Influenza Virus-Derived CD8 T Cell Epitopes: Implications for the Development of Universal Influenza Vaccines.

The influenza virus poses a global health burden. Currently, an annual vaccine is used to reduce influenza virus-associated morbidity and mortality. Most influenza vaccines have been developed to elicit neutralizing Abs against influenza virus. These Abs primarily target immunodominant epitopes derived from hemagglutinin (HA) or neuraminidase (NA) of the influenza virus incorporated in vaccines. However, HA and NA are highly variable proteins that are prone to antigenic changes, which can reduce vaccine efficacy. Therefore, it is essential to develop universal vaccines that target immunodominant epitopes derived from conserved regions of the influenza virus, enabling cross-protection among different virus variants. The internal proteins of the influenza virus serve as ideal targets for universal vaccines. These internal proteins are presented by MHC class I molecules on Ag-presenting cells, such as dendritic cells, and recognized by CD8 T cells, which elicit CD8 T cell responses, reducing the likelihood of disease and influenza viral spread by inducing virus-infected cell apoptosis. In this review, we highlight the importance of CD8 T cell-mediated immunity against influenza viruses and that of viral epitopes for developing CD8 T cell-based influenza vaccines.

CD8+T-cell response to mutated HLA-B*35-restricted Gag HY9 and HA9 epitopes from HIV-1 variants from Medellin, Colombia.

The HLA-B*35 alleles have been associated with a slow or rapid progression of HIV-1 infection. However, the mechanisms related to HIV-1 progression have yet to be entirely understood. Several reports indicate that the binding affinity between the HLA-I molecule and peptides could be associated with an increased CD8+ T-cell response. Novel HLA-B*35-restricted mutated variants have been described from HSNQVSQNY (HY9) and HPVHAGPIA (HA9) epitopes. Bioinformatic analysis has indicated that these mutated epitopes show low and high binding affinity towards HLA-B*35, respectively. However, the polyfunctionality of CD8+ T-cells stimulated with these mutated and wild-type epitopes has yet to be reported. The results suggest that the low-binding affinity H124 N/S125 N/N126S mutated peptide in the HY9 epitope induced a lower percentage of CD107a+CD8+ T-cells than the wild-type epitope. Instead, the high-binding affinity peptides I223V and I223A in the HA9 epitope induced a significantly higher frequency of polyfunctional CD8+ T-cells. Also, a higher proportion of CD8+ T-cells with two functions, with Granzyme B+ Perforin+ being the predominant profile, was observed after stimulation with mutated peptides associated with high binding affinity in the HA9 epitope. These results suggest that the high-affinity mutated peptides induced a more polyfunctional CD8+ T-cell response, which could be related to the control of viral replication.

An immunoinformatic investigation on Rift Valley fever virus protein reveals possible epitopes for vaccines.

INTRODUCTION: This immunoinformatic study identified potential epitopes from the envelopment polyprotein (Gn/Gc) of Rift Valley fever virus (RVFV), a pathogenic virus causing severe fever in humans and livestock. Effective vaccination is crucial for controlling RVFV outbreaks. The identification of suitable epitopes is crucial for the development of safe and effective vaccines. METHODOLOGY: Protein sequences were obtained from the UniProt database, and evaluated through VaxiJen v2.0 to predict the B and T-cell epitopes within the RVFV glycoprotein. Gn/Gc protein sequences were analyzed with bioinformatics tools and algorithms. The predicted T-cell and B-cell epitopes were evaluated for antigenicity, allergenicity, and toxicity by the VaxiJen v2.0 system, AllerTop v2.0, and ToxinPred server, respectively. RESULTS: We employed computational methods to screen the RVFV envelopment polyprotein encompassing N-terminal and C-terminal glycoprotein segments, to discover antigenic T- and B-cell epitopes. Our analysis unveiled multiple potential epitopes within the RVFV glycoprotein, specifically within the Gn/Gc protein sequences. Subsequently, we selected eleven cytotoxic T-lymphocytes (CTL) and four helper T-lymphocytes (HTL) for population coverage analysis, which collectively extended to cover 97.04% of the world's population, representing diverse ethnicities and regions. Notably, the CTL epitope VQADLTLMF exhibited binding affinity to numerous human leukocyte antigen (HLA) alleles. The identification of glycoprotein (Gn/Gc) epitopes through this immunoinformatic study bears significant implications for advancing the development of an effective RVFV vaccine. CONCLUSIONS: These findings provide valuable insights into the immunological aspects of the disease and may contribute towards the development of broad-spectrum antiviral therapies targeting other RNA viruses with similar polymerase enzymes.

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.

Immuno-informatics study identifies conserved T cell epitopes in non-structural proteins of Bluetongue virus serotypes: formulation of a computationally optimized next-generation broad-spectrum multi-epitope vaccine.

Introduction: Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines. Methodology: In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning. Results: We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system. Discussion: These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry.

T-Cell Epitope Mapping of SARS-CoV-2 Reveals Coordinated IFN-γ Production and Clonal Expansion of T Cells Facilitates Recovery from COVID-19.

BACKGROUND: T-cell responses can be protective or detrimental during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, the underlying mechanism is poorly understood. METHODS: In this study, we screened 144 15-mer peptides spanning the SARS-CoV-2 spike, nucleocapsid (NP), M, ORF8, ORF10, and ORF3a proteins and 39 reported SARS-CoV-1 peptides in peripheral blood mononuclear cells (PBMCs) from nine laboratory-confirmed coronavirus disease 2019 (COVID-19) patients (five moderate and four severe cases) and nine healthy donors (HDs) collected before the COVID-19 pandemic. T-cell responses were monitored by IFN-γ and IL-17A production using ELISA, and the positive samples were sequenced for the T cell receptor (TCR) ß chain. The positive T-cell responses to individual SARS-CoV-2 peptides were validated by flow cytometry. RESULTS: COVID-19 patients with moderate disease produced more IFN-γ than HDs and patients with severe disease (moderate vs. HDs, p < 0.0001; moderate vs. severe, p < 0.0001) but less IL-17A than those with severe disease (p < 0.0001). A positive correlation was observed between IFN-γ production and T-cell clonal expansion in patients with moderate COVID-19 (r = 0.3370, p = 0.0214) but not in those with severe COVID-19 (r = -0.1700, p = 0.2480). Using flow cytometry, we identified that a conserved peptide of the M protein (Peptide-120, P120) was a dominant epitope recognized by CD8+ T cells in patients with moderate disease. CONCLUSION: Coordinated IFN-γ production and clonal expansion of SARS-CoV-2-specific T cells are associated with disease resolution in COVID-19. Our findings contribute to a better understanding of T-cell-mediated immunity in COVID-19 and may inform future strategies for managing and preventing severe outcomes of SARS-CoV-2 infection.

Identification and Characterization of Antigen-Specific T-Cells in Viral Infections.

Identification and characterization of CD8+ T-cells is important to determine their role in protecting and clearing viral infections. Here we provide details of the peptide-MHC (pMHC) tetramers-based approach to identify antigen-specific T-cells in human and murine samples. This method provides ex vivo quantification and functional characterization of T-cells reactive to specific viral antigens derived from CMV and rotavirus in human blood and in murine intestinal lamina propria samples, respectively.

In Silico Tools for Predicting Novel Epitopes.

Identifying antigens within a pathogen is a critical task to develop effective vaccines and diagnostic methods, as well as understanding the evolution and adaptation to host immune responses. Historically, antigenicity was studied with experiments that evaluate the immune response against selected fragments of pathogens. Using this approach, the scientific community has gathered abundant information regarding which pathogenic fragments are immunogenic. The systematic collection of this data has enabled unraveling many of the fundamental rules underlying the properties defining epitopes and immunogenicity, and has resulted in the creation of a large panel of immunologically relevant predictive (in silico) tools. The development and application of such tools have proven to accelerate the identification of novel epitopes within biomedical applications reducing experimental costs. This chapter introduces some basic concepts about MHC presentation, T cell and B cell epitopes, the experimental efforts to determine those, and focuses on state-of-the-art methods for epitope prediction, highlighting their strengths and limitations, and catering instructions for their rational use.

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo.

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8+ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo. Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay. Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC50 value of 21 µm or lower. The peptides elicited an interferon-gamma response in vivo. Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

Engineered HCMV-infected APCs enable the identification of new immunodominant HLA-restricted epitopes of anti-HCMV T-cell immunity.

Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11SAL, with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65NLV epitope. A11SAL-specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.

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.

[IgE reactivity of sera from patients with autoimmune diseases against a multi-epitope protein constructed from T epitopes of Ascaris lumbricoides allergens]. / Reactividad IgE de sueros en pacientes con enfermedades autoinmunes, frente a una proteína multi-epítopes construida a partir de epítopes T de alérgenos de Ascaris lumbricoides.

OBJECTIVE: To evaluate the IgE reactivity of sera in patients suffering from type 1 diabetes (T1D), lupus nephritis (LN) and juvenile idiopathic arthritis (JIA) against a molecule constructed from T epitopes of A. lumbricoides allergens. METHODS: We designed and expressed a synthetic multi-epítope protein named MP1 from A. lumbricoides and house dust mites allergens. By indirect ELISA, we evaluated IgE-reactivity to MP1 and to the whole-body extract of Ascaris lumbricoides in 45 sera from Colombian Caribbean patients with lupus nephritis (LN; n=25), type 1 diabetes (T1D; n=10) and Juvenil idiopathic arthritis (JIA; n=10). Individuals with poly autoimmunity were excluded. All patients were referred to the study by their specialist doctor. RESULTS: IgE to whole-body extract of A. lumbricoides showed the following median concentrations.484.2 ng/ml (IQR: 203.4) in JIA patients, 325.6 ng/ml (IQR: 179.3) in individuals with LN, and 424.7 ng/ml (IQR: 80.1) in the T1D group. On the other hand, IgE-reactivity to MP1 was 126.4 ng/ml (IQR: 90.9) in JIA patients, 130.7 ng/ml (IQR: 94.8) in an individual with LN, and 148.8 ng/ml (IQR: 102.1) in the T1D group. Although no statistical differences were observed between patient groups, the IgE to MP1 in all patients (n: 45) (IgE median: 134.2 ng/ml; IQR: 100) were significantly less compared to Ascaris extract (IgE median: 380.7 ng/ml; IQR: 175.8); (W: 0.732; p-value: 1.034x10-7). CONCLUSIONS: These preliminary results suggest that MP1 showed antigenic properties with low IgE- reactivity, compared to Ascaris lumbricoides extracted in individuals with autoimmune diseases. Further studies are needed to understand better the immune response induced by this molecule.

OBJETIVO: Evaluar la reactividad IgE de sueros en pacientes que padecen diabetes tipo 1 (DT1), nefritis lúpica (NL) y artritis idiopática juvenil (AIJ) frente a una molécula construida a partir de epítopes T de alérgenos de A. lumbricoides. MÉTODOS: Se diseñó y expresó una proteína multi-epítopes sintética (MP1), a partir de alérgenos de A. lumbricoides y ácaros del polvo doméstico. Mediante ELISA indirecto, se evaluaron las reactividades IgE anti-MP1 y al extracto de cuerpo entero de Ascaris lumbricoides, en sueros de pacientes con nefritis lúpica (NL; n=25), diabetes tipo 1 (T1D; n=10) y artritis idiopática juvenil (AIJ; n=10), procedentes del Caribe colombiano. Se excluyeron los individuos con poliautoinmunidad. Todos los pacientes fueron remitidos al estudio por su médico especialista. RESULTADOS: La IgE frente al extracto de cuerpo completo de A. lumbricoides mostró concentraciones de 484,2 ng/ml (RIQ: 203,4) en pacientes con AIJ; 325,6 ng/ml (RIQ: 179,3) en individuos con NL; y 424,7 ng/ml (RIQ: 80,1) en el grupo con DT1. Por otra parte, la reactividad de IgE anti-MP1 fue de 126,4 ng/ml (RIQ: 90,9) en los pacientes con AIJ; 130,7 ng/ml (RIQ: 94,8) en los individuos con NL; y 148,8 ng/ml (RIQ: 102,1) en el grupo con DT1. Aunque no se observaron diferencias estadísticas entre los grupos de pacientes, la reactividad IgE anti- MP1 en todos los pacientes (n: 45) (mediana de IgE: 134,2 ng/ml; RIQ: 100), fue significativamente inferior en comparación con el extracto de Ascaris (mediana de IgE: 380,7 ng/ml; RIQ: 175,8); (W: 0,732; p-valor: 1,034x10-7). CONCLUSIONES: Estos resultados preliminares sugieren que MP1 mostró propiedades antigénicas con baja reactividad IgE, en comparación con el extracto de Ascaris lumbricoides en individuos con enfermedades autoinmunes. Se necesitan más estudios para comprender mejor la respuesta inmunitaria inducida por esta molécula.

[Ige reactivity of a recombinant multi-epitope protein designed from allergens of interest in the tropics - preliminary findings]. / Reactividad IgE de una proteína multi-epítope recombinante diseñada a partir de alérgenos de interés en el trópico - hallazgos preliminares.

OBJECTIVE: The objective of the present study was to design a multi-epitope protein from A. lumbricoides and APD allergens and to evaluate its IgE reactivity preliminarily. METHODS: Using computational tools, a molecule containing multiple "T" epitopes of allergens derived from A. lumbricoides and APD was designed "in silico" This multi-epitope protein (MP1) was expressed using an E. coli system and purified by affinity chromatography using Ni-NTA agarose. Anti-MP1 and anti-HDM extract IgE reactivity was evaluated by Dot-Blot and indirect ELISA from sera of HDM-allergic patients and non-allergic individuals from Barranquilla-Colombia. Allergic individuals had a positive skin test to a standardized battery of inhaled allergens (EUROLINE - Ref: DP 3704-1601-1 E) and mite- specific IgE. RESULTS: Multi-epitope (MP1) protein was expressed and purified with high purity. Dot-Blot result showed that all sera from allergic patients showed lower IgE reactivity to MP1 compared to HDM extract. By ELISA, significantly lower concentrations of anti-MP1 IgE (Median: 270.86 ng/ml; IQR: 90.3) were observed in contrast to anti-HDM IgE levels (Median: 988.5 ng/ml; IQR: 1117.6) in sera of patients allergic to HDM. CONCLUSIONS: A protein composed of multiple epitopes of A. lumbricoides and HDM allergens was designed, expressed, and purified. Preliminary Dot-Blot results suggest that this molecule shows hypoallergenic properties with very low IgE reactivity compared to mite extract. Further functional studies are needed to understand better the immune response induced by this molecule.

OBJETIVO: Diseñar una proteína multiepítope a partir de alérgenos de A. lumbricoides y APD; y evaluar preliminarmente su reactividad IgE. MÉTODOS: Mediante herramientas computacionales se diseñó In Silico, una molécula que contiene múltiples epítopos T, de alérgenos derivados de A. lumbricoides y APD. Esta proteína multiepítope (MP1) se expresó utilizando un sistema de E. coli, y se purificó mediante cromatografía de afinidad, empleando agarosa Ni-NTA. La reactividad IgE anti-MP1 y anti-extracto de APD, se evaluó mediante Dot-Blot y ELISA indirecta, a partir de suero de pacientes alérgicos a APD, e individuos no alérgicos procedentes de Barranquilla, Colombia. Los individuos alérgicos contaron con prueba cutánea positiva a una batería estandarizada de alérgenos inhalados (EUROLINE - Ref: DP 3704-1601-1 E) e IgE específica para ácaros. RESULTADOS: La proteína multiepítope MP1 se expresó y purificó con alta pureza. El resultado del Dot-Blot, mostró que todos los sueros de pacientes alérgicos tuvieron una reactividad IgE menor a MP1 en comparación al extracto de APD. Por ELISA, se observaron concentraciones significativamente menores de IgE anti-MP1 (Mediana: 270,86 ng/ml; RIQ: 90,3), en contraste a los niveles de IgE anti-APD (Mediana: 988,5 ng/ml; RIQ: 1117,6), en suero de pacientes alérgicos a APD. CONCLUSIONES: Se diseñó, expresó y purificó una proteína compuesta por múltiples epítopes de alérgenos de A. lumbricoides y APD. Los resultados preliminares de Dot-Blot sugieren que esta molécula muestra propiedad hipoalergénica con una reactividad IgE muy baja, en comparación con el extracto de ácaros. Se necesita continuar con estudios funcionales para comprender mejor la respuesta inmune inducida por esta molécula.

Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay.

BACKGROUND: The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. METHODS: A broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3-5 months. RESULTS: The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = - 0.21, - 0.21, - 0.27, - 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. CONCLUSION: The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Cross-Reactivity Assessment of Vaccine-Derived SARS-CoV-2 T Cell Responses against BA.2.86 and JN.1.

The SARS-CoV-2 Omicron sub-variants BA.2.86 and JN.1 contain multiple mutations in the spike protein that were not present in previous variants of concern and Omicron sub-variants. Preliminary research suggests that these variants reduce the neutralizing capability of antibodies induced by vaccines, which is particularly significant for JN.1. This raises concern as many widely deployed COVID-19 vaccines are based on the spike protein of the ancestral Wuhan strain of SARS-CoV-2. While T cell responses have been shown to be robust against previous SARS-CoV-2 variants, less is known about the impact of mutations in BA.2.86 and JN.1 on T cell responses. We evaluate the effect of mutations specific to BA.2.86 and JN.1 on experimentally determined T cell epitopes derived from the spike protein of the ancestral Wuhan strain and the spike protein of the XBB.1.5 strain that has been recommended as a booster vaccine. Our data suggest that BA.2.86 and JN.1 affect numerous T cell epitopes in spike compared to previous variants; however, the widespread loss of T cell recognition against these variants is unlikely.

Back to the Future: Immune Protection or Enhancement of Future Coronaviruses.

Before the emergence of SARS-CoV-1, MERS-CoV, and most recently, SARS-CoV-2, four other coronaviruses (the alpha coronaviruses NL63 and 229E and the beta coronaviruses OC43 and HKU1) had already been circulating in the human population. These circulating coronaviruses all cause mild respiratory illness during the winter seasons, and most people are already infected in early life. Could antibodies and/or T cells, especially against the beta coronaviruses, have offered some form of protection against (severe) COVID-19 caused by infection with SARS-CoV-2? Related is the question of whether survivors of SARS-CoV-1 or MERS-CoV would be relatively protected against SARS-CoV-2. More importantly, would humoral and cellular immunological memory generated during the SARS-CoV-2 pandemic, either by infection or vaccination, offer protection against future coronaviruses? Or rather than protection, could antibody-dependent enhancement have taken place, a mechanism by which circulating corona antibodies enhance the severity of COVID-19? Another related phenomenon, the original antigenic sin, would also predict that the effectiveness of the immune response to future coronaviruses would be impaired because of the reactivation of memory against irrelevant epitopes. The currently available evidence indicates that latter scenarios are highly unlikely and that especially cytotoxic memory T cells directed against conserved epitopes of human coronaviruses could at least offer partial protection against future coronaviruses.

An immunoinformatics and structural vaccinology approach to design a novel and potent multi-epitope base vaccine targeting Zika virus.

Zika virus is an infectious virus, that belongs to Flaviviridae family, which is transferred to humans through mosquito vectors and severely threatens human health; but, apart from available resources, no effective and secure vaccine is present against Zika virus, to prevent such infections. In current study, we employed structural vaccinology approach to design an epitope-based vaccine against Zika virus, which is biocompatible, and secure and might trigger an adaptive and innate immune response by using computational approaches. We first retrieved the protein sequence from National Center for Biotechnology Information (NCBI) database and carried out for BLAST P. After BLAST P, predicted protein sequences were shortlisted and checked for allergic features and antigenic properties. Final sequence of Zika virus, with accession number (APO40588.1) was selected based on high antigenic score and non-allergenicity. Final protein sequence used various computational approaches including antigenicity testing, toxicity evaluation, allergenicity, and conservancy assessment to identify superior B-cell and T-cell epitopes. Two B-cell epitopes, five MHC-six MHC-II epitopes and I were used to construct an immunogenic multi-epitope-based vaccine by using suitable linkers. A 50S ribosomal protein was added at N terminal to improve the immunogenicity of vaccine. In molecular docking, strong interactions were presented between constructed vaccine and Toll-like receptor 9 (- 1100.6 kcal/mol), suggesting their possible relevance in the immunological response to vaccine. The molecular dynamics simulations ensure the dynamic and structural stability of constructed vaccine. The results of C-immune simulation revealed that constructed vaccine activate B and T lymphocytes which induce high level of antibodies and cytokines to combat Zika infection. The constructed vaccine is an effective biomarker with non-sensitization, nontoxicity; nonallergic, good immunogenicity, and antigenicity, however, experimental assays are required to verify the results of present study.

Impact of predicted HLA class I immunopeptidome on viral reservoir in a cohort of people living with HIV in Italy.

The class I HLA genotype has been widely recognized as a factor influencing HIV disease progression in treatment-naïve subjects. However, little is known regarding its role in HIV disease course and how it influences the size of the viral reservoir once anti-retroviral therapy (ART) is started. Here, leveraging on cutting-edge bioinformatic tools, we explored the relationship between HLA class I and the HIV reservoir in a cohort of 90 people living with HIV (PLWH) undergoing ART and who achieved viral suppression. Analysis of HLA allele distribution among patients with high and low HIV reservoir allowed us to document a predominant role of HLA-B and -C genes in regulating the size of HIV reservoir. We then focused on the analysis of HIV antigen (Ag) repertoire, by investigating immunogenetic parameters such as the degree of homozygosity, HLA evolutionary distance and Ag load. In particular, we used two different bioinformatic algorithms, NetMHCpan and MixMHCpred, to predict HLA presentation of immunogenic HIV-derived peptides and identified HLA-B*57:01 and HLA-B*58:01 among the highest ranking HLAs in terms of total load, suggesting that their previously reported protective role against HIV disease progression might be linked to a more effective viral recognition and presentation to Cytotoxic T lymphocytes (CTLs). Further, we speculated that some peptide-HLA complexes, including those produced by the interaction between HLA-B*27 and the HIV Gag protein, might be particularly relevant for the efficient regulation of HIV replication and containment of the HIV reservoir. Last, we provide evidence of a possible synergistic effect between the CCR5 ∆32 mutation and Ag load in controlling HIV reservoir.