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.

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.

Prediction of promiscuous multiepitope-based peptide vaccine against RdRp of rotavirus using immunoinformatics studies

ABSTRACT Rotavirus, a dsRNA virus in the Reoviridae family, shows a segmented genome. The VP1 gene encodes the RNA-dependent RNA polymerase (RdRp). This study aims to develop a multiepitope-based vaccine targeting RdRp using immunoinformatic approaches. In this study, 100 available nucleotide sequences of VP1-Rotavirus belonging to different strains across the world were retrieved from NCBI database. The selected sequences were aligned, and a global consensus sequence was developed by using CLC work bench. The study involved immunoinformatic approaches and molecular docking studies to reveal the promiscuous epitopes that can be eventually used as active vaccine candidates for Rotavirus. In total, 27 highly immunogenic, antigenic, and non-allergenic T-cell and B-cell epitopes were predicted for the Multiepitope vaccine (MEV) against rotavirus. It was also observed that MEV can prove to be effective worldwide due to its high population coverage, demonstrating the consistency of this vaccine. Moreover, there is a high docking interaction and immunological response with a binding score of −50.2 kcal/mol, suggesting the vaccine's efficacy. Toll-like receptors (TLRs) also suggest that the vaccine is physiologically and immunologically effective. Collectively, our data point to an effective MEV against rotavirus that can effectively reduce viral infections and improve the health status worldwide.

Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine.

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Viral immunogenic footprints conferring T cell cross-protection to SARS-CoV-2 and its variants.

COVID-19 brought scenes from sci-fi movies into real life. Infected individuals include asymptomatic cases to severe disease leading to death, suggesting the involvement of the genetic constitution of populations and pathogens contributing to differential individuals' outcomes. To investigate shared immunogenic features between SARS-CoV-2 targets and other coronaviruses, we modeled their peptides in 3D structures of HLA-A*02:01 (pMHC), comparing their molecular surfaces These structures were also compared with a panel of epitopes from unrelated viruses, looking for potential triggers conferring cross-protection in uninfected individuals. As expected, SARS-CoV 1 and 2 peptides share molecular and physicochemical features, providing an explanation for the verified experimental immunogenicity among them. Surprisingly, even discordant sequences from human coronaviruses 229E, OC43 and epitopes from unrelated viruses involved in endemic human infections exhibit similar fingerprints of immunogenicity with SARS-CoV-2 peptides. The same approach indicates a conserved CD8+ T cell recognition between Wuhan SARS-CoV-2 sequences and altered peptides from Variants of Concern. Examination of structural data over epitope sequence analysis here could explain how previous infections may produce a heterologous immunity response in a global scale against emergent diseases such as Covid-19, mitigating its full lethal potential, and paves the way for the development of wide spectrum vaccine development.

A Recombinant Chimeric Protein-Based Vaccine Containing T-Cell Epitopes from Amastigote Proteins and Combined with Distinct Adjuvants, Induces Immunogenicity and Protection against Leishmania infantum Infection.

Currently, there is no licensed vaccine to protect against human visceral leishmaniasis (VL), a potentially fatal disease caused by infection with Leishmania parasites. In the current study, a recombinant chimeric protein ChimT was developed based on T-cell epitopes identified from the immunogenic Leishmania amastigote proteins LiHyp1, LiHyV, LiHyC and LiHyG. ChimT was associated with the adjuvants saponin (Sap) or monophosphoryl lipid A (MPLA) and used to immunize mice, and their immunogenicity and protective efficacy were evaluated. Both ChimT/Sap and ChimT/MPLA induced the development of a specific Th1-type immune response, with significantly high levels of IFN-γ, IL-2, IL-12, TNF-α and GM-CSF cytokines produced by CD4+ and CD8+ T cell subtypes (p < 0.05), with correspondingly low production of anti-leishmanial IL-4 and IL-10 cytokines. Significantly increased (p < 0.05) levels of nitrite, a proxy for nitric oxide, and IFN-γ expression (p < 0.05) were detected in stimulated spleen cell cultures from immunized and infected mice, as was significant production of parasite-specific IgG2a isotype antibodies. Significant reductions in the parasite load in the internal organs of the immunized and infected mice (p < 0.05) were quantified with a limiting dilution technique and quantitative PCR and correlated with the immunological findings. ChimT/MPLA showed marginally superior immunogenicity than ChimT/Sap, and although this was not statistically significant (p > 0.05), ChimT/MPLA was preferred since ChimT/Sap induced transient edema in the inoculation site. ChimT also induced high IFN-γ and low IL-10 levels from human PBMCs isolated from healthy individuals and from VL-treated patients. In conclusion, the experimental T-cell multi-epitope amastigote stage Leishmania vaccine administered with adjuvants appears to be a promising vaccine candidate to protect against VL.

Functional Profile of CD8+ T-Cells in Response to HLA-A*02:01-Restricted Mutated Epitopes Derived from the Gag Protein of Circulating HIV-1 Strains from Medellín, Colombia.

CD8+ T-cells play a crucial role in the control of HIV replication. HIV-specific CD8+ T-cell responses rapidly expand since the acute phase of the infection, and it has been observed that HIV controllers harbor CD8+ T-cells with potent anti-HIV capacity. The development of CD8+ T-cell-based vaccine against HIV-1 has focused on searching for immunodominant epitopes. However, the strong immune pressure of CD8+ T-cells causes the selection of viral variants with mutations in immunodominant epitopes. Since HIV-1 mutations are selected under the context of a specific HLA-I, the circulation of viral variants with these mutations is highly predictable based on the most prevalent HLA-I within a population. We previously demonstrated the adaptation of circulating strains of HIV-1 to the HLA-A*02 molecule by identifying mutations under positive selection located in GC9 and SL9 epitopes derived from the Gag protein. Also, we used an in silico prediction approach and evaluated whether the mutations found had a higher or lower affinity to the HLA-A*02. Although this strategy allowed predicting the interaction between mutated peptides and HLA-I, the functional response of CD8+ T-cells that these peptides induce is unknown. In the present work, peripheral blood mononuclear cells from 12 HIV-1+ HLA-A*02:01+ individuals were stimulated with the mutated and wild-type peptides derived from the GC9 and SL9 epitopes. The functional profile of CD8+ T-cells was evaluated using flow cytometry, and the frequency of subpopulations was determined according to their number of functions and the polyfunctionality index. The results suggest that the quality of the response (polyfunctionality) could be associated with the binding affinity of the peptide to the HLA molecule, and the functional profile of specific CD8+ T-cells to mutated epitopes in individuals under cART is maintained.

In Silico Design of Recombinant Chimera T Cell Peptide Epitope Vaccines for Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. Systemic VL is fatal if untreated and there are no prophylactic human vaccines available. Several studies suggest that Th1 cell-mediated immunity plays a major role in protecting against VL. In this chapter we describe a method for designing recombinant chimera vaccines in silico based on the prediction of T cell epitopes within protein antigens identified as potential protective immunogens. Development of a recombinant chimera protein (RCP) vaccine using T cell epitope peptides identified from four Leishmania proteins is used as an exemplar of this method.

Identifying molecularly defined antigens for a Histoplasma capsulatum-specific interferon gamma release assay.

BACKGROUND: In a previous work we showed the feasibility of an interferon gamma release assay (IGRA) for detecting latent infection by Histoplasma capsulatum. While in that proof-of-concept study we used crude fungal extracts as antigens, the newest IGRAs developed for other infections are based on molecularly defined antigens, mostly on mixtures of immunogenic peptides. AIMS: To identify proteins in H. capsulatum that might serve as molecularly defined antigens for an IGRA test. METHODS: We surveyed the literature looking for known H. capsulatum-immunogenic proteins and assayed two of them as antigens in an IGRA test, in a study that involved 80 volunteers. Furthermore, we used several bioinformatics tools to identify specific H. capsulatum proteins and to analyze possible strategies for the design of H. capsulatum-specific immunogenic peptides. RESULTS: Seven H. capsulatum-immunogenic proteins were retrieved from the literature. IGRA tests using either the heat shock protein 60 or the M antigen showed high sensitivities but low specificities, most likely due to the high sequence similarity with the corresponding orthologs in other pathogenic microorganisms. We identified around 2000 H. capsulatum-specific proteins, most of which remain unannotated. Class II T-cell epitope predictions for a small number of these proteins showed a great variability among different alleles, prompting for a "brute force" approach for peptide design. CONCLUSIONS: The H. capsulatum genome encodes a large number of distinctive proteins, which represent a valuable source of potential specific antigens for an IGRA test. Among them, the Cfp4 protein stands out as a very attractive candidate.

Immunogenicity of Potential CD4+ and CD8+ T Cell Epitopes Derived From the Proteome of Leishmania braziliensis.

Background: A safe and effective vaccine against human leishmaniasis still requires the identification of better antigens for immunization and adequate models to evaluate the immune response. To support vaccine development, this work tested the immunogenicity of 10 different peptides derived from the proteome of Leishmania braziliensis, which were selected by their in silico affinity to MHC complexes. Research design and Methods: Comparative cell proliferation assays were performed by culturing, in the presence of each peptide, PBMC cells from subclinical subjects (SC), cutaneous leishmaniasis patients with active disease (AD), post-treatment (PT) individuals, and healthy controls. Culture supernatants were then used for Th1, Th2, and Th17 cytokine measurements. Cells from selected PT samples were also used to assess the expression, by T cells, of the T-bet Th1 transcription factor. Results: A robust cell proliferation was observed for the SC group, for all the tested peptides. The levels of Th1 cytokines were peptide-dependent and had substantial variations between groups, where, for instance, IFN-γ and TNF levels were some of the highest, particularly on PT cultures, when compared to IL-2. On the other hand, Th2 cytokines displayed much less variation. IL-6 was the most abundant among all the evaluated cytokines while IL-4 and IL-10 could be found at much lower concentrations. IL-17 was also detected with variations in SC and AD groups. T-bet was up-regulated in CD4+ and CD8+ T cells from the PT group after stimulation with all peptides. Conclusions: The peptide epitopes can differentially stimulate cells from SC, AD, and PT individuals, leading to distinct immune responses.

Improved Immune Responses Against Zika Virus After Sequential Dengue and Zika Virus Infection in Humans.

The high levels of dengue-virus (DENV) seroprevalence in areas where the Zika virus (ZIKV) is circulating and the cross-reactivity between these two viruses have raised concerns on the risk of increased ZIKV disease severity for patients with a history of previous DENV infections. To determine the role of DENV preimmunity in ZIKV infection, we analyzed the T- and B-cell responses against ZIKV in donors with or without previous DENV infection. Using peripheral blood mononuclear cells (PBMCs) from donors living in an endemic area in Colombia, we have identified, by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay, most of the immunodominant ZIKV T-cell epitopes in the nonstructural (NS) proteins NS1, NS3, and NS5. Analyses of the T- and B-cell responses in the same donors revealed a stronger T-cell response against peptides conserved between DENV and ZIKV, with a higher level of ZIKV-neutralizing antibodies in DENV-immune donors in comparison with DENV-naïve donors. Strikingly, the potential for antibody-mediated enhancement of ZIKV infection was reduced in donors with sequential DENV and ZIKV infection in comparison with donors with DENV infection only. Altogether, these data suggest that individuals with DENV immunity present improved immune responses against ZIKV.

Bioinformatics Tools for the Prediction of T-Cell Epitopes.

T-cell responses are activated by specific peptides, called epitopes, presented on the cell surface by MHC molecules. Binding of peptides to the MHC is the most selective step in T-cell antigen presentation and therefore an essential factor in the selection of potential epitopes. Several in-vitro methods have been developed for the determination of peptide binding to MHC molecules, but these are all costly and time-consuming. In consequence, significant effort has been dedicated to the development of in-silico methods to model this event. Here, we describe two such tools, NetMHCcons and NetMHCIIpan, for the prediction of peptide binding to MHC class I and class II molecules, respectively, involved in the activation pathways of CD8+ and CD4+ T cells.

GLMEEMSAL epitope common in different isoforms of hMena elicits in vitro activation of cytotoxic T cells and stimulates specific antitumor immunity in BALB/c mice.

BACKGROUND: Alternative expression of human ortholog of murine Mena (hMena) hMena/hMena11a and hMena/hMenaΔv6 isoforms regulate the invasiveness and metastatic potential of tumor cells. It is then important to identify epitopes of these proteins that can elicit antitumor immune response to contribute to the elimination of cells with metastatic potential. METHODS: We assayed the capacity of the peptide GLMEEMSAL, common in hMena/hMena11a and hMena/hMenaΔv6 isoforms, to generate an antitumor immune response through an in vitro vaccination system with mature dendritic cells (MDC) loaded with this peptide and in vivo immunization using a tumor model with the mammary adenocarcinoma JC cell line to induce tumors in BALBc mice. RESULTS: MDC loaded with the peptide GLMEEMSAL elicited strong proliferation and activation of CD8+ T lymphocytes. The CTLs generated with this system were capable to lyse specifically BrCa and CeCa cell lines expressing either hMena/hMena11a or hMena/hMenaΔv6. Immunization with GLMEEMSAL provided protective and therapeutic antitumor activity as well as increased survival in BALB/c mice. CONCLUSION: These results are highly relevant for the use of common peptides among the different isoforms of hMena to develop immunotherapy protocols to counteract the growth and metastatic potential of tumors with over-expression of hMena.

Allergens of Blomia tropicalis: An Overview of Recombinant Molecules.

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis.

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1ß, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH+ and cured subjects. F2 also promoted the highest frequencies of CD3+CD4+IL-2+TNF-α-IFN-γ-, CD3+CD4+IL-2+TNF-α+IFN-γ-, CD3+CD4+IL-2+TNF-α-IFN-γ+, and CD3+CD4+IL-2+TNF-α+IFN-γ+ T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen (R = -0.428, P = 0.05) and liver sizes (R = -0.428, P = 0.05) and with increased hematocrit counts (R = 0.532, P = 0.015) in response to F1 domain, and with increased hematocrit (R = 0.512, P 0.02) and hemoglobin counts (R = 0.434, P = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 (R = -0.595, P = 0.005) and F2 (R = -0.462, P = 0.04). Conversely, F1 and F3 increased the CD3+CD8+IL-2+TNF-α-IFN-γ-, CD3+CD8+IL-2+TNF-α+IFN-γ-, and CD3+CD8+IL-2+TNF-α+IFN-γ+ T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4+-Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8+ T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico-predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4+ and CD8+ T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.

A recombinant chimeric protein composed of human and mice-specific CD4+ and CD8+ T-cell epitopes protects against visceral leishmaniasis.

In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4+ and CD8+ T-cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin-vaccinated mice presented significantly higher levels of antileishmanial IFN-γ, IL-12 and GM-CSF before and after challenge, which were associated with the reduction of IL-4 and IL-10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real-time PCR techniques were used. In addition, the protected animals presented higher levels of parasite-specific nitrite, as well as the presence of anti-Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL.

Combination of In Silico Methods in the Search for Potential CD4(+) and CD8(+) T Cell Epitopes in the Proteome of Leishmania braziliensis.

The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals.

Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis.

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.