Identification of immune biomarkers for use in early HIV detection and monitoring in sub-Saharan Africa = Identificación de biomarcadores de respuesta inmunitaria para la detección temprana y monitorización del VIH en África sub-Sahariana
[eng] BACKGROUND AND RATIONALE Acute HIV infection (AHI) is the period between the acquisition of the human immunodeficiency virus (HIV) and the development of HIV-specific antibodies that define seroconversion. AHI is characterised by high HIV viral replication and, in most cases, a transient non-s...
| Autor: | |
|---|---|
| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/122607 |
| Acceso en línea: | https://hdl.handle.net/2445/122607 http://hdl.handle.net/10803/565673 |
| Access Level: | acceso abierto |
| Palabra clave: | Malalties infeccioses Infeccions per VIH Marcadors bioquímics Àfrica subsahariana Communicable diseases HIV infections Biochemical markers Sub-Saharan Africa |
| Sumario: | [eng] BACKGROUND AND RATIONALE Acute HIV infection (AHI) is the period between the acquisition of the human immunodeficiency virus (HIV) and the development of HIV-specific antibodies that define seroconversion. AHI is characterised by high HIV viral replication and, in most cases, a transient non-specific febrile illness that typically occurs around 2 weeks after the HIV- transmission event. Primary HIV infection (PHI) is generally considered the period up to 6 months after infection and is a rapidly evolving phase characterized by the stepwise gain in positivity for the detection of HIV-RNA and HIV-specific antibodies. Different HIV antigen specificities appear in sequence after HIV transmission as do immunoglobulin G (IgG) subclass responses. As such, using different diagnostic tools, PHI has been categorised into ‘Fiebig stages’ that are useful in approximating infection date with relative accuracy. As a result of high viremia in bodily fluids, individuals are considered hyper-infectious during AHI. In areas of high HIV incidence, this phenomenon could contribute greatly to fuelling the worldwide HIV pandemic. Despite the importance of early diagnosis and treatment to reduce onward transmissions and prevent substantial irreversible immunological damage, AHI represents a ‘window period’ during which persons infected with HIV are commonly undiagnosed. Routine second generation HIV-rapid test algorithms provide negative or indeterminate results for up to 6-8 weeks after infection. During this time, HIV can only be diagnosed by detecting the presence of the virus itself. The current gold-standard test for confirming viremia is HIV-RNA viral load (VL) testing. However, technical and financial constraints make this technique very limited in low-income areas such as Sub-Saharan Africa, where the prevalence of AHI among febrile patients may reach 3%. VL testing is also used to monitor the efficacy of anti-retroviral treatment (ART). Achieving effective ART monitoring is a key determinant to ensure viral suppression and reach the UNAIDS 90-90-90 targets. Although considerable international efforts have resulted in a dramatic increase in ART coverage in the last years, relatively little progress has been achieved in the development of simple, accurate and affordable tools that allow proper surveillance of ART efficacy. VL monitoring is important for timely diagnosis of virological failure (VF) to allow early adherence interventions, prevent further transmissions and avoid delays in regimen switches that could lead to disease progression or emergence of drug resistances. Detecting virological failure depends on VL testing; whose availability is very limited in low and middle-income countries (LMIC) due to cost and operational constraints. The alternative to VL has often been clinical and/or immunological monitoring, which frequently results in patients remaining on failing ART as well as unnecessary regimen switches. Indeed, recent cross-sectional surveys reported that around 25%-35% of individuals on ART in Mozambique had detectable HIV viremia levels. As viremia increases during PHI, there is a striking cascade response of inflammatory cytokines. Significant efforts have been made to characterise host and viral proteins present during AHI aiming to identify biomarkers of progression or key pathological pathways that could be targeted to minimize HIV-induced immune damage over the course of infection. Subsets of T cells can be defined by their specificity, surface phenotype or degree of maturation, and any or all of these parameters can be affected by HIV infection. During PHI, many cells of the immune system show signs of extensive activation and a progressive loss of resting subsets. Generally, untreated HIV-infection is characterized by progressive CD4 T- cell depletion and CD8 T-cell expansion. The profound CD4 T-cell depletion is linked directly to the risk for opportunistic infections and mortality, while CD8 T-cell activation and exhaustion have been observed to be strong correlates of disease progression. Such alterations of CD4 and CD8 homeostatic mechanisms lead to progressive loss of the naïve and memory T-cell pool, resulting in an imbalance in T-cell phenotypes. Similarly, after HIV infection, accelerated aging of T cells or immunosenescence has been also associated with risk of adverse clinical events in HIV infected individuals. The challenges of identifying individuals during the AHI phase have resulted in a lack of critical information that constrains the development of therapeutic interventions. In this thesis, we provide a longitudinal characterization of T-cell subsets and the expression of soluble inflammatory biomarkers over the first year after PHI in a cohort of Mozambican adults and compare these changes with Chronic HIV-infection (CHI). Additionally, we assess the predictive power of these soluble biomarkers as surrogates of viremia for detection of AHI in seronegative febrile individuals and for identification of virological failure in ART- treated subjects. METHODS This thesis is based on research conducted at the Barcelona Institute for Global Health (ISGlobal)/ Hospital Clinic-Universitat de Barcelona in Spain, the AIDS Research Insitute/ Germans Trias i Pujol Research Institute (IGTP) and at the Centro de Investigaçao em Saude de Manhica (CISM) in Mozambique. To longitudinally analyze individuals during PHI, a screening based on reported-fever and pooled VL- testing was used to identify AHI in HIV-seronegative adults presenting at the Manhiça District Hospital (MDH), Mozambique. HIV-uninfected and chronically HIV infected individuals, both ART-naïve and on first-line ART, were also recruited at MDH in the context of the study. Plasma levels of inflammatory and immune biomarkers were subsequently determined by Luminex and ELISA, anti-HIV antibodies were analysed by flow-cytometry and Western Blot (WB) and T-cell phenotyping was performed through multi-panel flow- cytometry analysis. To evaluate biomarkers in treated HIV infected individuals, samples from a resistance survey study performed in 2013 were retrospectively analysed. The thesis is presented as a collection of four articles, of which three are accepted for publication in peer-reviewed international journals, and one is under review for publication. KEY RESULTS The findings from the studies that constitute this thesis provide a further characterization of the dynamics of immune response biomarkers over the different stages of HIV infection among adults in Mozambique. Immune response biomarkers across Fiebig staging at primary HIV infection A total of 85 AHI individuals were identified in our cohort as seronegative or indeterminate for rapid test and positive for plasma HIV-RNA among Mozambican adults seeking health care at the MDH. This represents an AHI prevalence of 3% among seronegative individuals reporting febrile illness. Soluble biomarkers, including inflammatory cytokines and general and HIV-specific antibody subtypes, were determined over different Fiebig stages at PHI, together with clinical and immunological characteristics. We compared cytokine levels between individuals at pre- (Fiebig I-IV) and post-seroconversion stages (Fiebig V-VI) at the screening visit. Thus, we identified a signature of four cytokines composed of BAFF, MCP-1, sCD163 and MIG that is highly associated with the PHI phase prior to development of the HIV-specific humoral response as determined by standard Western Blot serology. Longitudinal analysis of soluble and cellular biomarkers along the HIV infection process After longitudinal follow up of the PHI individuals, T-cell subsets and the expression of soluble inflammatory and immune biomarkers were characterized over the first year after of infection. Although plasma HIV viremia, CD4 and CD8 T cell counts undergo a rapid stabilization after HIV infection, several immunological parameters, including Th1Th17 CD4 T cells and activation or exhaustion of CD8 T cells continue to decrease even after 9 months post-infection. Importantly, no sign of immunosenescence was detected over the first year of HIV infection and no significant changes were observed in the Tregs population. Levels of IP-10, MCP-1, BAFF, sCD14, TNFR2 and TRAIL were significantly overexpressed at the first month of infection and underwent a prompt decrease in the following months. However, MIG and CD27 levels started to increase 1 month after infection and remained over- expressed for almost one year post-infection. Early levels of plasma TNFR2, sCD27, BAFF, IL- 10 and sCD14 cytokines were significantly associated with later levels of exhausted CD4 T- cells or with CD8 T-cell activation. Biomarkers as an accurate tool to identify acute HIV infection in febrile individuals In order to evaluate whether levels of a single or a combination of biomarkers had predictive accuracy to identify AHI among HIV-seronegative adults presenting with reported fever at the MDH, plasma levels of 49 inflammatory biomarkers from AHI (n=61) and non- HIV infected outpatients (n=65) were compared. The cytokine IP-10 demonstrated the best predictive accuracy for AHI detection (AUC=0.88 [95%CI 0.80-0.96]). A cut-off value of IP- 10≥161.6pg/mL provided a sensitivity of 95.5% (95%CI 85.5-99.5) and a specificity of 76.5% (95%CI 62.5-87.2) for AHI identification. Thus, an IP-10-based screening for subsequent AHI identification with VL could reduce the number of VL determinations necessary by 75%. After a cost-effectiveness analysis of this IP-10-based approach, we concluded that the implementation of an IP-10 screening test could avert from 21 to 84 new infections and save from US$176,609 to US$533,467 to the health system per 1,000 tested patients. IP-10 predictive power to detect virological failure in treated patients Due to the strong association with VL, we hypothesized that plasma IP-10 levels could be a surrogate marker of detectable viremia in ART-treated individuals. Consequently, we found that IP-10 levels were significantly higher in ART-treated subjects with detectable VL (108.2 pg/mL) as compared to those with undetectable VL (38.0 pg/mL) (U-test p<0.0001) in a cohort of 316 HIV-infected individuals on ART for more than a year. An IP-10 univariate model demonstrated high accuracy for prediction of detectable viremia (AUC=0.85 [95% Confidence Interval (CI) 0.80-0.90]). Using a cut-off value of IP-10≥44.2 pg/mL, the IP-10 model identified viremic ART-treated subjects with 91.9% sensitivity (95% CI 83.9-96.7) and 59.9% specificity (95% CI 52.0-67.4). Accordingly, we found that such IP-10 screening for potential virological failure would reduce by 43% the number of VL determinations required to monitor the same number of patients and this approach could potentially save 38% of VL-derived costs to the health system. CONCLUSIONS AND RECOMMENDATIONS AHI prevalence in febrile seronegative adults presenting at the MDH was found to be 3% as reported in 2008, having thus remained unchanged in this population in the last 5 years. After quantification of the soluble biomarkers across the different Fiebig stages described in PHI, we identified a signature of four cytokines composed of BAFF, MCP-1, sCD163 and MIG that was highly associated with the pre-seronversion phase as determined by WB serology. These effectors could provide clues for the development of vaccine or immunomodulatory strategies aimed at reducing the irreversible immune damage inflicted during PHI. Throughout characterization of cellular and soluble immune biomarkers along the different phases of HIV infection, we found that activated and effector memory CD8 T-cells together with Th1Th17 cells continued to decay several months after control of viremia. These findings indicate that balance in the T-cell compartments occurs months after viremia or CD4 count stabilize, suggesting persistent immune dysfunction in many different T cell subsets and raises the potential need for early initiation of therapy that could limit immunological damage. From the 49 soluble biomarkers that were assessed in febrile seronegative individuals, IP-10 demonstrated the best predictive power for AHI detection, providing a sensitivity of 95.5% and a specificity of 76.5%. Implementation of an IP-10-based screening for subsequent AHI diagnosis with VL is a cost effective strategy that could avert up to 84 new infections and save up to US$500,000 to the health system per 1,000 tested patients. IP-10 is also an accurate biomarker to screen individuals on ART for virological failure (VF), identifying 91.9% of patients with detectable viremia with a specificity of 59.9%. Employing IP-10 as a screening tool to target the individuals on ART most likely to require VL testing would reduce by 43% the number of VL determinations required to control viral suppression and it could save 38% of VL-derived costs to the health system. Thus, IP-10 quantification could be developed as a screening tool to identify both AHI in febrile seronegative individuals and VF in patients on ART with subsequent viral load testing. The implementation of these algorithms would facilitate AHI diagnosis and ART- monitoring in LMIC, as sub-Saharan Africa. Nevertheless, further research is necessary to explore the impact of other common HIV comorbidities on HIV-induced levels of IP-10, validate the IP-10 predictive power and optimize model cut-off values in other populations. |
|---|