Multidisciplinary analysis of HIV-1 elite controllers

Chronic HIV-1 infection is characterized by progressive depletion of CD4+ T lymphocytes, persistent immune activation and ongoing viral replication, leading to a profound immunodeficiency state if left untreated with antiretroviral therapy. However, a small percentage of infected individuals are able to maintain durable control of HIV replication and stable CD4 counts, in the absence of antiretroviral treatment (ART). This rare group of individuals are known as Elite Controllers (ECs) and represent evidence that control of infection without ART for years is possible, thereby providing an extraordinary insight into new vaccine and functional cure strategies. Despite extensive studies, the specific mechanisms by which ECs maintain control remain undefined. A better understanding of host factors that contribute to how ECs spontaneously control the infection is crucial for future therapeutic strategies. In Paper I, we showed that ECs possessed a richer gut microbiota compared to untreated HIV-infected individuals, and that several metabolic pathways were significantly different to untreated individuals. Specifically, the tryptophan catabolism pathway in ECs was very similar to healthy subjects, indicating a contributing factor for lower persistent immune activation usually observed in HIV-infected individuals. Our data suggest that the unique bacterial composition and metabolic profile of ECs may be involved in control of infection. Further, in Paper II, we used a modified antibody assay, LIPS, to perform antibody profiling against HIV-1 proteome in ECs. We found that LIPS detected a strong response against several HIV-1 fusion proteins in ECs compared to long-term treated individuals. Interestingly, the observed heterogeneity in antibody levels among ECs were not very different from untreated, viremic patients, indicating a non-homogenous patient group among ECs and a continuous viral expression with limited release of virus. By adapting a comprehensive analysis strategy of transcriptomics and targeted proteomics (Paper III), we demonstrated that more than 150 protein-coding genes and 33 soluble factors were differentially expressed in ECs compared to untreated patients. In particular, CXCR6 and SIGLEC1 (associated with viral entry and formation) were downregulated in ECs. Also, PD-1, an inhibitory receptor associated with T cell exhaustion, was significantly elevated in untreated vs both ECs and healthy subjects. The observed difference between ECs and untreated patients in molecular pathways regulating apoptosis, inflammation and cellular differentiation, suggests they play a synergistic role in HIV control. To further understand the differences in inhibitory receptor expression related to spontaneous HIV control, we assessed the expression of inhibitory molecules associated with T cell exhaustion on CD4+ T cells (Paper IV). We observed that ECs maintain a co-expression pattern of inhibitory receptors similar to healthy subjects and significantly different to both treated and untreated patients. We found that ECs harbor a “healthy” state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status. In summary, this thesis describes a comprehensive analysis of important immune factors that is associated with natural control of HIV infection in ECs. The multidisciplinary approach has provided a better understanding for the complexity of spontaneous HIV control and possible future therapeutic interventions

Richer gut microbiota with distinct metabolic profile in HIV infected Elite Controllers

Gut microbiota dysbiosis features progressive HIV infection and is a potential target for intervention. Herein, we explored the microbiome of 16 elite controllers (EC), 32 antiretroviral therapy naive progressors and 16 HIV negative controls. We found that the number of observed genera and richness indices in fecal microbiota were significantly higher in EC versus naive. Genera Succinivibrio, Sutterella, Rhizobium, Delftia, Anaerofilum and Oscillospira were more abundant in EC, whereas Blautia and Anaerostipes were depleted. Additionally, carbohydrate metabolism and secondary bile acid synthesis pathway related genes were less represented in EC. Conversely, fatty acid metabolism, PPAR-signalling and lipid biosynthesis proteins pathways were enriched in EC vs naive. The kynurenine pathway of tryptophan metabolism was altered during progressive HIV infection, and inversely associated with microbiota richness. In conclusion, EC have richer gut microbiota than untreated HIV patients, with unique bacterial signatures and a distinct metabolic profile which may contribute to control of HIV

<雜録>歐洲ニ於ケル農業的勞働關係

HIV infection provokes a myriad of pathological effects on the immune system where many markers of CD4+ T cell dysfunction have been identified. However, most studies to date have focused on single/double measurements of immune dysfunction, while the identification of pathological CD4+ T cell clusters that is highly associated to a specific biomarker for HIV disease remain less studied. Here, multi-parametric flow cytometry was used to investigate immune activation, exhaustion, and senescence of diverse maturation phenotypes of CD4+ T cells. The traditional method of manual data analysis was compared to a multidimensional clustering tool, FLOw Clustering with K (FLOCK) in two cohorts of 47 untreated HIV-infected individuals and 21 age and sex matched healthy controls. In order to reduce the subjectivity of FLOCK, we developed an "artificial reference", using 2% of all CD4+ gated T cells from each of the HIV-infected individuals. Principle component analyses demonstrated that using an artificial reference lead to a better separation of the HIV-infected individuals from the healthy controls as compared to using a single HIV-infected subject as a reference or analyzing data manually. Multiple correlation analyses between laboratory parameters and pathological CD4+ clusters revealed that the CD4/CD8 ratio was the preeminent surrogate marker of CD4+ T cells dysfunction using all three methods. Increased frequencies of an early-differentiated CD4+ T cell cluster with high CD38, HLA-DR and PD-1 expression were best correlated (Rho = -0.80, P value = 1.96×10-11) with HIV disease progression as measured by the CD4/CD8 ratio. The novel approach described here can be used to identify cell clusters that distinguish healthy from HIV infected subjects and is biologically relevant for HIV disease progression. These results further emphasize that a simple measurement of the CD4/CD8 ratio is a useful biomarker for assessment of combined CD4+ T cell dysfunction in chronic HIV disease

Quantitative humoral profiling of the HIV-1 proteome in elite controllers and patients with very long-term efficient antiretroviral therapy

A major challenge in evaluating the success of HIV eradication approaches is the need for accurate measurement of persistent HIV during effective antiretroviral therapy (ART). Previous studies have reported that the anti-HIV antibody assay "luciferase immuno-precipitation systems (LIPS)"can distinguish HIV-infected individuals harboring different sizes of the viral reservoirs. We performed antibody profiling of HIV-1 proteomes using LIPS in viremic progressors (n = 38), elite controllers (ECs; n = 19) and patients with fully suppressive long-term antiretroviral therapy (ART) (n = 19) (mean 17 years). IgG was quantified against six HIV-1 fusion proteins: p24, gp41, RT, Tat, integrase and protease. Lower antibody levels to all six-fusion proteins were observed in long-term ART patients compared to viremics (p &lt; 0.05). In contrast ECs had lower antibody levels only against Tat and Integrase (p &lt; 0.05). Principal component analysis and cluster-network analysis identified that 68% (13/19) of the long-term ART patients clustered together with 26% (5/19) ECs. The remaining ECs clustered together with the viremics indicating non-homogeneity among the ECs. The low anti-HIV levels in the long-term treated patients may indicate a restricted remaining viral replication. In contrast, the higher levels in ECs suggest a continuous viral expression with a limited concomitant release of extracellular virus.QC 20170512</p

Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells

Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a “healthy” state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status

Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers

A small subset of HIV-1 infected individuals, the "Elite Controllers" (EC), can control viral replication and restrain progression to immunodeficiency without antiretroviral therapy (ART). In this study, a cross-sectional transcriptomics and targeted proteomics analysis were performed in a well-defined Swedish cohort of untreated EC (n = 19), treatment naive patients with viremia (VP, n = 32) and HIV-1-negative healthy controls (HC, n = 23). The blood transcriptome identified 151 protein-coding genes that were differentially expressed (DE) in VP compared to EC. Genes like CXCR6 and SIGLEC1were downregulated in EC compared to VP. A definite distinction in gene expression between males and females among all patient-groups were observed. The gene expression profile between female EC and the healthy females was similar but did differ between male EC and healthy males. At targeted proteomics analysis, 90% (29/32) of VPs clustered together while EC and HC clustered separately from VP. Among the soluble factors, 33 were distinctive to be statistically significant (False discovery rate = 0.02). Cell surface receptor signaling pathway, programmed cell death, response to cytokine and cytokine-mediated signaling seem to synergistically play an essential role in HIV-1 control in EC.QC 20180316</p

Multiparametric Bioinformatics Distinguish the CD4/CD8 Ratio as a Suitable Laboratory Predictor of Combined T Cell Pathogenesis in HIV Infection

Abstract HIV disease progression is characterized by numerous pathological changes of the cellular immune system. Still, the CD4 cell count and viral load represent the laboratory parameters that are most commonly used in the clinic to determine the disease progression. In this study, we conducted an interdisciplinary investigation to determine which laboratory parameters (viral load, CD4 count, CD8 count, CD4 %, CD8 %, CD4/CD8) are most strongly associated with pathological changes of the immune system. Multiparametric flow cytometry was used to assess markers of CD4+ and CD8+ T cell activation (CD38, HLA-DR), exhaustion (PD-1, Tim-3), senescence (CD28, CD57), and memory differentiation (CD45RO, CD27) in a cohort of 47 untreated HIV-infected individuals. Using bioinformatical methods, we identified 139 unique populations, representing the “combined T cell pathogenesis,” which significantly differed between the HIV-infected individuals and healthy control subjects. CD38, HLA-DR, and PD-1 were particularly expressed within these unique T cell populations. The CD4/CD8 ratio was correlated with more pathological T cell populations (n = 10) and had a significantly higher average correlation coefficient than any other laboratory parameters. We also reduced the dimensionalities of the 139-unique populations by Z-transformations and principal component analysis, which still identified the CD4/CD8 ratio as the preeminent surrogate of combined T cell pathogenesis. Importantly, the CD4/CD8 ratio at baseline was shown to be significantly associated with CD4 recovery 2 y after therapy initiation. These results indicate that the CD4/CD8 ratio would be a suitable laboratory predictor in future clinical and therapeutic settings to monitor pathological T cell events in HIV infection.</jats:p

Box plots of all manual and FLOCK gated populations between HIV-infected and -uninfected subjects.

<p>Box plot representation of summary results of the two groups generated by the three methods used to investigate the same HIV immunopathogenesis dataset, manual data analysis (left), FLOCK data analysis using the single HIV reference (middle) and FLOCK data analysis using the artificial reference (right). The data is presented in a box and whisker plot where the horizontal line in the box is the median population occupation, the edges of the boxes are the 25th and 75th percentiles of the population occupation and the ‘whiskers’ represent the 10th and 90th percentiles of the population occupation, and the dots indicate outliers. The purple and grey boxes represent the HIV+ and healthy control group, respectively, where the green dots indicate outliers that are AIDS patients. Results of the multiple Mann-Whitney tests followed by Bonferroni adjustments between the HIV and healthy control group for each gated population is shown using P value significance codes found directly above: 0 *** 0.001 ** 0.01 * 0.05.</p

Summary of the HIV-infected cohort.

<p>Median (IQR) shown for all parameters; n: numbers.</p><p>Summary of the HIV-infected cohort.</p

Non-parametric Spearman rank tests correlation analysis of the manual, sFLOCK and aFLOCK immunopathological populations to the clinical parameters.

<p>^<b>a</b> Bonferroni corrections have been performed.</p><p>Non-parametric Spearman rank tests correlation analysis of the manual, sFLOCK and aFLOCK immunopathological populations to the clinical parameters.</p