Cell Epitopes Research Articles

Omicron BA.2 breakthrough infection elicits CD8+ T cell responses recognizing the spike of later Omicron subvariants.

Here, we examine peripheral blood memory T cell responses against the SARS-CoV-2 BA.4/BA.5 variant spike among vaccinated individuals with or without Omicron breakthrough infections. We provide evidence supporting a lack of original antigenic sin in CD8+ T cell responses targeting the spike. We show that BNT162b2-induced memory T cells respond to the BA.4/BA.5 spike. Among individuals with BA.1/BA.2 breakthrough infections, IFN-γ-producing CD8+ T cell responses against the BA.4/BA.5 spike increased. In a subgroup with BA.2 breakthrough infections, IFN-γ-producing CD8+ T cell responses against the BA.2-mutated spike region increased and correlated directly with responses against the BA.4/BA.5 spike, indicating that BA.2 spike-specific CD8+ T cells elicited by BA.2 breakthrough infection cross-react with the BA.4/BA.5 spike. We identified CD8+ T cell epitope peptides that are present in the spike of BA.2 and BA.4/BA.5 but not the original spike. These peptides are fully conserved in the spike of now-dominant XBB lineages. Our study shows that breakthrough infection by early Omicron subvariants elicits CD8+ T cell responses that recognize epitopes within the spike of newly emerging subvariants.

Identification and relative abundance of naturally presented and cross-reactive influenza A virus MHC class I-restricted T cell epitopes

ABSTRACT Cytotoxic T lymphocytes are key for controlling viral infection. Unravelling CD8+ T cell-mediated immunity to distinct influenza virus strains and subtypes across prominent HLA types is relevant for combating seasonal infections and emerging new variants. Using an immunopeptidomics approach, naturally presented influenza A virus-derived ligands restricted to HLA-A*24:02, HLA-A*68:01, HLA-B*07:02, and HLA-B*51:01 molecules were identified. Functional characterization revealed multifunctional memory CD8+ T cell responses for nine out of sixteen peptides. Peptide presentation kinetics was optimal around 12 h post infection and presentation of immunodominant epitopes shortly after infection was not always persistent. Assessment of immunogenic epitopes revealed that they are highly conserved across the major zoonotic reservoirs and may contain a single substitution in the vicinity of the anchor residues. These findings demonstrate how the identified epitopes promote T cell pools, possibly cross-protective in individuals and can be potential targets for vaccination.

Monkeypox: Can we count on the current smallpox immunization?

Two vaccines ACAM 2000 and JYNNEOS have obtained approval from the Food and Drug Administration as preventive measures against monkeypox, contributing significantly to the management of the monkeypox epidemic. Nonetheless, research has demonstrated that smallpox vaccination offers approximately 88.8% protection against monkeypox, while immunization with these vaccines generates relatively low levels of neutralizing antibodies. In this work, we performed a comprehensive comparison of antigens between the 2022–2023 monkeypox strains and the smallpox vaccine strains. Our analysis has revealed considerable amino acid changes in all 27 antigens, including core and envelope proteins. Amino acid substitutions within B cell epitopes were observed in 26 of these antigens, with at least half of the antigen substitutions occurring within B cell epitopes in 20 out of the 26 antigens analyzed. These findings may raise potential concerns regarding the efficacy of these vaccines.

A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection.

Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4+ T cell response that can protect female mice from mortality. We inoculated C57BL/6 Ifnar-/- mice with CVB3. We investigated the T cell response in the spleen and mesenteric lymph nodes in male and female mice following infection. We found that CVB3 can induce expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4+ T cells before infection increased mortality in female mice, indicating that CD4+ T cells play a protective role against CVB3 in our model. Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease.

Short-chain fatty acids ameliorate experimental anti-glomerular basement membrane disease

BackgroundShort-chain fatty acids (SCFAs), as the link between gut microbiota and the immune system, had been reported to be protective in many autoimmune diseases by the modulation of T cell differentiation. The pathogenic role of autoreactive Th1 and Th17 cells and the protective role of Treg cells in the pathogenesis of anti-GBM disease have been fully demonstrated. Thus, the present study aimed to investigate the therapeutic effects of SCFAs in a rat model of anti-GBM disease. Materials and methodsExperimental anti-GBM disease was constructed by immunizing Wistar Kyoto rats with a nephrogenic T cell epitope α3127–148, and intervened by sodium acetate, sodium propionate, or sodium butyrate, 150 mM in the drinking water from day 0 to 42. Kidney injury was accessed by the biochemical analyzer, immunofluorescence, and immunohistochemistry. Antibody response was detected by ELISA. T cell clustering and proliferation were detected by flow cytometry. Human kidney 2 (HK2) cells were stimulated in vitro and cytokines were assessed by quantitative real-time PCR. ResultsTreatment with sodium acetate, sodium propionate, or sodium butyrate ameliorated the severity of kidney impairment in rats with anti-GBM glomerulonephritis. In the sodium butyrate-treated rats, the urinary protein, serum creatinine, and blood urea nitrogen levels were significantly lower; the percentage of crescent formation in glomeruli was significantly reduced; and the kidneys showed reduced IgG deposition, complement activation, T cell, and macrophage infiltration as well as the level of circulating antibodies against anti-α3(IV)NC1. The treatment of sodium butyrate reduced the α3127–148-specific T cell activation and increased the Treg cells differentiation and the intestinal beneficial bacteria flora. It also alleviated the damage of HK2 cells treated with inflammatory factors and complement. ConclusionTreatment with SCFAs, especially butyrate, alleviated anti-GBM nephritis in rat model, indicating its potential therapeutic effects in clinical usage.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Identification of a Spike-Specific CD8+ T-Cell Epitope Following Vaccination Against the Middle East Respiratory Syndrome Coronavirus in Humans.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

New bacteriophage-derived lysins, LysJ and LysF, with the potential to control Bacillus anthracis

Bacillus anthracis is an etiological agent of anthrax, a severe zoonotic disease that can be transmitted to people and cause high mortalities. Bacteriophages and their lytic enzymes, endolysins, have potential therapeutic value in treating infections caused by this bacterium as alternatives or complements to antibiotic therapy. They can also be used to identify and detect B. anthracis. Endolysins of two B. anthracis Wbetavirus phages, J5a and F16Ba which were described by us recently, differ significantly from the best-known B. anthracis phage endolysin PlyG from Wbetavirus genus bacteriophage Gamma and a few other Wbetavirus genus phages. They are larger than PlyG (351 vs. 233 amino acid residues), contain a signal peptide at their N-termini, and, by prediction, have a different fold of cell binding domain suggesting different structural basis of cell epitope recognition. We purified in a soluble form the modified versions of these endolysins, designated by us LysJ and LysF, respectively, and depleted of signal peptides. Both modified endolysins could lyse the B. anthracis cell wall in zymogram assays. Their activity against the living cells of B. anthracis and other species of Bacillus genus was tested by spotting on the layers of bacteria in soft agar and by assessing the reduction of optical density of bacterial suspensions. Both methods proved the effectiveness of LysJ and LysF in killing the anthrax bacilli, although the results obtained by each method differed. Additionally, the lytic efficiency of both proteins was different, which apparently correlates with differences in their amino acid sequence.Key points• LysJ and LysF are B. anthracis-targeting lysins differing from lysins studied so far• LysJ and LysF could be overproduced in E. coli in soluble and active forms• LysJ and LysF are active in killing cells of B. anthracis virulent strains

In silico analysis of virulence factors of Streptococcus uberis for a chimeric vaccine design

Streptococcus uberis is one of the causative agents of bovine mastitis, which has detrimental effects on animal health and the dairy industry. Despite decades of research, the requirement for effective vaccines against the disease remains unmet. The goal of this study was to create a multi-epitope vaccine using five virulence factors of S. uberis through the reverse vaccinology approach, which has been employed due to its high efficiency and applicability. Plasminogen activator A (PauA), glyceraldehyde-3-phosphate dehydrogenase C (GapC), C5a peptidase, S. uberis adhesion molecule (SUAM), and sortase A (SrtA) were selected for the T cytotoxic (CTL) and B cell epitope analyses as they were extensively studied in S. uberis or other pathogens. Eighteen CTL and ten B cell epitopes that were antigenic, non-toxic, and non-allergenic were selected in order to design a chimeric vaccine candidate that in silico analysis revealed to be potentially immunogenic, non-allergenic, and stable. Molecular docking analysis of the vaccine candidate with Toll-like receptor (TLR) 2 and TLR 4 revealed stable interactions between the candidate and the immune receptors. Meanwhile, the stability of the docked complexes was confirmed using normal mode analysis. Additionally, in silico immune simulation of the vaccine candidate demonstrated the stimulation of primary immune responses, indicating that the chimeric protein can hold promise as a viable vaccine candidate for preventing S. uberis mastitis. Moreover, the current study can provide a background for designing epitope-based vaccines based on the explored epitopes.

Nitrated T cell epitope linked vaccine targeting CD47 elicits antitumor immune responses and acts synergistically with vaccine targeting PDL1

Despite the clinical breakthrough made by immune checkpoint blockades (ICB) in cancer immunotherapy, immunosuppressed tumor microenvironment (TME) remains a major impediment in the efficacy of ICB immunotherapy. In this study, we constructed a Nitrated T cell epitope (NitraTh) linked vaccine targeting CD47, namely CD47-NitraTh. CD47-NitraTh could repress the progression of tumor by inducing tumor-specific immune response. Furthermore, combination vaccination with CD47-NitraTh and PDL1-NitraTh could reconstruct tumor associated macrophage, enhance macrophage-mediated phagocytosis for tumor cells, and promote the activation of tumor infiltrating T cells. Notably, by activating chemokine signaling pathway, NitraTh based vaccines reversed immunosuppressed TME, resulting in improved therapeutic outcome for tumor. With the advantage of reversing immunosuppressed TME, NitraTh based vaccine seems an optimal immunotherapy strategy for patients who are not sensitive to antibody based ICB.

PIRCHE-II Risk and Acceptable Mismatch Profile Analysis in Solid Organ Transplantation.

To optimize outcomes in solid organ transplantation, the HLA genes are regularly compared and matched between the donor and recipient. However, in many cases a transplant cannot be fully matched, due to widespread variation across populations and the hyperpolymorphism of HLA alleles. Mismatches of the HLA molecules in transplanted tissue can be recognized by immune cells of the recipient, leading to immune response and possibly organ rejection. These adverse outcomes are reduced by analysis using epitope-focused models that consider the immune relevance of the mismatched HLA.PIRCHE, an acronym for Predicted Indirectly ReCognizable HLAEpitopes, aims to categorize and quantify HLA mismatches in a patient-donor pair by predicting HLA-derived T cell epitopes. Specifically, the algorithm predicts and counts the HLA-derived peptides that can be presented by the host HLA, known as indirectly-presented T cell epitopes. Looking at the immune-relevant epitopes within HLA allows a more biologically relevant understanding of immune response, and provides an expanded donor pool for a more refined matching strategy compared with allele-level matching. This PIRCHE algorithm is available for analysis of single transplantations, as well as bulk analysis for population studies and statistical analysis for comparison of probability of organ availability and risk profiles.

In silico identification of drug targets and vaccine candidates against Bartonella quintana: a subtractive proteomics approach.

The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts. Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana. To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively. This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.

To stay or not to stay intact as an allergen: the endolysosomal degradation assay used as tool to analyze protein immunogenicity and T cell epitopes.

Antigen uptake and processing of exogenous proteins is critical for adaptive immunity, particularly for T helper cell activation. Proteins undergo distinct proteolytic processing in endolysosomal compartments of antigen-presenting cells. The resulting peptides are presented on MHC class II molecules and specifically recognized by T cells. The in vitro endolysosomal degradation assay mimics antigen processing by incubating a protein of interest with a protease cocktail derived from the endolysosomal compartments of antigen presenting cells. The kinetics of protein degradation is monitored by gel electrophoresis and allows calculation of a protein's half-life and thus endolysosomal stability. Processed peptides are analyzed by mass spectrometry and abundant peptide clusters are shown to harbor T cell epitopes. The endolysosomal degradation assay has been widely used to study allergens, which are IgE-binding proteins involved in type I hypersensitivity. In this review article, we provide the first comprehensive overview of the endolysosomal degradation of 29 isoallergens and variants originating from the PR-10, Ole e 1-like, pectate lyase, defensin polyproline-linked, non-specific lipid transfer, mite group 1, 2, and 5, and tropomyosin protein families. The assay method is described in detail and suggestions for improved standardization and reproducibility are provided. The current hypothesis implies that proteins with high endolysosomal stability can induce an efficient immune response, whereas highly unstable proteins are degraded early during antigen processing and therefore not efficient for MHC II peptide presentation. To validate this concept, systematic analyses of high and low allergenic representatives of protein families should be investigated. In addition to purified molecules, allergen extracts should be degraded to analyze potential matrix effects and gastrointestinal proteolysis of food allergens. In conclusion, individual protein susceptibility and peptides obtained from the endolysosomal degradation assay are powerful tools for understanding protein immunogenicity and T cell reactivity. Systematic studies and linkage with in vivo sensitization data will allow the establishment of (machine-learning) tools to aid prediction of immunogenicity and allergenicity. The orthogonal method could in the future be used for risk assessment of novel foods and in the generation of protein-based immunotherapeutics.

Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.

How to Predict Binding Specificity and Ligands for New MHC-II Alleles with MixMHC2pred.

MHC-II molecules are key mediators of antigen presentation in vertebrate species and bind to their ligands with high specificity. The very high polymorphism of MHC-II genes within species and the fast-evolving nature of these genes across species has resulted in tens of thousands of different alleles, with hundreds of new alleles being discovered yearly through large sequencing projects in different species. Here we describe how to use MixMHC2pred to predict the binding specificity of any MHC-II allele directly from its amino acid sequence. We then show how both MHC-II ligands and CD4+ T cell epitopes can be predicted in different species with our approach. MixMHC2pred is available at http://mixmhc2pred.gfellerlab.org/ .

An Immunoinformatic-Based In Silico Identification on the Creation of a Multiepitope-Based Vaccination Against the Nipah Virus.

The zoonotic viruses pose significant threats to public health. Nipah virus (NiV) is an emerging virus transmitted from bats to humans. The NiV causes severe encephalitis and acute respiratory distress syndrome, leading to high mortality rates, with fatality rates ranging from 40% to 75%. The first emergence of the disease was found in Malaysia in 1998-1999 and later in Bangladesh, Cambodia, Timor-Leste, Indonesia, Singapore, Papua New Guinea, Vietnam, Thailand, India, and other South and Southeast Asian nations. Currently, no specific vaccines or antiviral drugs are available. The potential advantages of epitope-based vaccines include their ability to elicit specific immune responses while minimizing potential side effects. The epitopes have been identified from the conserved region of viral proteins obtained from the UniProt database. The selection of conserved epitopes involves analyzing the genetic sequences of various viral strains. The present study identified two B cell epitopes, seven cytotoxic T lymphocyte (CTL) epitopes, and seven helper T lymphocyte (HTL) epitope interactions from the NiV proteomic inventory. The antigenic and physiological properties of retrieved protein were analyzed using online servers ToxinPred, VaxiJen v2.0, and AllerTOP. The final vaccine candidate has a total combined coverage range of 80.53%. The tertiary structure of the constructed vaccine was optimized, and its stability was confirmed with the help of molecular simulation. Molecular docking was performed to check the binding affinity and binding energy of the constructed vaccine with TLR-3 and TLR-5. Codon optimization was performed in the constructed vaccine within the Escherichia coli K12 strain, to eliminate the danger of codon bias. However, these findings must require further validation to assess their effectiveness and safety. The development of vaccines and therapeutic approaches for virus infection is an ongoing area of research, and it may take time before effective interventions are available for clinical use.

Tetanus-diphtheria vaccine can prime SARS-CoV-2 cross-reactive T cells.

Vaccines containing tetanus-diphtheria antigens have been postulated to induce cross-reactive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which could protect against coronavirus disease (COVID-19). In this work, we investigated the capacity of Tetanus-diphtheria (Td) vaccine to prime existing T cell immunity to SARS-CoV-2. To that end, we first collected known SARS-CoV-2 specific CD8+ T cell epitopes targeted during the course of SARS-CoV-2 infection in humans and identified as potentially cross-reactive with Td vaccine those sharing similarity with tetanus-diphtheria vaccine antigens, as judged by Levenshtein edit distances (≤ 20% edits per epitope sequence). As a result, we selected 25 potentially cross-reactive SARS-CoV-2 specific CD8+ T cell epitopes with high population coverage that were assembled into a synthetic peptide pool (TDX pool). Using peripheral blood mononuclear cells, we first determined by intracellular IFNγ staining assays existing CD8+ T cell recall responses to the TDX pool and to other peptide pools, including overlapping peptide pools covering SARS-CoV-2 Spike protein and Nucleocapsid phosphoprotein (NP). In the studied subjects, CD8+ T cell recall responses to Spike and TDX peptide pools were dominant and comparable, while recall responses to NP peptide pool were less frequent and weaker. Subsequently, we studied responses to the same peptides using antigen-inexperienced naive T cells primed/stimulated in vitro with Td vaccine. Priming stimulations were carried out by co-culturing naive T cells with autologous irradiated peripheral mononuclear cells in the presence of Td vaccine, IL-2, IL-7 and IL-15. Interestingly, naive CD8+ T cells stimulated/primed with Td vaccine responded strongly and specifically to the TDX pool, not to other SARS-CoV-2 peptide pools. Finally, we show that Td-immunization of C57BL/6J mice elicited T cells cross-reactive with the TDX pool. Collectively, our findings support that tetanus-diphtheria vaccines can prime SARS-CoV-2 cross-reactive T cells and likely contribute to shape the T cell responses to the virus.

Multi-epitopes vaccine design for surface glycoprotein against SARS-CoV-2 using immunoinformatic approach

BackgroundThe recent COVID vaccinations have successfully reduced death and severity but did not stop the transmission of viruses by the emerging SARS-CoV-2 strain. There is a need for better and long-lasting dynamic vaccines for numerous prevailing strains and the evolving SARS-CoV-2 virus, necessitating the development of broad-spectrum strains being used to stop infection by reducing the spread rate and re-infection. The spike (S) glycoprotein is one of the proteins expressed commonly in the early phases of SARS-CoV-2 infection. It has been identified as the most immunogenic protein of SARS-CoV-2. MethodsIn this study, advanced bioinformatics techniques have been exploited to design the novel multi-epitope vaccine using conserved S protein portions from widespread strains of SARS-CoV-2 to predict B cell and T cell epitopes. These epitopes were selected based on toxicity, antigenicity score and immunogenicity. Epitope combinations were used to construct the maximum potent multi-epitope construct with potential immunogenic features. EAAAK, AAY, and GPGPG were used as linkers to construct epitopes. ResultsThe developed vaccine has shown positive results. After the chimeric vaccine construct was cloned into the PET28a (+) vector for expression screening in Escherichia coli, the potential expression of the construct was identified. ConclusionThe construct vaccine performed well in computer-based immune response simulation and covered a variety of allelic populations. These computational results are more helpful for further analysis of our contract vaccine, which can finally help control and prevent SARS-CoV-2 infections worldwide.

Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

Integrated computational approaches assisted development of a novel multi-epitope vaccine against MDR Streptococcus pseudopneumoniae.

The emergence of antibiotic resistance (AR) in bacteria is becoming an alarming health concern because it allows them to adapt themselves to changing environments. It is possible to prevent the spread of AR in many ways, such as reducing antibiotic misuse in human and veterinary medicine. Streptococcus pseudopneumoniae is one of these AR bacterial species that can cause pneumonia in humans and is responsible for high mortality and morbidity rates. It is oval shaped gram-positive bacterium that shows resistance to several antibiotics like penicillin, tetracycline, ciprofloxacin, erythromycin, and co-trimoxazale and no approved vaccine is available to overcome diseases of the pathogen. Thus, substantial efforts are necessary to select protective antigens from a whole genome of pathogens that are easily tested experimentally. The in silico designed vaccine was safe and potent in immunizing individuals against the aforementioned pathogens. Herein, we utilized a subtractive genomic approach to identify potential epitope-based vaccine candidates against S. pseudopneumoniae. In total, 50850 proteins were retrieved from the NCBI, representing the complete genome of S. pseudopneumoniae. Out of the total, CD-HIT analysis identified 1022 proteins as non-redundant and 49828 proteins as redundant and further subjected for subcellular localization in which bulk of proteins was located in the cytoplasm, with seven extracellular proteins (penicillin-binding protein, alpha-amylase, solute-binding protein, hypothetical protein, CHAP domain-containing protein, polysaccharide deacetylase family protein, hypothetical protein). Six immune cells epitopes (SNLQSENDRL, RNDSLQKQAR, NPTTTSEGF, KVKKKNNKK, AYSQGSQKEH, and SVVDQVSGDF) were predicted with the help of the IEDB server. To design a multi-epitopes vaccine these immune cell epitopes were together by GPGPG and adjuvant linker to enhance immune response efficacy. The 3D structure of the designed vaccine was modeled and conducted molecular docking and dynamic simulation studies were to check the binding efficacy with immune cells receptor and dynamic behavior of the docked complex. Finally, we concluded that the designed vaccine construct can provoke a proper and protective immune response against S. pseudopneumoniae.