Programme 4: Immunopathogenesis of HIV infection
Chair: Pepe Alcamí
Co-Chair: Ezequiel Ruiz-Mateos
Despite extensive investigation in the field of HIV pathogenesis, new questions are continuously emerging. This research area faces new challenges in the context of highly effective antiretroviral therapy: mechanisms involved in HIV control, immune damage in the absence of viral replication, virus host interactions at genetic level and the discovery of new restriction mechanisms. Besides, after the success of combination antiretroviral therapy (cART), the next great frontier is to cure infected persons, a formidable challenge.
Based on this scenario, the Immunopathogenesis Program of the RIS focuses on the following objectives:
1. To study the nature of immune response leading viral control and to understand the mechanisms of viral escape from these responses.
A small proportion of HIV-infected patients present apparent control of viral replication, or a delayed progression to immune deficiency or, in contrast, an atypical rapid progression to AIDS. All these examples are named extreme phenotypes. These patients have been extensively studied and specific genetic backgrounds and immunological mechanisms have been identified. The knowledge of the immune response and the mechanisms of control and immune escape in HIV infection are not only essential milestones to fully understand HIV pathogenesis, but also a major step in the development of an HIV vaccine.
2. To investigate the mechanisms of immune damage persistence despite cART.
Immune damage induced by HIV infection is only partially restored by cART-mediated suppression of viral replication. Even in those subjects experiencing optimal CD4 recovery, immune activation and a low-grade of systemic inflammation persist, being both factors involved in their higher risk of age-associated comorbidities and mortality. This situation is even aggravated in cases of poor CD4 recovery.
In particular, two clinical scenarios regarding immune damage study will be addressed:
- Late presentation (diagnosis in an advanced stage of the infection, below 200 cells/ul) is highly frequent in our context, and probably, is accompanied by a deeper and/or irreversible immune damage, which in some cases also affects the mechanisms underlying CD4 recovery under cART. Comprehensive studies on the nature of immune damage associated to late presentation and their potential poor CD4 recovery are urgently needed.
- Patients infected through vertical transmission (mother to child infection during pregnancy, delivery or breastfeeding): Although nearly no new cases of vertical HIV-infection occur in Spain, more than 1000 perinatal HIV infections are included in the Spanish cohort of patients CoRISpe, and the future clinical progression of these young HIV infected subjects is still unknown.
3. To characterize the pathogenic properties of the viral envelope.
The HIV envelope complex of proteins (gp120/gp41) is key for the recognition and binding to the receptor and co-receptors in target cells, mainly lymphocytes CD4, thus governing the early events of viral infection and fusion with the cellular membrane. Among viral factors, the HIV envelope glycoprotein (Env) seems to be highly relevant for the immunological outcome of patients after cART.
4. To develop new strategies against HIV persistence to achieve functional AIDS cure.
In spite of tremendous advances in a relatively short period of time, we do not yet have a cure for HIV infection. According to current knowledge, HIV persistence during long-term effective cART may be associated with different mechanisms, including the presence of a reservoir of long-lived latently infected cells and failure of host-responses to clear infected cells.
For a functional cure, we are working in the strategy of shock and kill or kick and kill. The objective is decreasing the pool of latent reservoirs inducing HIV gene expression in latently infected cells together with an intensive antiviral regimen. This could be combined with immune-based strategies aimed at enhancing the clearance of latently infected cells that have been induced to express HIV antigens. Several compounds named “latency reversal agents” (LRA) have been tested. In addition, a recent approach is the use of immunomodulatory agents to prevent or reduce the formation of viral reservoirs in acute infection.
These objectives are based on previous scientific background of RIS members but also in different structures that have been set up in the last years:
- Specific cohorts of patients with extreme phenotypes, mainly Long Term non-progressors and Elite controller patients.
- A centralized BioBank, representing the collaborative effort of all the groups involved in the network.
- A Scientific Repository with biological reagents of a high scientific value that includes a bank of envelopes from different clades, tropisms and patients with slow or rapid progression to AIDS.
Work Package 1 (WP1): Cohorts and patients with extreme phenotypes
Leader: Ezequiel Ruiz-Mateos
Patients with extreme phenotypes provide essential clues to understand HIV immunopathogenesis. These patients can control viral replication longer or have a delayed progression to immune deficiency or, on the contrary, can develop atypical rapid progression to AIDS.
This WP will provide a platform to design, coordinate, manage and analyse data from the cohorts of individuals with extreme phenotypes and the related samples stored in the HIV BioBank (WP2). There are two consolidated cohorts of special relevance due to immunologic and virologic characteristics of a subset of these patients, that can help us to find correlates of HIV-disease protection which will be important for the development of future HIV vaccines and immunotherapies:
- Long-Term Non Progressors Cohort (LTNP-RIS): LTNP-RIS patients are defined as those maintaining high levels of CD4 cells (above 500 cells/µl) and low levels of viral load (below 10,000 copies/mL) for more than 10 years without antiretroviral treatment.
- Controllers Cohort (EC-RIS): ECRIS Cohort brings together data from different groups of patients characterized by low viral load during at least one year in the absence of antiretroviral therapy (HIV-controllers): the Viremic Controllers (<2000 HIV-RNA copies/mL), and patients with undetectable viral load, the Elite Controllers (<50 HIV-RNA copies/mL).
In addition, two cohorts will be stablished in collaboration with CoRIS (P1):
- Late Presenters Cohort (LatePres-RIS): This cohort will include patients diagnosed in an advanced stage of the disease (below 200 cells/µl). It will be an essential source of patients for another cohort characterized for a poor immune reconstitution after the start of antiretroviral treatment.
- Acute/Recent Infection Cohort (PHI-RIS): As inclusion criteria, the seroconversion or the infection must have been documented in the first six months of infection. However, we distinguish between recent infection (<6 months) and acute infection (<30 days). The latter requires a very active recruitment structure. This cohort is of special interest for different RIS Programmes. In P2, the study of patients since seroconversion will have great significance from the epidemiological point of view. In P4, it will be possible to analyze virological and immunological factors in a very early stage of the infection. Finally, this cohort would be essential to P3 and P5, to study the efficacy of antiretrovirals and therapeutic vaccines at this stage of infection, and the influence of early treatment in viral reservoirs.
Work Package 2 (WP2): Biobank and repository
Leader: Mª Ángeles Muñoz-Fernández
The HIV BioBank is one of the central platforms of the RIS network, representing the collaborative effort of all the groups involved in the network and it has been very well valuated at internal and international evaluations. In addition, RIS has generated two more structures, a Repository with biological reagents of a high scientific value, and a Laboratory Platform dedicated to standardize immunological, molecular, cellular and histological protocols and new techniques.
Since its creation, HIV BioBank has maintained an ongoing commitment to quality and, for this reason, in 2008 was certified in the Quality Management Standard UNE-EN-ISO 9001 that guarantees the excellence of this biobank in the development of its processes and the quality of their products and services.
HIV BioBank collects, manages and stores blood, tissue and feces samples from 52 Spanish hospitals under conditions that guarantee their availability, quality and traceability. The donation of samples by HIV-1 infected patients are divided in 7 cohorts according to their characteristics: HIV infected adults (CoRIS), rapid progressors (RP), long term non progressors (LTNP), elite controllers (EC-RIS), acute/recent infection (PHI-RIS), vertical transmission (CoRISpe), and HIV/VHC coinfection. In this new period, the necessary infrastructure to set up samples of a new cohort of people starting Pre-Exposure Prophylaxis (PrEP) will be prepared. BioBank maintains a tight collaboration with P1 and P4 in charge of several of the cohorts, linking the samples with their associated datasets.
BioBank applies Standard Operating Procedures (SOP) for the preservation of viability, functionality, structural integrity and stability of the samples, and meets specific quality criteria and compliance with all necessary regulatory and statutory obligations.
One of the objective of the HIV BioBank is to provide samples to the applicant researchers from the RIS network or other national or international research programmes, whose requests have been approved by Clinical Research Ethics Committee (CEIC) of the institution where the study will be place, and that have been positively evaluated by the Scientific and Ethics Committes of the BioBank. In this sense, the BioBank has collaborated in the past with all the RIS programmes, increasing this collaboration with new projects every year.
Moreover, HIV BioBank offers services regarding the management of private collections for research projects and clinical trials. Amongst others, the services include: advice in legal and ethical matters, creation and management of new collections, designing informed consents and documents to store samples, management of preservation and conservation of samples, registration of the identifying information associated to biological material, storage of pre-existing collections…
- Repository: The aim of the repository is to put at the researchers disposition a collection of biological reagents of a high scientific value associated with donor´s clinical, functional and genetic information. This material is stored and maintained under conditions that guarantee its availability, viability and outstanding quality. In particular, new viral isolates and clones of particular interest are being generated by the different groups of P4 and stored in the viral repository: different HIV clades, variants resistant to different antiretrovirals and viruses with different tropism. Besides, groups working in viral entry and pathogenic effects of the envelope are generating a new collection of viral envelopes cloned in expression vectors and recombinant viruses.
- Laboratory platform: This platform, in association with researchers of the ImmunoBiology Lab, is standardizing protocols and techniques to give support to all the Programmes, and in particular to WP3, WP4, WP5 and WP6 of P4. The laboratory platform has cell culture, immunology and virology laboratories. The platform has standardized some techniques such as ELISPOT, ELISA, HIV R5 or X4 isolation, histological staining, quantification of proviral HIV-1 DNA by doplet digital PCR, DNA extraction from peripheral blood,... DNA/RNA/protein extraction from tissue (frozen and paraffin-embedded), amplification of genomic DNA, Quantitative Real-Time PCR, miRNA, siRNA, exosomas extractions, polymorphisms studies, DNA genotyping…
For more information: http://hivhgmbiobank.com/
Work Package 3 (WP3): Mechanisms of HIV control
Leader: Norma Rallón
The main objective of this WP is to better characterize Elite Controllers (EC) cohort to find special phenotypes among these patients regarding the ability to maintain a long-term control of HIV replication and a stable CD4 level, with the final goal to analyze the correlates associated with these special phenotypes. Specifically, our lines of research are:
- Elite Controllers (EC) special phenotypes identification. The current cohort of HIV controllers (EC-RIS cohort) is being used to establish a subcohort of patients (LTEC) able to maintain long-term control of HIV (more than five years) and stable CD4+ cell counts. These patients are being classified in two groups: LTEC with the highest level of suppression (HIV-RNA < 1 copy/mL) and LTEC with stable CD4+ cell counts. Besides, EC who spontaneously eradicate HCV infection are being identified from the EC-RIS cohort (supercontrollers).
- Virological characteristics associated with the EC special phenotypes are being studied with the goal to analyzing the correlates associated with these special phenotypes. In particular, we are studying the viral genomes, viral evolution and reservoirs.
- Immune correlates associated with the EC special phenotypes. We are interested in the specific immune response to the virus in these patients (in particular T lymphocyte response to HIV and HCV and role of dendritic cells) and the homeostasis (state of physiological balance) of T lymphocytes.
- Other host correlates associated with the EC special phenotypes. Microbes in HIV infection are likely playing an important role through microbial translocation and microbiome dysbiosis. In microbial translocation, bacteria or bacterial products translocate from intestine to systemic circulation, contributing to inflammation and pathogenesis. In microbiome dysbiosis, there is an alteration of microbial populations in the gastrointestinal tract, which contributes to inflammation and mucosal immune dysfunction. Therefore, we are studying both processes in these patients, together with soluble markers of inflammation, host genetic background and metabolomic profile.
- Clinical features in the EC special phenotypes. In this line of research, we are studying the prevalence of non-AIDS clinical events in these patients, and a set of variables that discriminate the presence or absence of clinical events.
Work Package 4 (WP4): Mechanisms of immune damage and recovery
Leader: Yolanda Pacheco
In this WP, we are performing basic research including immunological and virological characterization and functional studies, with a collaborative approach that integrates different groups in this Programme (P4) to comprehensive tackle mechanisms of immune damage and recovery through two cohorts:
- Late presenters. These patients are diagnosed in an advanced stage of the infection (below 200 CD4 T-cells/l), and many of them are characterized by poor CD4 recovery after beginning antiretroviral treatment and achieve viral control. A deeper evaluation of these last subjects will provide the earliest molecular signatures of poor CD4 recovery (during late presentation). This study will be supported by CoRIS (P1) and other WPs of P4: cohorts with extreme phenotypes (WP1) and RIS BioBank (WP2).
- Adolescents infected through vertical transmission. Several factors support that these patients infected through vertical transmission (mother to child transmission of infection during pregnancy, delivery or breastfeeding) could have specific mechanisms of immune damage: a) they were infected when their immune system was still maturating, b) a long-term exposure to the infection usually parallel to a long-term exposure to antiviral drugs, c) usual exposition to suboptimal doses of these drugs. However, this population has also shown a high regenerative potential. Therefore, adolescents infected through vertical transmission are a group of special interest to analyze the magnitude and reversibility of immune damage and the residual immunodeficiency. This study will be also supported by CoRISpe (P2, WP1) and RIS BioBank (P4, WP2).
From the immunological point of view, we are characterizing different immune cell sets and studying potential homeostatic (state of physiological balance) disturbances and its relationship with systemic inflammation. From the virological point of view, we are analyzing the involvement of the HIV envelope in the mechanisms of immune damage and recovery and the size of viral reservoirs. A systems biology approach to these data will be used to analyze the relevance of and relationships between different parameters.
The collaboration with other Programmes, like P1 (CoRIS) and P2 (CoRISpe), is also supporting the analysis of the clinical impact of such explored mechanisms of immune damage and recovery. Particularly, we are interested in the impact of immune activation, inflammation and viral factors in the further clinical progression of these cohorts, mainly in the development of oncologic and cardiovascular events.
Work Package 5 (WP5): The viral envelope as a pathogenic factor
Leader: Cecilio López-Galíndez
HIV envelope (Env) is the most important protein for the initial events of HIV life cycle. We think that viral pathogenesis and the clinical profile of HIV infected patients could be related to the ability of HIV-Env to trigger signals (for cell survival or for cell death) during the first HIV-cell contacts. To this aim, and with a multidisciplinary approach, we are studying a selection of viral envelopes to correlate their characteristics with HIV virulence.
Over the last four years, different groups of the RIS have isolated, cloned and expressed Envs from different HIV infected individuals, and produced a catalog of standardized techniques to characterize the function of Env. The different envelopes we are studying are:
- HIV Envs from patients with extreme phenotypes. We are characterizing viral envelopes from:
- Long-term non progressors and Elite controllers. To this aim we will collaborate with WP1 (cohorts with extreme phenotypes) and WP2 (BioBank) from P4 and P1 (CoRIS).
- Rapid progressors. In collaboration with P4 (WP1, WP2) and P1 (CoRIS).
- Late presenters with poor CD4 recovery under antiretroviral treatment (cART) vs late presenters with adequate CD4 recovery under cART. In collaboration with WP4 (mechanisms of immune damage and recovery) of P4 and with P1 (CoRIS).
- Envelopes from different clusters of HIV epidemics in Spain. We will focus on the analysis of envelopes from emerging subtypes F1, an expanding cluster in north-west of Spain that is replacing the circulating B-subtype. The causes for the emergence of this cluster are unknown. However, we know that a large proportion of transmitted F viruses are carrying an R5X4 phenotype and the F-infected patients present a rapid progression. Therefore, we hypothesize that HIV variants from this cluster display factors of virulence that could be due to their envelope characteristics. In this line of work, we are comparing the properties of F envelopes with envelopes from "classical" non-cluster F clade and B clades from the general Spanish population.
- Ancestral envelopes. We are comparing envelopes of ancestral and modern viruses.
- Envelopes from transmitted/founder (T/F) viruses. Infection by HIV-1 most often results from the successful transmission of a single virus, termed the transmitted/founder (T/F) virus. In this line of work, we are comparing the envelopes of T/F vs chronic viruses. This task is being performed in collaboration with P1 (CoRIS) and the cohort of acute/recent infection.
All these collections of cloned envelopes will be included in the Repository of the RIS (WP2). They are available for all groups involved in the WP in order to perform a systematic approach for the study of the immuno-pathological mechanisms, the cellular processes, and the signaling properties that mediate viral pathogenesis.
Work Package 6 (WP6): HIV reservoirs and strategies for cure
Leader: Mayte Coiras
Antiretroviral treatment (ART) is not enough to completely eliminate the viruses from the organism which persist in the viral reservoirs and, therefore, the cure for HIV infection remains elusive. At present, different therapeutic approaches to achieve a functional cure (long term control of HIV-1 replication and disease progression in the absence of ART) are being studied.
In this WP, we are working in the following lines:
- Standardization of protocols to quantify the reservoir (the proviral load) and latency models in primary cells. In order to get comparable results between all the participant groups, we are standardizing these protocols in peripheral blood CD4+ lymphocytes and tissues of infected patients (rectal biopsies). The latency models will be used to test the efficacy of different drugs in vitro and provide clinical trials with the appropriate tools to evaluate ex vivo the impact of such strategies on reservoir size.
- Strategies to diminish the reservoir size through latency reversal agents (LRA). The strategy of “shock and kill” or “kick and kill” aims at decreasing the pool of latent reservoirs inducing activation of HIV gene expression in latently infected cells together with an intensive antiviral regimen. This could be combined with immune-based strategies aimed at enhancing the clearance of latently infected cells that have been induced to express HIV antigens. Several LRA compounds have already been tested, and although clinical trials have shown the capacity of these compounds to trigger HIV reactivation, a clinical benefit defined by a reduction in the proviral load and reservoir size has not been proved to be effective so far. Combination of different LRAs will be tested in order to determine synergy between them.
- Treatment with immunomodulators to reduce the formation of viral reservoirs in acute infection. Viral reservoirs are established very early after infection. The immunomodulators strategy is based on the ability of these drugs to preserve and/or enhance the antiviral function of the innate immune system, the first line of defense of the immune system. This may affect the size of the reservoir since the beginning of the infection.
- Viral reservoir in patients with vertical transmission of HIV. We are studying viral reservoirs in the Spanish cohort of paediatric patients (CoRISpe), a cohort with more than 1000 perinatally HIV-infected patients. We are especially interested in perinatally HIV-1 infected children initiating very early antiretroviral therapy and children carrying delta32 heterozigosity in the CCR5 receptor gene.
- Viral reservoirs in HIV-infected patients with co-infection of hepatitis viruses such as HBV, HCV and HDV to determine how the co-infection with these viruses may affect the size of the reservoir.
- Nanoparticles. We are interested in developing nanoparticles as a tool to deliver miRNA or LRAs in order to silence or reactivate latent proviruses.