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.

DNA methylation changes on immune cells in Systemic Lupus Erythematosus.

DNA methylation as a process that regulates gene expression is crucial in immune cells biology. Global and gene specific methylation changes have been described in autoimmunity, especially in Systemic Lupus Erythematosus. These changes not only contribute to the understanding of the disease, but also some have been proposed as diagnostic or disease activity biomarkers. The present review compiles the most recent discoveries on this field on each type of immune cells, including specific changes in signalling pathways, genes of interest and its possible applications on diagnosis or treatment.

Identificación de mutaciones asociadas con resistencia a inhibidores de la transferencia de cadena de integrasa en pacientes VIH-1 positivos naive para tratamiento antirretroviral en Medellín, Colombia / Identification of mutations associated with resistance to Integrase Strand Transfer Inhibitors (INSTI) in ART-naïve HIV- 1 patients in Medellin, Colombia

Objetivo: Estimar las frecuencias de mutaciones y de polimorfismos adicionales asociados con resistencia a los fármacos inhibidores de la integrasa del virus de inmunodeficiencia humana tipo 1 (VIH-1). Metodología: Estudio descriptivo, de corte transversal, en individuos VIH-1 positivos de la ciudad de Medellín, quienes no habían recibido tratamiento antirretroviral. En ellos se determinó, a través del método de 2-dideoxinucleótidos y el sistema ABI3730XL, la secuencia del gen de la integrasa del VIH-1 a partir del ARN viral circulante, la cual fue analizada en la base de datos de resistencia a medicamentos antirretrovirales de la Universidad de Stanford y según reportes de literatura científica. Resultados: Se encontraron las siguientes mutaciones (con sus respectivas frecuencias): una mutación mayor, E138K (1/46), tres mutaciones accesorias G163E (3/46), L74I (3/50) y E157Q (2/48), una mutación no polimórfica A128T (1/49) y otras dos mutaciones potencialmente asociadas con resistencia a inhibidores de integrasa S230N (9/39) y S119P/R/T (4/47, 2/47 y 14/47, respectivamente). Conclusiones: En las secuencias analizadas, llama la atención la presencia de al menos una mutación asociada a resistencia a inhibidores de integrasa en el 14% de los individuos estudiados, sugiriendo una pobre presión selectiva de este tipo de fármacos en la población viral circulante en la zona.

Aim: To estimate the frequencies of major and accessory mutations, as well as additional polymorphisms associated with resistance to human immunodeficiency virus type 1 (VIH-1) integrase strand transfer inhibitors. Materials and methods: Descriptive cross-sectional study, focused on HIV-1 positive individuals from Medellín, recruited between 2013 and 2015, and that had not received antiretroviral therapy. In these patients, the sequence from HIV-1 integrase was determined from circulating viral RNA through Sanger chain termination method with the ABI3730XL system, and the sequences were analyzed using the HIV Drug Resistance Database from the University of Stanford, together with previous literature reports. Results: The following mutations associated with resistance to integrase strand transfer inhibitors, along with its respective frequencies, were found: one major mutation, E138K (1/46), three accessory mutations, G163E (3/46), L74I (3/50) and E157Q (2/48); one non-polymorphic mutation, A128T (1/49); and two mutations potentially associated with resistance to integrase strand transfer inhibitors, S230N (9/39) and S119P/R/T (4/47, 2/47 and 14/47, respectively). Conclusions: In the sequences analyzed, it is noteworthy the presence of at least one mutation related with resistance to integrase inhibitors in 14% of the studied patients, suggesting a poor selective pressure of this kind of drugs in the circulating viral population in our region.

Inmunología de la leptospirosis / Immunology of leptospirosis

Resumen Leptospirosis es una enfermedad re-emergente de distribución mundial ocasionada por espiroquetas patogénicas del género Leptospira que afectan humanos, animales domésticos o silvestres. Las manifestaciones clínicas de la enfermedad son diversas y son el resultado de la interacción de la respuesta inmune del hospedador y las condiciones de virulencia propias de las especies patógenas. Aunque se desconocen muchos aspectos de la inmunidad en la infección por Leptospira spp, es reconocido que los hospe deros susceptibles presentan diferencias en su respuesta inmune, como la activación/evasión del sistema del complemento, la activación de sub poblaciones celulares, la producción de citoquina y el desarrollo de anti cuerpos. El estudio del perfil inmunológico en pacientes con leptospirosis ha sido documentado y contribuye en la identificación de biomarcadores asociados con severidad. Esta revisión presenta algunos de los eventos relacionados con la respuesta inmune desde el ingreso de la bacteria en la fase inicial de la infección hasta su multiplicación y generación de enfer medad en el humano.

Abstract Leptospirosis is a re-emergent disease of worldwide distribution caused by pathogenic spirochetes of the Leptospira genus that affect humans, do mestic and wild animals. The clinical manifestations of the disease are diverse and are the result of the interaction of the immune response of the host and the virulence conditions of the pathogenic species. Although many aspects of immunity in infection with Leptospira spp are unknown, it is recognized that susceptible hosts show differences in their immune res ponse, such as activation / evasion of the complement system, activation of cellular subpopulations, production of cytokines, development of anti bodies. Study of the immunological profile in patients with leptospirosis has been documented and contributes in the identification of bio-markers associated with severity. This review presents updated events related to the immune response from the entry of the bacteria in the initial phase of the infection to its multiplication and generation of human disease.

Polyfunctional CD8+ T-Cell Response to Autologous Peptides from Protease and Reverse Transcriptase of HIV-1 Clade B.

BACKGROUND: The diversity of the HIV proteome influences the cellular response and development of an effective vaccine, particularly due to the generation of viral variants with mutations located within CD8+ T-cell epitopes. These mutations can affect the recognition of the epitopes, that may result in the selection of HIV variants with mutated epitopes (autologous epitopes) and different CD8+ T-cell functional profiles. OBJECTIVE: To determine the phenotype and functionality of CD8+ T-cell from HIV-infected Colombian patients in response to autologous and consensus peptides derived from HIV-1 clade B protease and reverse transcriptase (RT). METHODS: By flow cytometry, we compared the ex vivo CD8+ T-cell responses from HIV-infected patients to autologous and consensus peptides derived from HIV-1 clade B protease and RT, restricted by HLA-B*35, HLA-B*44 and HLA-B*51 alleles. RESULTS: Although autologous peptides restricted by HLA-B*35 and HLA-B*44 did not show any differences compared with consensus peptides, we observed the induction of a higher polyfunctional profile of CD8+ T-cells by autologous peptides restricted by HLA-B*51, particularly by the production of interferon-γ and macrophage inflammatory protein-1ß. The response by different memory CD8+ T-cell populations was comparable between autologous vs. consensus peptides. In addition, the magnitude of the polyfunctional response induced by the HLA-B*51-restricted QRPLVTIRI autologous epitope correlated with low viremia. CONCLUSION: Autologous peptides should be considered for the evaluation of HIV-specific CD8+ Tcell responses and to reveal some relevant epitopes that could be useful for therapeutic strategies aiming to promote polyfunctional CD8+ T-cell responses in a specific population of HIV-infected patients.

Cytotoxic CD4+ T-cells during HIV infection: Targets or weapons?

Classically, CD4+ T-cells have been referred as cytokine-producing cells and important players in immune responses by providing soluble factors that potentiate several effector immune functions. However, it is now evident that CD4+ T-cells can also elaborate cytotoxic responses, inducing apoptosis of target cells. Cytotoxic CD4+ T cells (CD4+ CTLs), exhibit cytolytic functions that resemble those of CD8+ T-cells; in fact, there is evidence suggesting that they may have a role in the control of viral infections. In this article, we discuss the role of CD4+ CTLs during HIV infection, where CD4+ CTLs have been associated with viral control and slow disease progression. In addition, we address the implication of CD4+ CTLs in the context of antiretroviral therapy and the partial reconstitution of CD8+ T-cells effector function.

Micronutrientes: un eslabón clave en la inmunopatogénesis de la lepra / Micronutrients: a key link in the immunopathogenesis of leprosy

The purpose of this work was to analyze the prevalence of fetal mortality (FM) in mothers in early adolescence (10-14 years), late adolescence (15-19 years) and in adults (20-34 years), during the period 2014-2016, in the North Department of Santander-Colombia. The factors taken into account were: gestation time, fetal weight, childbirth, basic causes, area of residence, and educational level of the mothers. Method: The study was retrospective, correlational, analytical-comparative. The database was from a secondary public access source of the National Administrative Department of Statistics (DANE-Colombia). The analysis was performed using the following tests: chi-square, Kruskal-Wallis H, Cramer's V coefficient, Goodman and Kruskal's gamma, Tukey's post-hoc procedures and the Bonferroni method based on Student's t-test. Results: The prevalence of FM for the years 2014-2016 was 10.0 per 1000 live births in mothers in early adolescence, 19.2 in mothers in late adolescence and 18.6 in adult mothers. It emerged that the prevalence of FM in pregnancies of under 22 weeks was higher in adult mothers, before delivery and during childbirth (chi-square = 32.023; p = 0.021), and there was a slight negative relationship between mother's age and weight of the fetus (gamma = -0.186; p = 0.014). The prevalence of FM was higher in adult mothers residing in the municipal district (chi-square = 80.18; p = 0.000), in mothers with primary, secondary and professional-level basic education (chi-square = 105.56; p = 0.000), and greater in adult mothers due to obstetric complications and birth trauma

La lepra es una enfermedad infecciosa crónica causada por Mycobacterium leprae, la cual tiene una notoria afinidad por la piel y los troncos nerviosos periféricos. Esta enfermedad se caracteriza por tener una clínica polimorfa que depende de la respuesta inmune del hospedero. La inmunopatogénesis de esta enfermedad aún representa un reto para los investigadores, y un eslabón faltante en su comprensión es el estudio de los micronutrientes, los cuales se ha demostrado que tienen la capacidad de modular la respuesta inmune innata y adaptativa. El objetivo de esta revisión es describir y relacionar algunos nutrientes, como las vitaminas A, D, E, C y B6, el folato, el zinc y el hierro, con la respuesta inmune en la lepra. Además, proponemos que algunos micronutrientes (vitaminas A, D y C y zinc) serían importantes para mitigar la aparición de reacciones lepróticas por medio de la modulación de la respuesta inmune en el hospedero infectado por M. leprae, y que micronutrientes como las vitaminas A, D, B6 y D, el folato, el hierro y el zinc serían importantes para reducir la incidencia de la lepra, dado que promoverían una mejor respuesta inmune en convivientes. Por lo tanto, el estudio del estado nutricional y el aporte suplementario con micronutrientes en convivientes y en afectados con lepra sería clave en la eliminación de esta enfermedad que ha deformado cuerpos y ha destruido sueños a lo largo de los siglos.

Potential immune escape mutations under inferred selection pressure in HIV-1 strains circulating in Medellín, Colombia.

The introduction of highly active antiretroviral therapy (HAART) has significantly improved life expectancy of HIV-infected patients; nevertheless, it does not eliminate the virus from hosts, so a cure for this infection is crucial. Some strategies have employed the induction of anti-HIV CD8+ T cells. However, the high genetic variability of HIV-1 represents the biggest obstacle for these strategies, since immune escape mutations within epitopes restricted by Human Leukocyte Antigen class I molecules (HLA-I) abrogate the antiviral activity of these cells. We used a bioinformatics pipeline for the determination of such mutations, based on selection pressure and docking/refinement analyses. Fifty HIV-1 infected patients were recruited; HLA-A and HLA-B alleles were typified using sequence-specific oligonucleotide approach, and viral RNA was extracted for the amplification of HIV-1 gag, which was bulk sequenced and aligned to perform selection pressure analysis, using Single Likelihood Ancestor Counting (SLAC) and Fast Unconstrained Bayesian Approximation (FUBAR) algorithms. Positively selected sites were mapped into HLA-I-specific epitopes, and both mutated and wild type epitopes were modelled using PEP-FOLD. Molecular docking and refinement assays were carried out using AutoDock Vina 4 and FlexPepDock. Five positively selected sites were found: S54 at HLA-A*02 GC9, T84 at HLA-A*02 SL9, S125 at HLA-B*35 HY9, S173 at HLA-A*02/B*57 KS12 and I223 at HLA-B*35 HA9. Although some mutations have been previously described as immune escape mutations, the majority of them have not been reported. Molecular docking/refinement analysis showed that one combination of mutations at GC9, one at SL9, and eight at HY9 epitopes could act as immune escape mutations. Moreover, HLA-A*02-positive patients harbouring mutations at KS12, and HLA-B*35-positive patients with mutations at HY9 have significantly higher plasma viral loads than patients lacking such mutations. Thus, HLA-A and -B alleles could be shaping the genetic diversity of HIV-1 through the selection of potential immune escape mutations.

Mycobacterium leprae-induced nerve damage: direct and indirect mechanisms.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. This disease is characterized by skin and peripheral nerve trunk damage. The mechanisms responsible for the observed nerve damage in leprosy could be directly related to the ability of M. leprae to infect Schwann cells, leading to triggering of signaling events. Therefore, we hypothesize that in response to M. leprae infection, activation of the Notch signaling pathway in Schwann cells could play a crucial role in glial cell dedifferentiation. On the other hand, nerve damage evidenced in this disease may be additionally explained by indirect mechanisms such as the immune response and genetic susceptibility of the host. The understanding of the mechanisms leading to nerve damage induced by M. leprae infection will allow us to generate valuable tools for the early detection of leprosy as well as for the mitigation of the effects of this disabling disease.

A hypothetical role for Notch signaling pathway in immunopathogenesis of leprosy.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae mainly affecting skin and peripheral nerves. Leprosy has a broad range of clinical manifestations that range from mild (tuberculoid leprosy) to severe (lepromatous leprosy) forms, and are highly dependent on the host's immune response. Among the immune response elements involved in the pathogenesis of leprosy are the Toll-like receptors (TLRs), vitamin D receptor (VDR), natural killer cells (NK), and T cells. These innate and adaptive immune response elements may be related to the Notch signaling pathway, which is involved in immune cell growth, differentiation, and proliferation. We hypothesize that failure in Notch signaling in leprosy patients may be associated to: 1) compromising NK cell maturation, lysing of infected cells, and CD4+ Th1 differentiation. 2) VDR alterations and TLR polymorphisms may affect expression of Notch Delta-like ligands (DLL) in antigen presenting cells (APCs). 3) altered DLL expression by APCs could compromise CD4+ T cell differentiation towards the Th1 and Th17 effector phenotypes; and finally 4) expression of Notch Jagged ligands would induce CD4+ T cell differentiation towards Th2 effector phenotype and alternative activation of macrophages. Altogether, these signaling failures could favor proliferation of M. leprae in the host. Therefore, evidence of the proposed immunologic failures in leprosy patients would be essential for the better understanding of immunopathogenesis of this disease, and would ultimately enable detection of susceptible individuals, providing a valuable tool for prevention of this debilitating disease.

Role of CD8+ T Cells in the Selection of HIV-1 Immune Escape Mutations.

Human immunodeficiency virus type-1 (HIV-1) infection represents one of the biggest public health problems worldwide. The immune response, mainly the effector mechanisms mediated by CD8+ T cells, induces the selection of mutations that allows the virus to escape the immune control. These mutations are generally selected within CD8+ T cell epitopes restricted to human leukocyte antigen class I (HLA-I), leading to a decrease in the presentation and recognition of the epitope, decreasing the activation of CD8+ T cells. However, these mutations may also affect cellular processing of the peptide or recognition by the T cell receptor. Escape mutations often carry a negative impact in viral fitness that is partially or totally compensated by the selection of compensatory mutations. The selection of either escape mutations or compensatory mutations may negatively affect the course of the infection. In addition, these mutations are a major barrier for the development of new therapeutic strategies focused on the induction of specific CD8+ T cell responses.

The APPEESFRS Peptide, Restricted by the HLA-B*35:01 Molecule, and the APPEESFRF Variant Derived from an Autologous HIV-1 Strain Induces Polyfunctional Responses in CD8+ T Cells.

Numerous reports have focused on consensus peptides to determine CD8+ T-cell responses; however, few studies evaluated the functional profile using peptides derived from circulating strains of a specific region. We determined the effector profile and maturation phenotype of CD8+ T-cells targeting the consensus APPEESFRS (AS9) epitope and its variant APPEESFRF (AF9), previously identified. The free energy of binding, maturation phenotype, and polyfunctional profile of both peptides were similar. The magnitude of CD8+ T-cell responses to AF9 was greater than the one elicited by AS9, although the difference was not significant. The polyfunctional profile of AF9 was characterized by CD107a/interleukin-2 (IL-2)/macrophage inflammatory protein beta (MIP1ß) and by interferon gamma (IFNγ)/MIP1ß/tumor necrosis factor alpha (TNFα) in response to AS9. TNFα production was significantly higher in response to AF9 than to AS9, and there was a negative correlation between the absolute number of CD8+ T-cell-producing TNFα and the plasma human immunodeficiency virus (HIV) load, suggesting a role of this cytokine in the control of HIV replication.

Selection pressure in CD8⁺ T-cell epitopes in the pol gene of HIV-1 infected individuals in Colombia. A bioinformatic approach.

One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells.

Resistencia a antirretrovirales: bases moleculares e implicaciones farmacológicas / Resistance to antiretrovirals: molecular bases and pharmacological implications

Una de las principales características del virus de la inmunodeficiencia humana es la alta diversidad genética, dada, en parte, por la baja fidelidad de la transcriptasa reversa, lo cual lleva a la generación de variantes virales con mutaciones asociadas a evasión de la respuesta inmune, cambio del tropismo celular o resistencia a medicamentos antirretrovirales. La terapia antirretroviral altamente activa es un esquema farmacológico contra el virus que utiliza dos o más familias de antirretrovirales, que pretende llevar la replicación viral a niveles indetectables, reduciendo entonces la morbimortalidad y aumentando la calidad y expectativa de vida de los individuos infectados. Las mutaciones que confieren resistencia a estos fármacos pueden ser fijadas en el genoma viral y ser transmitidas a nuevos hospederos, aportando así a la circulación de una población viral resistente a estos medicamentos que resulta en falla virológica. En esta revisión se describen los mecanismos más comunes asociados con resistencia a antirretrovirales y las mutaciones reportadas en la literatura.

One of the main characteristics of HIV is its high genetic diversity given in part by the low fidelity of the reverse transcriptase, which leads to the generation of viral variants bearing mutations associated with the evasion of the immune response, changes in the cellular tropism and/or antiretroviral resistance. Highly active antiretroviral therapy (HAART) is a pharmacologic scheme against HIV, which comprises two or more antiretroviral drug families, HAART aims to suppress viral replication which in turn decrease the morbidity and mortality of infected individuals, and increase their life expectancy and improve their quality of life. Mutations that confer resistance to antiretrovirals can be fixed in the viral genome and be transmitted to new hosts contributing to the movement of a viral population resistant to these drugs that results in virologic failure. In this review we describe the most common mechanisms associated with antiretroviral resistance, mutations reported in the literature and bioinformatics tools used for their determination.

Antígenos leucocitarios humanos y su asociación con resistencia/susceptibilidad a la infección por el VIH-1 / Human leukocyte antigens associated to resistance/susceptibility to HIV infection

El curso clínico de la infección por el virus de inmunodeficiencia humana tipo 1 es un proceso variable y complejo que depende de componentes virales y del hospedero. En la mayoría de los individuos infectados, la respuesta inmune generada en las fases iniciales de la infección logra controlar la replicación viral por mecanismos efectores innatos, de anticuerpos neutralizantes específicos y particularmente de la actividad de los linfocitos T CD8+ (LT CD8+). A pesar de generarse una respuesta inmune específica, esta se vuelve ineficaz en las etapas crónicas de la infección debido a cambios en los péptidos virales blanco, los cuales conducen a una pérdida del reconocimiento del antígeno presentado; dichos cambios son dados por la baja fidelidad de la transcriptasa reversa y la selección de cuasi-especies por la presión inmunológica. Durante la activación de los LT CD8+ es importante la señal ejercida por el péptido viral, el cual se presenta en el contexto de una molécula del complejo mayor de histocompatibilidad clase I (CMH-I). Estudios de correlación entre el CMH-I y la resistencia/susceptibilidad (R/S) al VIH se han centrado en cuatro aspectos: 1) la expresión de alelos específicos; 2) el grado de homocigocidad/heterocigocidad; 3) la exposición a diversos aloantígenos; 4) la relación con receptores KIR. En esta revisión se aborda el fenómeno de resistencia/susceptibilidad a la infección por el VIH-I relacionado con el CMH-I, cuyo entendimiento favorecerá el desarrollo de herramientas novedosas de intervención terapéutica.

The clinical course of infection with human immunodeficiency virus type-1 (HIV-1) is a variable and complex process that depends on viral and host components. In the majority of infected individuals, the immune response is generated from the initial phases of infection, achieving the control of the viral replication through innate effector mechanisms, neutralizing specific antibodies and particularly through cytotoxic CD8+T cell activity. Despite the generation of these specific cellular and humoral responses, it becomes ineffective in chronic stages of infection because of changes in viral peptide targets, the low fidelity of the reverse transcriptase and the immune pressure. During the activation of CD8+ T cells, the signal delivered by the viral peptide presented in the context of the class I major histocompatibility complex (MHC-I) molecules, is essential. Correlation studies between the MHC-I and the resistance/ susceptibility (R/S) to HIV infection have focused on four aspects, namely: 1) the expression of specific alleles; 4) the degree of homozygosity/heterozygosity; 3) the degree of exposure to different alloantigens; 4) the relation with KIR receptors. In this review, we focus on resistance/susceptibility to HIV-1 infection, particularly related to the MHC, hoping to have a better understanding of this phenomenon that may allow the development of novel therapeutic intervention tools.