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.

Multitranscript analysis reveals an effect of 2-deoxy-d-glucose on gene expression linked to unfolded protein response and integrated stress response in primary human monocytes and monocyte-derived macrophages.

BACKGROUND: Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) binds to hexokinase in a non-competitive manner and phosphoglucose isomerase in a competitive manner, blocking the initial steps of the glycolytic pathway. Although 2-DG stimulates endoplasmic reticulum (ER) stress, activating the unfolded protein response to restore protein homeostasis, it is unclear which ER stress-related genes are modulated in response to 2-DG treatment in human primary cells. Here, we aimed to determine whether the treatment of monocytes and monocyte-derived macrophages (MDMs) with 2-DG leads to a transcriptional profile specific to ER stress. METHODS: We performed bioinformatics analysis to identify differentially expressed genes (DEGs) in previously reported RNA-seq datasets of 2-DG treated cells. RT-qPCR was performed to verify the sequencing data on cultured MDMs. RESULTS: A total of 95 common DEGs were found by transcriptional analysis of monocytes and MDMs treated with 2-DG. Among these, 74 were up-regulated and 21 were down-regulated. Multitranscript analysis showed that DEGs are linked to integrated stress response (GRP78/BiP, PERK, ATF4, CHOP, GADD34, IRE1α, XBP1, SESN2, ASNS, PHGDH), hexosamine biosynthetic pathway (GFAT1, GNA1, PGM3, UAP1), and mannose metabolism (GMPPA and GMPPB). CONCLUSIONS: Results reveal that 2-DG triggers a gene expression program that might be involved in restoring protein homeostasis in primary cells. GENERAL SIGNIFICANCE: 2-DG is known to inhibit glycolysis and induce ER stress; however, its effect on gene expression in primary cells is not well understood. This work shows that 2-DG is a stress inducer shifting the metabolic state of monocytes and macrophages.

Dynamics of humoral immune response in SARS-CoV-2 infected individuals with different clinical stages.

Background: The COVID-19 pandemic remains a global health problem. As in other viral infections, the humoral immune response against SARS-CoV-2 is thought to be crucial for controlling the infection. However, the dynamic of B cells in the clinical spectrum of this disease is still controversial. This study aimed to characterize B cell subsets and neutralizing responses in COVID-19 patients according to disease severity through a one-month follow-up. Methods: A cohort of 71 individuals with SARS-CoV-2 infection confirmed by RT-PCR were recruited and classified into four groups: i) asymptomatic; ii) symptomatic outpatients; iii) hospitalized in ward, and iv) intensive care unit patients (ICU). Samples were taken at days 0 (inclusion to the study), 7 and 30. B cell subsets and neutralizing antibodies were assessed using multiparametric flow cytometry and plaque reduction neutralization, respectively. Results: Older age, male gender and body mass index over 25 were common factors among hospitalized and ICU patients, compared to those with milder clinical presentations. In addition, those requiring hospitalization had more comorbidities. A significant increase in the frequencies of CD19+ cells at day 0 was observed in hospitalized and ICU patients compared to asymptomatic and symptomatic groups. Likewise, the frequency of plasmablasts was significantly increased at the first sample in the ICU group compared to the asymptomatic group, but then waned over time. The frequency of naïve B cells decreased at days 7 and 30 compared to day 0 in hospitalized and ICU patients. The neutralizing antibody titers were higher as the severity of COVID-19 increased; in asymptomatic individuals, it was strongly correlated with the percentage of IgM+ switched memory B cells, and a moderate correlation was found with plasmablasts. Conclusion: The humoral immune response is variable among SARS-CoV-2 infected people depending on the severity and time of clinical evolution. In severe COVID-19 patients, a higher plasmablast frequency and neutralizing antibody response were observed, suggesting that, despite having a robust humoral immunity, this response could be late, having a low impact on disease outcome.

Identification of CD8+ T cell subsets that normalize in early-treated people living with HIV receiving antiretroviral therapy.

BACKGROUND: Although combined antiretroviral therapy (cART) has decreased the mortality associated with HIV infection, complete immune reconstitution is not achieved despite viral suppression. Alterations of CD8+ T cells and some of their subpopulations, such as interleukin (IL)-17-producing cells, are evidenced in treated individuals and are associated with systemic inflammation and adverse disease outcomes. We sought to evaluate if different CD8+ T cell subsets are differentially normalized during a clinical follow-up of people living with HIV (PLWH) receiving suppressive cART. METHODS: We explored the changes in the frequencies, activation/exhaustion phenotypes (HLA-DR, CD38, PD-1, and TIM-3), and function (total and HIV-specific cells expressing CD107a, perforin, granzyme B, interferon [IFN]-γ and IL-17) of CD8+ T cells from early-treated PLWH receiving cART in a 1-year follow-up, using a multidimensional flow cytometry approach. RESULTS: Despite continuous cART-induced viral suppression and recovery of CD4+ T cells, after a 1-year follow-up, the CD8+ T cell counts, CD4:CD8 ratio, PD-1 expression, and IL-17 production by CD8+ T cells exhibited incomplete normalization compared with seronegative controls. However, the proportion of CD8+ T cells with an exhausted phenotype (co-expressing PD-1 andTIM-3), and cells co-expressing cytotoxic molecules (Perforin and Granzyme B), reached normalization. CONCLUSIONS: Although suppressive cART achieves normalization of CD4+ T cell counts, only particular subsets of CD8+ T cells are more rapidly normalized in PLWH receiving cART, which could be routinely used as biomarkers for therapy efficiency in these patients.

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.

Implicaciones de los virus Zika y Chikungunya en el semen durante la transmisión sexual / Implications of Zika and Chikungunya viruses in semen during sexual transmission

Introducción: Los brotes de enfermedades causados por los virus Zika (VZIK) y Chikungunya (VCHIK) representan un problema de salud pública para muchos países tropicales y subtropicales. Objetivo: Discutir las implicaciones del hallazgo del VZIK y del VCHIK en el semen, y su relación con la transmisión sexual y la fertilidad masculina. Métodos: Se realizó una revisión narrativa de la literatura usando artículos indexados en PubMed (Medline), Embase y Scopus. Información, análisis y síntesis: Si bien los mosquitos del género Aedes son el vector principal y transmiten ambos virus, la transmisión sexual es una vía de infección significativa del VZIK y una posible ruta alterna para el VCHIK. La diseminación de estas arbovirosis vía linfática y sanguínea contribuye a la infección de diversos tejidos, incluyendo el tracto reproductivo masculino, donde el VZIK puede persistir. La infección de los testículos y quizás también de las glándulas accesorias del sistema reproductor masculino, se asocia con síntomas genitourinarios o alteraciones espermáticas, relacionadas con la detección del virus por largos periodos. Aunque no hay evidencia contundente sobre la presencia del VCHIK en el tracto genital masculino, se ha hallado en orina y semen. Además, se ha sugerido una posible persistencia en macrófagos que pueden infiltrar diferentes tejidos periféricos y cumplir una función de reservorio. Conclusiones: Hay presencia y persistencia de los virus Zika y Chikungunya en el tracto reproductor masculino. La infección en el semen se asocia con la transmisión sexual del virus, y con la alteración en la producción y calidad de los espermatozoides, con consecuencias clínicas graves en la salud sexual y reproductiva de los hombres infectados(AU)

Introduction: Disease outbreaks caused by Zika (ZIKV) and Chikungunya (CHIKV) viruses represent a public health problem for many tropical and subtropical countries. Objective: To discuss the implications of finding ZIKV and CHIKV in semen, and their relationship to sexual transmission and male fertility. Methods: A narrative review of the literature was carried out using articles indexed in PubMed (Medline), Embase and Scopus. Information, Analysis and Synthesis: Although Aedes mosquitoes are the primary vector and transmit both viruses, sexual transmission is a significant route of infection for ZIKV and a possible alternate route for CHIKV. Spread of these arboviruses via lymphatic and blood routes contributes to infection of various tissues, including the male reproductive tract, where ZIKV may persist. Infection of the testes and probably of the accessory glands of the male reproductive system is associated with genitourinary symptoms or sperm alterations, related to the detection of the virus for long periods. Although there is no conclusive evidence of the presence of CHIKV in the male genital tract, it has been found in urine and semen. In addition, a possible persistence in macrophages that can infiltrate different peripheral tissues and function as reservoir has been suggested. Conclusions: Zika and Chikungunya viruses can be present and persist in the male reproductive tract. Infection in semen is associated with sexual transmission of the virus and with alterations in the production and quality of spermatozoa, with serious clinical consequences in the sexual and reproductive health of infected men(AU)

Immune characterization of a Colombian family cluster with SARS-CoV-2 infection. / Caracterización inmunológica de un grupo familiar colombiano con infección por SARS-CoV-2

INTRODUCTION: Immunological markers have been described during COVID-19 and persist after recovery. These immune markers are associated with clinical features among SARSCoV-2 infected individuals. Nevertheless, studies reporting a comprehensive analysis of the immune changes occurring during SARS-CoV-2 infection are still limited. OBJECTIVE: To evaluate the production of proinflammatory cytokines, the antibody response, and the phenotype and function of NK cells and T cells in a Colombian family cluster with SARS-CoV-2 infection. MATERIALS AND METHODS: Proinflammatory cytokines were evaluated by RT-PCR and ELISA. The frequency, phenotype, and function of NK cells (cocultures with K562 cells) and T-cells (stimulated with spike/RdRp peptides) were assessed by flow cytometry. Anti-SARS-CoV-2 antibodies were determined using indirect immunofluorescence and plaque reduction neutralization assay. RESULTS: During COVID-19, we observed a high proinflammatory-cytokine production and a reduced CD56bright-NK cell and cytotoxic response. Compared with healthy controls, infected individuals had a higher frequency of dysfunctional CD8+ T cells CD38+HLA-DR-. During the acute phase, CD8+ T cells stimulated with viral peptides exhibited a monofunctional response characterized by high IL-10 production. However, during recovery, we observed a bifunctional response characterized by the co-expression of CD107a and granzyme B or perforin. CONCLUSION: Although the proinflammatory response is a hallmark of SARS-CoV-2 infection, other phenotypic and functional alterations in NK cells and CD8+ T cells could be associated with the outcome of COVID-19. However, additional studies are required to understand these alterations and to guide future immunotherapy strategies.

Introducción. Se han descrito diferentes marcadores inmunológicos durante la COVID-19, los cuales persisten incluso después de la convalecencia y se asocian con los estadios clínicos de la infección. Sin embargo, aún son pocos los estudios orientados al análisis exhaustivo de las alteraciones del sistema inmunológico en el curso de la infección. Objetivo. Evaluar la producción de citocinas proinflamatorias, la reacción de anticuerpos, y el fenotipo y la función de las células NK y los linfocitos T en una familia colombiana con infección por SARS-CoV-2. Materiales y métodos. Se evaluaron las citocinas proinflamatorias mediante RT-PCR y ELISA; la frecuencia, el fenotipo y la función de las células NK (en cocultivos con células K562) y linfocitos T CD8+ (estimulados con péptidos spike/RdRp) mediante citometría de flujo, y los anticuerpos anti-SARS-CoV-2, mediante inmunofluorescencia indirecta y prueba de neutralización por reducción de placa. Resultados. Durante la COVID-19 hubo una producción elevada de citocinas proinflamatorias, con disminución de las células NK CD56bright y reacción citotóxica. Comparados con los controles sanos, los individuos infectados presentaron con gran frecuencia linfocitos T CD8+ disfuncionales CD38+HLA-DR-. Además, en los linfocitos T CD8+ estimulados con péptidos virales, predominó una reacción monofuncional con gran producción de IL-10 durante la fase aguda y una reacción bifuncional caracterizada por la coexpresión de CD107a y granzima B o perforina durante la convalecencia. Conclusión. Aunque la reacción inflamatoria caracteriza la infección por SARS-CoV-2, hay otras alteraciones fenotípicas y funcionales en células NK y linfocitos T CD8+ que podrían asociarse con la progresión de la infección. Se requieren estudios adicionales para entender estas alteraciones y guiar futuras estrategias de inmunoterapia.

Immune characterization of a Colombian family cluster with SARS-CoV-2 infection / Caracterización inmunológica de un grupo familiar colombiano con infección por SARS-CoV-2

Abstract | Introduction: Immunological markers have been described during COVID-19 and persist after recovery. These immune markers are associated with clinical features among SARS- CoV-2 infected individuals. Nevertheless, studies reporting a comprehensive analysis of the immune changes occurring during SARS-CoV-2 infection are still limited. Objective: To evaluate the production of proinflammatory cytokines, the antibody response, and the phenotype and function of NK cells and T cells in a Colombian family cluster with SARS-CoV-2 infection. Materials and methods: Proinflammatory cytokines were evaluated by RT-PCR and ELISA. The frequency, phenotype, and function of NK cells (cocultures with K562 cells) and T-cells (stimulated with spike/RdRp peptides) were assessed by flow cytometry. Anti-SARS-CoV-2 antibodies were determined using indirect immunofluorescence and plaque reduction neutralization assay. Results: During COVID-19, we observed a high proinflammatory-cytokine production and a reduced CD56bright-NK cell and cytotoxic response. Compared with healthy controls, infected individuals had a higher frequency of dysfunctional CD8+ T cells CD38+HLA-DR-. During the acute phase, CD8+ T cells stimulated with viral peptides exhibited a monofunctional response characterized by high IL-10 production. However, during recovery, we observed a bifunctional response characterized by the co-expression of CD107a and granzyme B or perforin. Conclusion: Although the proinflammatory response is a hallmark of SARS-CoV-2 infection, other phenotypic and functional alterations in NK cells and CD8+ T cells could be associated with the outcome of COVID-19. However, additional studies are required to understand these alterations and to guide future immunotherapy strategies.

Resumen | Introducción. Se han descrito diferentes marcadores inmunológicos durante la COVID-19, los cuales persisten incluso después de la convalecencia y se asocian con los estadios clínicos de la infección. Sin embargo, aún son pocos los estudios orientados al análisis exhaustivo de las alteraciones del sistema inmunológico en el curso de la infección. Objetivo. Evaluar la producción de citocinas proinflamatorias, la reacción de anticuerpos, y el fenotipo y la función de las células NK y los linfocitos T en una familia colombiana con infección por SARS-CoV-2. Materiales y métodos. Se evaluaron las citocinas proinflamatorias mediante RT-PCR y ELISA; la frecuencia, el fenotipo y la función de las células NK (en cocultivos con células K562) y linfocitos T CD8+ (estimulados con péptidos spike/RdRp) mediante citometría de flujo, y los anticuerpos anti-SARS-CoV-2, mediante inmunofluorescencia indirecta y prueba de neutralización por reducción de placa. Resultados. Durante la COVID-19 hubo una producción elevada de citocinas proinflamatorias, con disminución de las células NK CD56 bright y reacción citotóxica. Comparados con los controles sanos, los individuos infectados presentaron con gran frecuencia linfocitos T CD8+ disfuncionales CD38+HLA-DR-. Además, en los linfocitos T CD8+ estimulados con péptidos virales, predominó una reacción monofuncional con gran producción de IL-10 durante la fase aguda y una reacción bifuncional caracterizada por la coexpresión de CD107a y granzima B o perforina durante la convalecencia. Conclusión. Aunque la reacción inflamatoria caracteriza la infección por SARS-CoV-2, hay otras alteraciones fenotípicas y funcionales en células NK y linfocitos T CD8+ que podrían asociarse con la progresión de la infección. Se requieren estudios adicionales para entender estas alteraciones y guiar futuras estrategias de inmunoterapia.

Antibody Responses in COVID-19: A Review.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide as a severe pandemic. Although its seroprevalence is highly variable among territories, it has been reported at around 10%, but higher in health workers. Evidence regarding cross-neutralizing response between SARS-CoV and SARS-CoV-2 is still controversial. However, other previous coronaviruses may interfere with SARS-CoV-2 infection, since they are phylogenetically related and share the same target receptor. Further, the seroconversion of IgM and IgG occurs at around 12 days post onset of symptoms and most patients have neutralizing titers on days 14-20, with great titer variability. Neutralizing antibodies correlate positively with age, male sex, and severity of the disease. Moreover, the use of convalescent plasma has shown controversial results in terms of safety and efficacy, and due to the variable immune response among individuals, measuring antibody titers before transfusion is mostly required. Similarly, cellular immunity seems to be crucial in the resolution of the infection, as SARS-CoV-2-specific CD4+ and CD8+ T cells circulate to some extent in recovered patients. Of note, the duration of the antibody response has not been well established yet.

NK Cell Activity and CD57+/NKG2Chigh Phenotype Are Increased in Men Who Have Sex With Men at High Risk for HIV.

Introduction: The HIV-exposed seronegative (HESN) status is for individuals who remain seronegative despite repeated exposure to HIV. One of the main cohorts within this group is men who have sex with men (MSM). Studies of this cohort have revealed different immunological and genetic mechanisms that can explain the phenomenon of natural HIV resistance. NK cells' higher effector capacity is related to natural resistance to HIV. Besides, a new population of NK cells with adaptive features was described recently. These cells are increased in some HESN cohorts and appear to be involved in better control of viral replication in primarily HIV-infected subjects. The present study evaluated the role of NK cells in the natural resistance to HIV-1 infection in MSM. Methodology: Phenotypic and functional features were evaluated in NK cells from two groups of MSM, at different risks of HIV infection, according to the number of sexual partners. The production of IFN-γ and ß-chemokines was included in the analysis, as well as the cytotoxic capacity and adaptive NK cell frequency. Genetic features, such as HLA and KIR allele frequencies, were also explored. Results: High-risk MSM exhibit an increased frequency of fully mature and CD57+/NKG2Chigh NK cells. These individuals also show higher cytotoxic capacity and IFN-γ production in response to K562 stimuli. NK cells with a CD107a+/IFN-γ+ functional profile were found more frequently and displayed higher IFN-γ production capacity among high-risk MSM than among low-risk MSM. The protective allele HLA-B∗18 was only present in the high-risk MSM group as well as HLA-B∗ 39. The protective phenotype KIR3DL1/S1-HLA-B∗Bw4, in a homozygous state, was particularly abundant in the high-risk population. Notably, some of these functional features were related to higher frequencies of mature and CD57+/NKG2Chigh NK cells, which, in turn, were associated with a higher number of sexual partners. Conclusion: The changes observed in the NK cell compartment can be driven by the magnitude of sexual exposure and immunological challenges of high-risk individuals, which could influence their resistance/susceptibility to HIV infection.

SARS-CoV-2 and the testis: similarity with other viruses and routes of infection.

Since the start of the latest coronavirus (SARS-CoV-2) outbreak, the number of infected individuals and cases of coronavirus disease (COVID-19) has been increasing exponentially worldwide. Of interest is existing evidence that orchitis can develop due coronavirus infection. It is therefore not unreasonable to believe that SARS-CoV-2 could be transmitted by semen. Consequently, it is of paramount importance that individuals who could potentially be infected take all possible care to mitigate the likely risk of passing on the infection through sexual intercourse.

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.

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.

Chikungunya Virus and Zika Virus, Two Different Viruses Examined with a Common Aim: Role of Pattern Recognition Receptors on the Inflammatory Response.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are 2 reemerging arboviruses that have been the focus of public health institutions worldwide, since the last decades and following a spate of outbreaks in tropical and subtropical areas. The disease caused by both viruses manifests itself first as an acute stage of severe inflammation into the infected tissues, which later progresses to arthritis and chronic polyarthralgia in the case of CHIKV or congenital microcephaly and neurological disorders such as Guillain-Barré syndrome in the case of ZIKV. This review aims to summarize on current knowledge of the role of different pattern recognition receptors that leads to an elevated production and secretion of antiviral response (interferon) and severe inflammation in response to CHIKV and ZIKV infection.

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.

An altered cytotoxic program of CD8+ T-cells in HIV-infected patients despite HAART-induced viral suppression.

Despite the suppression of viral replication induced by the highly active anti-retroviral therapy (HAART), an increased immune activation and inflammatory state persists in HIV-infected patients, contributing to lower treatment response and immune reconstitution, and development of non-AIDS conditions. The chronic activation and inflammation affect the functionality and differentiation of CD8+ T-cells, particularly reducing their cytotoxic capacity, which is critical in the control of HIV replication. Although previous studies have shown that HAART induce a partial immune reconstitution, its effect on CD8+ T-cells cytotoxic function, as well as its relationship with the inflammatory state, is yet to be defined. Here, we characterized the functional profile of polyclonal and HIV-specific CD8+ T cells, based on the expression of cell activation and differentiation markers, in individuals chronically infected with HIV, under HAART. Compared with seronegative controls, CD8+ T-cells from patients on HAART exhibited a low degranulation capacity (surface expression of CD107a), with consequent low secreted levels and high intracellular expression of granzyme B and perforin. This degranulation defect was particularly observed in those cells expressing the activation marker HLA-DR, which were further characterized as effector memory cells with high expression of CD57. The expression of CD107a, but not of granzyme B and perforin, in CD8+ T-cells from HIV-infected patients on HAART reached levels similar to those in seronegative controls when the treatment duration was higher than 25 months. In addition, the expression of CD107a was negatively correlated with the expression of exhaustion markers on CD8+ T-cells and the plasma inflammatory molecule sCD14. Thus, despite HAART-induced viral suppression, CD8+ T-cells from HIV-infected patients have an alteration in their cytotoxic program. This defect is associated with the cellular activation, differentiation and exhaustion state, as well as with the inflammation levels, and can be partially recovered with a long and continuous treatment.