TULIP: A transformer-based unsupervised language model for interacting peptides and T cell receptors that generalizes to unseen epitopes.

The accurate prediction of binding between T cell receptors (TCR) and their cognate epitopes is key to understanding the adaptive immune response and developing immunotherapies. Current methods face two significant limitations: the shortage of comprehensive high-quality data and the bias introduced by the selection of the negative training data commonly used in the supervised learning approaches. We propose a method, Transformer-based Unsupervised Language model for Interacting Peptides and T cell receptors (TULIP), that addresses both limitations by leveraging incomplete data and unsupervised learning and using the transformer architecture of language models. Our model is flexible and integrates all possible data sources, regardless of their quality or completeness. We demonstrate the existence of a bias introduced by the sampling procedure used in previous supervised approaches, emphasizing the need for an unsupervised approach. TULIP recognizes the specific TCRs binding an epitope, performing well on unseen epitopes. Our model outperforms state-of-the-art models and offers a promising direction for the development of more accurate TCR epitope recognition models.

Construction of multi-epitope vaccine against the Rhipicephalus microplus tick: an immunoinformatics approach.

Rhipicephalus microplus, known as the hard tick, is a vector for the parasites Babesia spp. and Anaplasma marginale, both of which can cause significant financial losses to the livestock industry. There is currently no effective vaccine for R. microplus tick infestations, despite the identification of numerous prospective tick vaccine candidates. As a result, the current research set out to develop an immunoinformatics-based strategy using existing methods for designing a multi-epitope based vaccination that is not only effective but also safe and capable of eliciting cellular and humoral immune responses. First, R. microplus proteins Bm86, Subolesin, and Bm95 were used to anticipate and link B and T-cell epitopes (HTL and CTL) to one another. Antigenicity testing, allergenicity assessment, and toxicity screening were just a few of the many immunoinformatics techniques used to identify potent epitopes. Multi-epitope vaccine design was chosen based on the antigenic score 0.935 that is promising vaccine candidate. Molecular docking was used to determine the nature of the interaction between TLR2 and the vaccine construct. Finally, molecular dynamic simulation was used to assess the stability and compactness of the resulting vaccination based on docking scores. The developed vaccine was shown to be stable, have immunogenic qualities, be soluble, and to have high expression by in silico cloning. These findings suggest that experimental investigation of the multi-epitope based vaccine designed in the current study will produce achievable vaccine candidates against R. microplus ticks, enabling more effective control of infestations.

Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations.

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

Development and application of EpitopeScan, a Python3 toolset for mutation tracking in SARS-CoV-2 immunogenic epitopes.

Introduction: Outbreaks of coronaviruses and especially the recent COVID-19 pandemic emphasize the importance of immunological research in this area to mitigate the effect of future incidents. Bioinformatics approaches are capable of providing multisided insights from virus sequencing data, although currently available software options are not entirely suitable for a specific task of mutation surveillance within immunogenic epitopes of SARS-CoV-2. Method: Here, we describe the development of a mutation tracker, EpitopeScan, a Python3 package with command line and graphical user interface tools facilitating the investigation of the mutation dynamics in SARS-CoV-2 epitopes via analysis of multiple-sequence alignments of genomes over time. We provide an application case by examining three Spike protein-derived immunodominant CD4+ T-cell epitopes restricted by HLA-DRB1*04:01, an allele strongly associated with susceptibility to rheumatoid arthritis (RA). Mutations in these peptides are relevant for immune monitoring of CD4+ T-cell responses against SARS-CoV-2 spike protein in patients with RA. The analysis focused on 2.3 million SARS-CoV-2 genomes sampled in England. Results: We detail cases of epitope conservation over time, partial loss of conservation, and complete divergence from the wild type following the emergence of the N969K Omicron-specific mutation in November 2021. The wild type and the mutated peptide represent potential candidates to monitor variant-specific CD4+ T-cell responses. EpitopeScan is available via GitHub repository https://github.com/Aleksandr-biochem/EpitopeScan.

Nonconserved epitopes dominate reverse preexisting T cell immunity in COVID-19 convalescents.

The herd immunity against SARS-CoV-2 is continuously consolidated across the world during the ongoing pandemic. However, the potential function of the nonconserved epitopes in the reverse preexisting cross-reactivity induced by SARS-CoV-2 to other human coronaviruses is not well explored. In our research, we assessed T cell responses to both conserved and nonconserved peptides shared by SARS-CoV-2 and SARS-CoV, identifying cross-reactive CD8+ T cell epitopes using enzyme-linked immunospot and intracellular cytokine staining assays. Then, in vitro refolding and circular dichroism were performed to evaluate the thermal stability of the HLA/peptide complexes. Lastly, single-cell T cell receptor reservoir was analyzed based on tetramer staining. Here, we discovered that cross-reactive T cells targeting SARS-CoV were present in individuals who had recovered from COVID-19, and identified SARS-CoV-2 CD8+ T cell epitopes spanning the major structural antigens. T cell responses induced by the nonconserved peptides between SARS-CoV-2 and SARS-CoV were higher and played a dominant role in the cross-reactivity in COVID-19 convalescents. Cross-T cell reactivity was also observed within the identified series of CD8+ T cell epitopes. For representative immunodominant peptide pairs, although the HLA binding capacities for peptides from SARS-CoV-2 and SARS-CoV were similar, the TCR repertoires recognizing these peptides were distinct. Our results could provide beneficial information for the development of peptide-based universal vaccines against coronaviruses.

Identification of HLA-A*11:01 and A*02:01-Restricted EBV Peptides Using HLA Peptidomics.

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.

Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.

Design of multi-epitope chimeric vaccine against Monkeypox virus and SARS-CoV-2: A vaccinomics perspective.

The current era we experience is full with pandemic infectious agents that no longer threatens the major local source but the whole globe. Almost the most emerging infectious agents are severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), followed by monkeypox virus (MPXV). Since no approved antiviral drugs nor licensed active vaccines are yet available, we aimed to utilize immunoinformatics approach to design chimeric vaccine against the two mentioned viruses. This is the first study to deal with design divalent vaccine against SARS-CoV-2 and MPXV. ORF8, E and M proteins from Omicron SARS-CoV-2 and gp182 from MPXV were used as the protein precursor from which multi-epitopes (inducing B-cell, helper T cells, cytotoxic T cells and interferon-É£) chimeric vaccine was contrived. The structure of the vaccine construct was predicted, validated, and docked to toll-like receptor-2 (TLR-2). Moreover, its sequence was also used to examine the immune simulation profile and was then inserted into the pET-28a plasmid for in silico cloning. The vaccine construct was probable antigen (0.543) and safe (non-allergen) with strong binding energy to TLR-2 (-1169.8 kcal/mol) and found to have significant immune simulation profile. In conclusion, the designed chimeric vaccine was potent and safe against SARS-CoV-2 and MPXV, which deserves further consideration.

A bioinformatic analysis of T-cell epitope diversity in SARS-CoV-2 variants: association with COVID-19 clinical severity in the United States population.

Long-term immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires the identification of T-cell epitopes affecting host immunogenicity. In this computational study, we explored the CD8+ epitope diversity estimated in 27 of the most common HLA-A and HLA-B alleles, representing most of the United States population. Analysis of 16 SARS-CoV-2 variants [B.1, Alpha (B.1.1.7), five Delta (AY.100, AY.25, AY.3, AY.3.1, AY.44), and nine Omicron (BA.1, BA.1.1, BA.2, BA.4, BA.5, BQ.1, BQ.1.1, XBB.1, XBB.1.5)] in analyzed MHC class I alleles revealed that SARS-CoV-2 CD8+ epitope conservation was estimated at 87.6%-96.5% in spike (S), 92.5%-99.6% in membrane (M), and 94.6%-99% in nucleocapsid (N). As the virus mutated, an increasing proportion of S epitopes experienced reduced predicted binding affinity: 70% of Omicron BQ.1-XBB.1.5 S epitopes experienced decreased predicted binding, as compared with ~3% and ~15% in the earlier strains Delta AY.100-AY.44 and Omicron BA.1-BA.5, respectively. Additionally, we identified several novel candidate HLA alleles that may be more susceptible to severe disease, notably HLA-A*32:01, HLA-A*26:01, and HLA-B*53:01, and relatively protected from disease, such as HLA-A*31:01, HLA-B*40:01, HLA-B*44:03, and HLA-B*57:01. Our findings support the hypothesis that viral genetic variation affecting CD8 T-cell epitope immunogenicity contributes to determining the clinical severity of acute COVID-19. Achieving long-term COVID-19 immunity will require an understanding of the relationship between T cells, SARS-CoV-2 variants, and host MHC class I genetics. This project is one of the first to explore the SARS-CoV-2 CD8+ epitope diversity that putatively impacts much of the United States population.

Epitope mapping and a candidate vaccine design from canine distemper virus.

Background: Canine distemper (CD) is a worldwide spread disease that has been described in 12 families of mammals, especially in the Carnivora order, being better studied in domestic canines where vaccination represents the best means of control. CD is controlled by vaccination, but many cases of the disease still occur in vaccinated animals. Aim: The aim of this work was to study antigen-specific epitopes that can subsidize the development of a new vaccine approach. Methods: Mapping of T cell reactive epitopes for CD virus (CDV) was carried out through enzyme-linked immunospot assays using 119 overlapped synthetic peptides from the viral hemagglutinin protein, grouped in 22 pools forming a matrix to test the immune response of 32 animals. Results: Evaluations using the criteria established to identify reactive pools, demonstrated that 26 animals presented at least one reactive pool, that one pool was not reactive to any animal, and six pools were the most frequent among the reactive peptides. The crisscrossing of the most reactive pools in the matrix revealed nine peptides considered potential candidate epitopes for T cell stimulation against the CDV and those were used to design an in-silico protein, containing also predicted epitopes for B cell stimulation, and further analyzed using immune epitope databases to ensure protein quality and stability. Conclusion: The final in silico optimized protein presents characteristics that qualify it to be used to develop a new prototype epitope-based anti-CDV vaccine.

Exploring the S Protein of SARS-CoV-2 to Design a Novel Multi-Epitope Vaccine against COVID-19 Based on Immunoinformatics Approaches.

BACKGROUND: Developing a novel COVID-19 multi-epitope vaccine (CoVMEV) is essential to containing the SARS-CoV-2 pandemic. METHODS: The virus's immunodominant B and T cell epitopes from the S protein were found and joined to create the CoVMEV. Bioinformatics techniques were used to investigate the secondary and tertiary structures, as well as the physical and chemical properties of CoVMEV. RESULTS: CoVMEV exhibited high antigenicity and immunogenicity scores, together with good water solubility and stability. Toll-like receptor 2 (TLR2) and toll-like receptor4 (TLR4), which are critical in triggering immunological responses, were also strongly favoured by CoVMEV. Molecular dynamics simulation and immune stimulation studies revealed that CoVMEV effectively activated T and B lymphocytes, and increased the number of active CD8+ T cells than similar vaccines. CONCLUSION: CoVMEV holds promise as a potential vaccine candidate for COVID-19, given its robust immunogenicity, stability, antigenicity, and capacity to stimulate a strong immune response. This study presents a significant design concept for the development of peptidyl vaccines targeting SARS-CoV-2. Further investigation and clinical trials will be crucial in assessing the efficacy and safety of CoVMEV as a potential vaccine for COVID-19.

T-cell immunity against Severe Acute Respiratory Syndrome Coronavirus 2 proteins in patients with type 1 diabetes.

AIMS: Individuals with type 1 diabetes (T1D) do not appear to have an elevated risk of severe Coronavirus Disease 19 (COVID-19). Pre-existing immune reactivity to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in unexposed individuals may serve as a protective factor. Hence, our study was designed to evaluate the existence of T cells with reactivity against SARS-CoV-2 antigens in unexposed patients with T1D. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were collected from SARS-CoV-2 unexposed patients with T1D and healthy control subjects. SARS-CoV-2 specific T cells were identified in PBMCs by ex-vivo interferon (IFN)γ-ELISpot and flow cytometric assays. The epitope specificity of T cells in T1D was inferred through T Cell Receptor sequencing and GLIPH2 clustering analysis. RESULTS: T1D patients unexposed to SARS-CoV-2 exhibited higher rates of virus-specific T cells than controls. The T cells primarily responded to peptides from the ORF7/8, ORF3a, and nucleocapsid proteins. Nucleocapsid peptides predominantly indicated a CD4+ response, whereas ORF3a and ORF7/8 peptides elicited both CD4+ and CD8+ responses. The GLIPH2 clustering analysis of TCRß sequences suggested that TCRß clusters, associated with the autoantigens proinsulin and Zinc transporter 8 (ZnT-8), might share specificity towards ORF7b and ORF3a viral epitopes. Notably, PBMCs from three T1D patients exhibited T cell reactivity against both ORF7b/ORF3a viral epitopes and proinsulin/ZnT-8 autoantigens. CONCLUSIONS: The increased frequency of SAR-CoV-2- reactive T cells in T1D patients might protect against severe COVID-19 and overt infections. These results emphasise the long-standing association between viral infections and T1D.

Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies.

SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) αß sequences and mRNA expression of the S-responded T cells were investigated using single-cell TCR- and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide pools were reconstituted into a reporter T cell line for the determination of epitopes and restricting HLAs. Among them, we could determine 78 S epitopes, most of which were conserved in variants of concern (VOCs). After the 2nd vaccination, T cell clonotypes highly responsive to recall S stimulation were polarized to follicular helper T (Tfh)-like cells in donors exhibiting sustained anti-S antibody titers (designated as 'sustainers'), but not in 'decliners'. Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which cross-reacted with environmental or symbiotic microbes. However, these clonotypes contracted after vaccination. Conversely, S-reactive clonotypes dominated after vaccination were undetectable in pre-vaccinated T cell pool, suggesting that highly responding S-reactive T cells were established by vaccination from rare clonotypes. These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination may contribute to the longevity of anti-S antibody titers.

In-silico formulation of a next-generation polyvalent vaccine against multiple strains of monkeypox virus and other related poxviruses.

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings.

Enhancing antitumor response by efficiently generating large-scale TCR-T cells targeting a single epitope across multiple cancer antigens.

The need to contrive interventions to curb the rise in cancer incidence and mortality is critical for improving patients' prognoses. Adoptive cell therapy is challenged with quality large-scale production, heightening its production cost. Several cancer types have been associated with the expression of highly-immunogenic CTAG1 and CTAG2 antigens, which share common epitopes. Targeting two antigens on the same cancer could improve the antitumor response of TCR-T cells. In this study, we exploited an efficient way to generate large-fold quality TCR-T cells and also demonstrated that the common epitopes of CTAG1 and CTAG2 antigens provide an avenue for improved cancer-killing via dual-antigen-epitope targeting. Our study revealed that xeno/sera-free medium could expand TCR-T cells to over 500-fold, posing as a better replacement for FBS-supplemented media. Human AB serum was also shown to be a good alternative in the absence of xeno/sera-free media. Furthermore, TCR-T cells stimulated with beads-coated T-activator showed a better effector function than soluble T-activator stimulated TCR-T cells. Additionally, TCR-T cells that target multiple antigens in the same cancer yield better anticancer activity than those targeting a single antigen. This showed that targeting multiple antigens with a common epitope may enhance the antitumor response efficacy of T cell therapies.

Characteristics of Norovirus capsid protein-specific CD8+ T-Cell responses in previously infected individuals.

Norovirus (NV) infection causes acute gastroenteritis in children and adults. Upon infection with NV, specific CD8+ T cells, which play an important role in anti-infective immunity, are activated in the host. Owing to the NV's wide genotypic variability, it is challenging to develop vaccines with cross-protective abilities against infection. To aid effective vaccine development, we examined specific CD8+ T-cell responses towards viral-structural protein (VP) epitopes, which enable binding to host susceptibility receptors. We isolated peripheral blood mononuclear cells from 196 participants to screen and identify predominant core peptides towards NV main and small envelope proteins using ex vivo and in vitro intracellular cytokine staining assays. Human leukocyte antigen (HLA) restriction characteristics were detected using next-generation sequencing. Three conservative immunodominant VP-derived CD8+ T-cell epitopes, VP294-102 (TDAARGAIN), VP2153-161 (RGPSNKSSN), and VP1141-148 (FPHIIVDV), were identified and restrictively presented by HLA-Cw * 0102, HLA-Cw * 0702, and HLA-A *1101 alleles, separately. Our findings provide useful insights into the development of future vaccines and treatments for NV infection.

Designing a smallpox B-cell and T-cell multi-epitope subunit vaccine using a comprehensive immunoinformatics approach.

Smallpox is a highly contagious human disease caused by the variola virus. Although the disease was eliminated in 1979 due to its highly contagious nature and historical pathogenicity, with a mortality rate of up to 30%, this virus is an important candidate for biological weapons. Currently, vaccines are the critical measures to prevent this virus infection and spread. In this study, we designed a peptide vaccine using immunoinformatics tools, which have the potential to activate human immunity against variola virus infection efficiently. The design of peptides derives from vaccine-candidate proteins showing protective potential in vaccinia WR strains. Potential non-toxic and nonallergenic T-cell and B-cell binding and cytokine-inducing epitopes were then screened through a priority prediction using special linkers to connect B-cell epitopes and T-cell epitopes, and an appropriate adjuvant was added to the vaccine construction to enhance the immunogenicity of the peptide vaccine. The 3D structure display, docking, and free energy calculation analysis indicate that the binding affinity between the vaccine peptide and Toll-like receptor 3 is high, and the vaccine receptor complex is highly stable. Notably, the vaccine we designed is obtained from the protective protein of the vaccinia and combined with preventive measures to avoid side effects. This vaccine is highly likely to produce an effective and safe immune response against the variola virus infection in the body. IMPORTANCE: In this work, we designed a vaccine with a cluster of multiple T-cell/B-cell epitopes, which should be effective in inducing systematic immune responses against variola virus infection. Besides, this work also provides a reference in vaccine design for preventing monkeypox virus infection, which is currently prevalent.

Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Advancing molecular modeling and reverse vaccinology in broad-spectrum yellow fever virus vaccine development.

Yellow fever outbreaks are prevalent, particularly in endemic regions. Given the lack of an established treatment for this disease, significant attention has been directed toward managing this arbovirus. In response, we developed a multiepitope vaccine designed to elicit an immune response, utilizing advanced immunoinformatic and molecular modeling techniques. To achieve this, we predicted B- and T-cell epitopes using the sequences from all structural (E, prM, and C) and nonstructural proteins of 196 YFV strains. Through comprehensive analysis, we identified 10 cytotoxic T-lymphocyte (CTL) and 5T-helper (Th) epitopes that exhibited overlap with B-lymphocyte epitopes. These epitopes were further evaluated for their affinity to a wide range of human leukocyte antigen system alleles and were rigorously tested for antigenicity, immunogenicity, allergenicity, toxicity, and conservation. These epitopes were linked to an adjuvant ( ß -defensin) and to each other using ligands, resulting in a vaccine sequence with appropriate physicochemical properties. The 3D structure of this sequence was created, improved, and quality checked; then it was anchored to the Toll-like receptor. Molecular Dynamics and Quantum Mechanics/Molecular Mechanics simulations were employed to enhance the accuracy of docking calculations, with the QM portion of the simulations carried out utilizing the density functional theory formalism. Moreover, the inoculation model was able to provide an optimal codon sequence that was inserted into the pET-28a( +) vector for in silico cloning and could even stimulate highly relevant humoral and cellular immunological responses. Overall, these results suggest that the designed multi-epitope vaccine can serve as prophylaxis against the yellow fever virus.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.