responses to blood-stage malaria proteins are requisite for the inhibition of parasite invasion. Plasmodium falciparum merozoite surface protein 3 (MSP3) is a secretory, expressed abundantly, merozoite surface protein that is important for the parasite invasion process. It has been shown to induce antibody responses during natural infections and is, therefore, considered to be the potential vaccine candidates against Plasmodium. Elucidating the immunogenicity and prevalence of anti-parasite antibodies is important in identifying potential targets as candidates for malarial diagnosis and anti‐malarial vaccine. The present study concerns the presence of antibodies against the MSP3 proteins of human malaria parasite- P. falciparum in infected individuals from endemic regions of India. Seventy-one anonymized P. falciparum infected serum samples were procured from the malaria fever clinic of ICMR-National Institute of Malaria Research (NIMR), New Delhi to detect the presence of antibodies against MSP3 protein by ELISA. The IgM antibody response against recombinant MSP3 was detected at significantly higher levels during acute malaria. The protein was found to be immunogenic and did not demonstrate any cross-reactivity with the serum of uninfected individuals or individuals infected with other Plasmodium species. The protein has hydrophilic regions in its N- and C-terminus which may contain immunogenic linear and conformational B-cell epitopes. The results from this study suggest that the MSP3 is immunogenic and likely a potential candidate for antibody-based diagnosis or vaccine development against the blood-stage of P. falciparum.Graphical abstract

Neospora caninum SRS2 Protein: Essential Vaccination Targets and Biochemical Features for Next-Generation Vaccine Design.

Vaccination is a standout preventive measure to combat neosporosis among cattle herds. The present in silico study was done to evaluate the physicochemical properties and potent immunogenic epitopes of N. caninum SRS2 protein as a possible vaccine candidate. Web-based tools were used to predict physicochemical properties, antigenicity, allergenicity, solubility, posttranslational modification (PTM) sites, transmembrane domains and signal peptide, and secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind mouse major histocompatibility complex (MHC) and cytotoxic T lymphocyte (CTL). The protein had 401 residues with a molecular weight of 42 kDa, representing aliphatic index of 69.35 (thermotolerant) and GRAVY score of -0.294 (hydrophilic). There were 53 PTM sites without a signal peptide in the sequence. Secondary structure comprised mostly by extended strand, followed by helices and coils. The Ramachandran plot of the refined model showed 90.2%, 8.8%, 0.5%, and 0.5% residues in the favored, additional allowed, generously allowed, and disallowed regions, correspondingly. Additionally, various potential B-cell (linear and conformational), CTL, and MHC-binding epitopes were predicted for N. caninum SRS2. These epitopes could be further utilized in the multiepitope vaccine constructs directed against neosporosis.

Potential Immunogenic Activity of Computationally Designed mRNA- and Peptide-Based Prophylactic Vaccines against MERS, SARS-CoV, and SARS-CoV-2: A Reverse Vaccinology Approach.

The continued emergence of human coronaviruses (hCoVs) in the last few decades has posed an alarming situation and requires advanced cross-protective strategies against these pandemic viruses. Among these, Middle East Respiratory Syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) have been highly associated with lethality in humans. Despite the challenges posed by these viruses, it is imperative to develop effective antiviral therapeutics and vaccines for these human-infecting viruses. The proteomic similarity between the receptor-binding domains (RBDs) among the three viral species offers a potential target for advanced cross-protective vaccine designs. In this study, putative immunogenic epitopes including Cytotoxic T Lymphocytes (CTLs), Helper T Lymphocytes (HTLs), and Beta-cells (B-cells) were predicted for each RBD-containing region of the three highly pathogenic hCoVs. This was followed by the structural organization of peptide- and mRNA-based prophylactic vaccine designs. The validated 3D structures of these epitope-based vaccine designs were subjected to molecular docking with human TLR4. Furthermore, the CTL and HTL epitopes were processed for binding with respective human Lymphocytes Antigens (HLAs). In silico cloning designs were obtained for the prophylactic vaccine designs and may be useful in further experimental designs. Additionally, the epitope-based vaccine designs were evaluated for immunogenic activity through immune simulation. Further studies may clarify the safety and efficacy of these prophylactic vaccine designs through experimental testing against these human-pathogenic coronaviruses.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86–100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Injectable Slow-Release Hydrogel Formulation of a Plant Virus-Based COVID-19 Vaccine Candidate.

Cowpea mosaic virus (CPMV) is a potent immunogenic adjuvant and epitope display platform for the development of vaccines against cancers and infectious diseases, including coronavirus disease 2019. However, the proteinaceous CPMV nanoparticles are rapidly degraded in vivo. Multiple doses are therefore required to ensure long-lasting immunity, which is not ideal for global mass vaccination campaigns. Therefore, we formulated CPMV nanoparticles in injectable hydrogels to achieve slow particle release and prolonged immunostimulation. Liquid formulations were prepared from chitosan and glycerophosphate (GP) before homogenization with CPMV particles at room temperature. The formulations containing high-molecular-weight chitosan and 0–4.5 mg mL–1 CPMV gelled rapidly at 37 °C (5–8 min) and slowly released cyanine 5-CPMV particles in vitro and in vivo. Importantly, when a hydrogel containing CPMV displaying severe acute respiratory syndrome coronavirus 2 spike protein epitope 826 (amino acid 809–826) was administered to mice as a single subcutaneous injection, it elicited an antibody response that was sustained over 20 weeks, with an associated shift from Th1 to Th2 bias. Antibody titers were improved at later time points (weeks 16 and 20) comparing the hydrogel versus soluble vaccine candidates; furthermore, the soluble vaccine candidates retained Th1 bias. We conclude that CPMV nanoparticles can be formulated effectively in chitosan/GP hydrogels and are released as intact particles for several months with conserved immunotherapeutic efficacy. The injectable hydrogel containing epitope-labeled CPMV offers a promising single-dose vaccine platform for the prevention of future pandemics as well as a strategy to develop long-lasting plant virus-based nanomedicines.

In silico designed Staphylococcus aureus B-cell multi-epitope vaccine did not elicit antibodies against target antigens suggesting multi-domain approach

The vaccine development strategies have evolved from using an entire organism as an immunogen to a single antigen and further towards an epitope. Since an epitope is a relatively tiny and immunologically relevant part of an antigen, it has the potential to stimulate more robust and specific immune responses while causing minimal adverse effects. As a result, the recent focus of vaccine development has been to develop multi-epitope vaccines that can target multiple virulence mechanisms. Accordingly, we designed multi-epitope vaccine candidates B (multi-B-cell epitope immunogen) and CTB-B (an adjuvant – cholera toxin subunit B (CTB) – attached to immunogen B) against S. aureus by employing immunoinformatics approaches. The designed vaccines are composed of B-cell epitope segments (20-mer) of the eight well-characterized S. aureus virulence factors, namely ClfB, FnbpA, Hla, IsdA, IsdB, LukE, SdrD, and SdrE connected in series. The designed vaccines were expressed, purified, and administered to C57BL/6 mice with Freund adjuvant to evaluate the immunogenicity and protective efficacy. The results revealed that the immunized mice showed high IgG titers for the immunogen, and the antibody titers increased significantly following the second immunization. However, the generated antibodies did not protect the mice from infection. The interaction of anti-B antibodies with source virulence factors showed that the generated antibodies have no binding affinity with any of the corresponding virulence factors. Our results demonstrate the limitation of the in silico designed B-cell multi-epitope vaccine and suggest that a protein domain carrying both linear and conformational B-cell epitopes might be a better choice for developing an effective multi-epitope vaccine against S. aureus.

Designing Personalized Antigen-Specific Immunotherapies for Autoimmune Diseases-The Case for Using Ignored Target Cell Antigen Determinants.

We have proposed that antigen-specific immunotherapies (ASIs) for autoimmune diseases could be enhanced by administering target cell antigen epitopes (determinants) that are immunogenic but ignored by autoreactive T cells because these determinants may have large pools of naïve cognate T cells available for priming towards regulatory responses. Here, we identified an immunogenic preproinsulin determinant (PPIL4-20) that was ignored by autoimmune responses in type 1 diabetes (T1D)-prone NOD mice. The size of the PPIL4-20-specific splenic naive T cell pool gradually increased from 2–12 weeks in age and remained stable thereafter, while that of the major target determinant insulin B-chain9-23 decreased greatly after 12 weeks in age, presumably due to recruitment into the autoimmune response. In 15–16 week old mice, insulin B-chain9-23/alum immunization induced modest-low level of splenic T cell IL-10 and IL-4 responses, little or no spreading of these responses, and boosted IFNγ responses to itself and other autoantigens. In contrast, PPIL4-20/alum treatment induced robust IL-10 and IL-4 responses, which spread to other autoantigens and increased the frequency of splenic IL-10-secreting Treg and Tr-1-like cells, without boosting IFNγ responses to ß-cell autoantigens. In newly diabetic NOD mice, PPIL4-20, but not insulin B-chain9-23 administered intraperitoneally (with alum) or intradermally (as soluble antigen) supplemented with oral GABA induced long-term disease remission. We discuss the potential of personalized ASIs that are based on an individual’s naïve autoantigen-reactive T cell pools and the use of HLA-appropriate ignored autoantigen determinants to safely enhance the efficacy of ASIs.

Computational analysis of proteome of Foot-and-mouth disease Virus for the prediction of immunogenic epitopes

ObjectiveFoot and Mouth Disease virus (FMDV) commonly affects cloven hoofed animals. Infection with one serotype does not confer immunity to others and currently available vaccines do not provide effective T-cell immunity. The objective of this work is to predict the Cytotoxic T lymphocyte (CTL) epitopes which could be used as vaccine components against FMD virus. Material and methodsProteomes of three Indian FMDV vaccine strains, Type A IND40/00, Type A IND17/82 and Asia-1 IND63/72 were retrieved. BoLa-T2b allele restricted CTL epitopes were predicted using Immune Epitope Data Base Analysis Resource (IEDB-AR) and NetMHCpan 2.8 tool. The predicted overlapping epitopes were further analysed for antigenicity prediction, binding to other BoLA allele and consensus epitope analysis. ResultsA total of 207 epitopes were predicted by IEDB-AR tool. Among this, 72, 68, 67, epitopes were predicted in Asia 1 IND 63/72, Type A IND40/00 and Type A IND17/82 respectively. Totally 489 epitopes were predicted by NetMHCpan 2.0 tool. Of which, 164, 163 and 162 epitopes were predicted in Type A IND17/82, Asia 1 IND63/72 and Type A IND40/00 respectively. In total 29 consensus epitopes were predicted during this analysis, among this, 28 epitopes are present to occur in all the three Indian strains that were tested. ConclusionsThe 29 consensus epitopes predicted in this study could be used for developing a potent vaccine for both Asia I and Type A strains of FMD. Further in vitro and in vivo studies are needed to confirm the CTL efficacy of this peptide against FMDV.

When Is It Appropriate to Take Off the Mask? Signaling Pathways That Regulate ß(1,3)-Glucan Exposure in Candida albicans.

Candida spp. are an important source of systemic and mucosal infections in immune compromised populations. However, drug resistance or toxicity has put limits on the efficacy of current antifungals. The C. albicans cell wall is considered a good therapeutic target due to its roles in viability and fungal pathogenicity. One potential method for improving antifungal strategies could be to enhance the detection of fungal cell wall antigens by host immune cells. ß(1,3)-glucan, which is an important component of fungal cell walls, is a highly immunogenic epitope. Consequently, multiple host pattern recognition receptors, such as dectin-1, complement receptor 3 (CR3), and the ephrin type A receptor A (EphA2) are capable of recognizing exposed (unmasked) ß(1,3)-glucan moieties on the cell surface to initiate an anti-fungal immune response. However, ß(1,3)-glucan is normally covered (masked) by a layer of glycosylated proteins on the outer surface of the cell wall, hiding it from immune detection. In order to better understand possible mechanisms of unmasking ß(1,3)-glucan, we must develop a deeper comprehension of the pathways driving this phenotype. In this review, we describe the medical importance of ß(1,3)-glucan exposure in anti-fungal immunity, and highlight environmental stimuli and stressors encountered within the host that are capable of inducing changes in the levels of surface exposed ß(1,3)-glucan. Furthermore, particular focus is placed on how signal transduction cascades regulate changes in ß(1,3)-glucan exposure, as understanding the role that these pathways have in mediating this phenotype will be critical for future therapeutic development.

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people.

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8+ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8+ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8+ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8+ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8+ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2320-331 and A11/PB2323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M41-49, A11/NS1186-195 and A11/NP511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8+ T cell epitopes has implications for understanding how CD8+ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.

L- to d-Amino Acid Substitution in the Immunodominant LCMV-Derived Epitope gp33 Highlights the Sensitivity of the TCR Recognition Mechanism for the MHC/Peptide Structure and Dynamics.

Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8+ T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wild-type peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2Db, predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2Db/gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2Db in complex with APLs, in which position 6 was subjected to an l- to d-amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the l- to d-modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.

Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection.

Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three −20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.