Transmission and Evolution of Drug Resistant HIV-1 Variants

__Abstract__ In at least 10% of newly diagnosed patients, HIV-1 variants harbouring resistance mutations in protease and reverse transcripase are detected. This thesis describes the impact of such resistance mutations on the transmission efficacy of HIV, the evolution of drug resistant HIV variants after transmission and the clinical impact of transmitted drug resistant viruses

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. D

Lipid-specific IgMs induce antiviral responses in the CNS: implications for progressive multifocal leukoencephalopathy in multiple sclerosis

Progressive multi-focal leukoencephalopathy (PML) is a potentially fatal encephalitis caused by JC polyomavirus (JCV). PML principally affects people with a compromised immune system, such as patients with multiple sclerosis (MS) receiving treatment with natalizumab. However, intrathecal synthesis of lipid-reactive IgM in MS patients is associated with a markedly lower incidence of natalizumab-associated PML compared to those without this antibody repertoire. Here we demonstrate that a subset of lipid-reactive human and murine IgMs induce a functional anti-viral response that inhibits replication of encephalitic Alpha and Orthobunyaviruses in multi-cellular central nervous system cultures. These lipid-specific IgMs trigger microglia to produce IFN-β in a cGAS-STING-dependent manner, which induces an IFN-α/β-receptor 1-dependent antiviral response in glia and neurons. These data identify lipid-reactive IgM as a mediator of anti-viral activity in the nervous system and provide a rational explanation why intrathecal synthesis of lipid-reactive IgM correlates with a reduced incidence of iatrogenic PML in MS

Establishing mixed neuronal and glial cell cultures from embryonic mouse brains to study infection and innate immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods

Infection with the frequently transmitted HIV-1 M41L variant has no influence on selection of tenofovir resistance

Objectives: In similar to 10% of newly diagnosed HIV-1 patients, drug-resistant viral variants are detected. In such transmitted HIV-1 variants, the thymidine analogue mutation (TAM) M41L is frequently observed as a single resistance mutation and these viral variants often belong to phylogenetic transmission clusters. The presence of at least three TAMs, in particular patterns with M41L/L210W, impairs the efficacy of the extensively used drug tenofovir. We investigated whether the presence of a single M41L mutation at baseline influences the selection of resistance to tenofovir and emtricitabine in vitro and in vivo. Methods: The impact of M41L on the development of drug resistance to tenofovir and emtricitabine was determined by extensive in vitro selection experiments and investigation of the virological outcome of patients on a first-line regimen. Results: The presence of a single M41L mutation did not influence the selected mutational profile or the genetic barrier to resistance to tenofovir and/or emtricitabine during long-term in vitro selection experiments. In vivo, virological outcome of first-line regimens containing tenofovir and emtricitabine was comparable between patients diagnosed with HIV-1 harbouring M41L (n = 17, 16 were part of one transmission cluster) and WT virus (n = 248). Conclusions: Detection of a single M41L reverse transcriptase mutation at baseline did not influence the development of resistance in vitro or virological outcome on tenofovir-containing regimens in patients belonging to a large transmission cluster. Our results indicate that a high genetic barrier regimen may not be required when patients are diagnosed with HIV variants containing a single M41L mutation in reverse transcriptase

Persistence of frequently transmitted drug-resistant HIV-1 variants can be explained by high viral replication capacity

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1). To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. Conclusions: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity