Investigating the consequences of interference between multiple CD8+ T cell escape mutations in early HIV infection

During early human immunodeficiency virus (HIV) infection multiple CD8+ T cell responses are elicited almost simultaneously. These responses exert strong selective pressures on different parts of HIV's genome, and select for mutations that escape recognition and are thus beneficial to the virus. Some studies reveal that the later these escape mutations emerge, the more slowly they go to fixation. This pattern of escape rate decrease(ERD) can arise by distinct mechanisms. In particular, in large populations with high beneficial mutation rates interference among different escape strains – an effect that can emerge in evolution with asexual reproduction and results in delayed fixation times of beneficial mutations compared to sexual reproduction – could significantly impact the escape rates of mutations. In this paper, we investigated how interference between these concurrent escape mutations affects their escape rates in systems with multiple epitopes, and whether it could be a source of the ERD pattern. To address these issues, we developed a multilocus Wright-Fisher model of HIV dynamics with selection, mutation and recombination, serving as a null-model for interference. We also derived an interference-free null model assuming initial neutral evolution before immune response elicitation. We found that interference between several equally selectively advantageous mutations can generate the observed ERD pattern. We also found that the number of loci, as well as recombination rates substantially affect ERD. These effects can be explained by the underexponential decline of escape rates over time. Lastly, we found that the observed ERD pattern in HIV infected individuals is consistent with both independent, interference-free mutations as well as interference effects. Our results confirm that interference effects should be considered when analyzing HIV escape mutations. The challenge in estimating escape rates and mutation-associated selective coefficients posed by interference effects cannot simply be overcome by improved sampling frequencies or sizes. This problem is a consequence of the fundamental shortcomings of current estimation techniques under interference regimes. Hence, accounting for the stochastic nature of competition between mutations demands novel estimation methodologies based on the analysis of HIV strains, rather than mutation frequencies

Within-epitope interactions affect CTL-escape estimation

As human immunodeficiency virus (HIV) begins to replicate within hosts, immune responses are elicited against it. Escape mutations in viral epitopes-immunogenic peptide parts presented on the surface of infected cells-allow HIV to partially evade these responses, and thus rapidly go to fixation. The faster they go to fixation, i.e., the higher their escape rate, the larger the selective pressure exerted by the immune system is assumed to be. This relation underpins the rationale for using escapes to assess the strength of immune responses. However, escape rate estimates are often obtained by employing an aggregation procedure, where several mutations that affect the same epitope are aggregated into a single, composite epitope mutation. The aggregation procedure thus rests upon the assumption that all within-epitope mutations have indistinguishable effects on immune recognition. In this study, we investigate how violation of this assumption affects escape rate estimates. To this end, we extend a previously developed simulation model of HIV that accounts for mutation, selection, and recombination to include different distributions of fitness effects (DFEs) and inter-mutational genomic distances. We use this discrete time Wright-Fisher based model to simulate early within-host evolution of HIV for DFEs and apply standard estimation methods to infer the escape rates. We then compare true with estimated escape rate values. We also compare escape rate values obtained by applying the aggregation procedure with values estimated without use of that procedure. We find that across the DFEs analyzed, the aggregation procedure alters the detectability of escape mutations: large-effect mutations are overrepresented while small-effect mutations are concealed. The effect of the aggregation procedure is similar to extracting the largest-effect mutation appearing within an epitope. Furthermore, the more pronounced the over-exponential decay of the DFEs, the more severely true escape rates are underestimated. We conclude that the aggregation procedure has two main consequences. On the one hand, it leads to a misrepresentation of the DFE of fixed mutations. On the other hand, it conceals within-epitope interactions that may generate irregularities in mutation frequency trajectories that are thus left unexplained

A de novo approach to inferring within-host fitness effects during untreated HIV-1 infection

Funder: Isaac Newton Trust; funder-id: http://dx.doi.org/10.13039/501100004815Funder: Li Ka Shing Foundation; funder-id: http://dx.doi.org/10.13039/100007421Funder: Division of Intramural Research, National Institute of Allergy and Infectious Diseases; funder-id: http://dx.doi.org/10.13039/100006492Funder: Helsingin Yliopisto; funder-id: http://dx.doi.org/10.13039/100007797In the absence of effective antiviral therapy, HIV-1 evolves in response to the within-host environment, of which the immune system is an important aspect. During the earliest stages of infection, this process of evolution is very rapid, driven by a small number of CTL escape mutations. As the infection progresses, immune escape variants evolve under reduced magnitudes of selection, while competition between an increasing number of polymorphic alleles (i.e., clonal interference) makes it difficult to quantify the magnitude of selection acting upon specific variant alleles. To tackle this complex problem, we developed a novel multi-locus inference method to evaluate the role of selection during the chronic stage of within-host infection. We applied this method to targeted sequence data from the p24 and gp41 regions of HIV-1 collected from 34 patients with long-term untreated HIV-1 infection. We identify a broad distribution of beneficial fitness effects during infection, with a small number of variants evolving under strong selection and very many variants evolving under weaker selection. The uniquely large number of infections analysed granted a previously unparalleled statistical power to identify loci at which selection could be inferred to act with statistical confidence. Our model makes no prior assumptions about the nature of alleles under selection, such that any synonymous or non-synonymous variant may be inferred to evolve under selection. However, the majority of variants inferred with confidence to be under selection were non-synonymous in nature, and in most cases were have previously been associated with either CTL escape in p24 or neutralising antibody escape in gp41. We also identified a putative new CTL escape site (residue 286 in gag), and a region of gp41 (including residues 644, 648, 655 in env) likely to be associated with immune escape. Sites inferred to be under selection in multiple hosts have high within-host and between-host diversity although not all sites with high between-host diversity were inferred to be under selection at the within-host level. Our identification of selection at sites associated with resistance to broadly neutralising antibodies (bNAbs) highlights the need to fully understand the role of selection in untreated individuals when designing bNAb based therapies

The role of early cytotoxic lymphocyte (CTL) escape in the pathogenesis of HIV-1 subtype C infection

Includes abstract.Includes bibliographical references.This study investigated the frequency and timing of cytotoxic T-lympthocyte (CTL) escape and its pathogenic consequences on HIV-1 subtype C disease progression

Biological And Clinical Markers Of Neuronal Injury In Primary And Chronic Hiv-1 Infection

The use of antiretroviral therapy (ART) has shifted the neurological manifestations of HIV-1 infection toward mild but debilitating HIV-associated neurocognitive disorder (HAND). Through two studies, we sought to characterize neuronal injury during primary and chronic HIV infection and to describe its relationship with HAND. The aim of the first study was to quantify cerebrospinal fluid (CSF) and neuroimaging biomarkers of neuronal injury in primary HIV infection (PHI). We compared CSF levels of neurofilament light chain (NFL), tau, and amyloid proteins in 92 subjects with PHI and 25 controls and examined relationships with disease progression and neuroinflammation, neuropsychological testing, and proton-magnetic resonance spectroscopy (MRS). We hypothesized that PHI is characterized by increased CSF NFL that correlates with neuronal inflammation, and that tau and amyloid levels are normal in PHI. NFL was elevated in PHI (p=0.0004) and correlated with CSF neopterin (r=0.38, p=0.0005), IP-10 (r=0.39, p=0.002), WBCs (r=0.32, p=0.004), and CSF:plasma albumin ratio (r=0.60, p\u3c0.0001). NFL correlated with decreased N-acteylaspartate and glutamate in the anterior cingulate, frontal white matter, and parietal gray matter (r\u3e0.30, p\u3c0.05). Beta-amyloid was elevated in PHI (p=0.0005) and correlated with time infected (r=0.34, p=0.003). Neither marker correlated with neuropsychological abnormalities. T-tau and amyloid precursor proteins did not differ between groups. The aim of the second study was to characterize HIV-infected patients with neuro-symptomatic CSF `escape,\u27 defined as detectable CSF HIV RNA in the setting of treatment-suppressed plasma levels or CSF RNA \u3e1 log higher than plasma RNA. We conducted a retrospective case series of virologically controlled HIV-infected patients on ART with progressive neurological abnormalities who were determined to have CSF `escape\u27 at 4 urban medical centers in the United States and Europe. We recorded levels of CSF HIV RNA and inflammatory markers, clinical signs and symptoms, and magnetic resonance imaging (MRI) findings. We hypothesized that individuals with this condition would have inflammation in CSF and MRI studies, that CSF virus would be resistant to the ART regimen, and that symptoms would improve when ART was changed based upon central nervous system (CNS) drug penetration and resistance genotyping. 10 patients presented with sensory, motor, and cognitive abnormalities. Median CSF HIV RNA was 3900 copies/mL; median plasma HIV RNA was 62 copies/mL. Median CD4+ T cell count was 482 cells/mm3. All patients had been controlled \u3c500 copies/mL for median 27.5 months and 5/10 had been suppressed \u3c50 copies/mL for median 19.5 months. Patients were on a stable ART regimen for median 21 months. All had CSF pleocytosis or elevated CSF protein; 7/8 had MRI abnormalities; and 6/7 harbored CSF resistance mutations. Following optimization of ART, 8/9 patients improved clinically. Although these processes occur at distinct time points in the disease, both neuronal injury during PHI and the development of symptomatic CSF `escape\u27 in chronic, well-treated infection are associated with, and possibly caused by, mechanisms involving immune activation and inflammation within the CNS. The inflammatory milieu induced by the activity of HIV in invading cells and triggering an immune response has important implications throughout the time course of infection, and may be particularly important for understanding the pathophysiology of HAND

A virologist’s guide to hide and seek : evasion of innate immunity by primate lentiviruses

HIV is the cause of a chronic, incurable infection in 37 million people worldwide in 2014. This thesis investigates how the immune system detects HIV and how in turn HIV avoids detection by the immune system. The understanding of the viral evasion mechanisms that prevent immune detection (“Hide and Seek”) is important to successfully develop future vaccines and cure strategies for HIV. Primate lentiviruses belong to the retrovirus family and include the human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency viruses (SIV). HIV infects cells of the immune system, including subsets of T cells and dendritic cells (DC). Upon cell entry, the detection of the virus by cellular pattern recognition receptors triggers an intracellular cascade of innate antiviral defense mechanisms. In DCs, these mechanisms include the secretion of interferon α and the induction of cellular restriction factors, among these members of the APOBEC3 family that inhibit viral replication. As demonstrated in Paper III, low doses of interferon α protected DCs from HIV-1 infection and limited viral spread from DCs to T cells by inducing an increase in APOBEC3G, F and A expression. DCs are professional antigen presenting cells that present antigen to cells of the innate and adaptive immune system. Invariant natural killer T cells (iNKT) cells are innate T cells that recognize endogenous and exogenous lipid antigens presented by CD1d. Activated iNKT cells regulate the immune response by producing cytokines that recruit and activate innate and adaptive immune cells. Previous studies have shown that the HIV-1 accessory proteins Vpu and Nef interfere with CD1d cell surface expression in infected DCs, thus inhibiting the effective activation of iNKT cells. The results of Paper II demonstrated that infected DCs respond to HIV-1 infection by increasing CD1d surface levels and enhanced presentation of the endogenous lipid GlcCer. This enabled iNKT cell activation by HIV-infected DCs. However, HIV-1 counteracts iNKT cell activation by reducing CD1d cell surface expression using the HIV-1 proteins Nef and Vpu. In Paper I, efforts to elucidate the mechanism of CD1d antagonism by Vpu identified a highly conserved C-terminal APW motif in HIV-1 group M subtype B Vpu proteins that was necessary for CD1d downregulation. Moreover, we identified this immune evasion mechanism to be a conserved function of diverse HIV-1 and related SIV Vpus. These findings emphasize the role of CD1d-mediated immunity in the antiviral defense against HIV-1 and support the need for further studies investigating the therapeutic potential of Vpu inhibition in the future. Previous studies found that innate cellular immune responses are altered in chronic HIV-1 infection. Our results in Paper IV from an occupational cohort in Guinea-Bissau suggest that this is a general phenomenon of chronic HIV infection as NK and iNKT cells were partly lost and the remaining populations displayed elevated activation levels in chronic HIV-1, HIV-2, and dual infections

Mechanisms and Factors that Influence High Frequency Retroviral Recombination

With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development

Regulation of non-responsiveness and death in cytotoxic T cells by the agonistic potency of MHC : Peptide ligands

CD8+ T lymphocytes are important for immunological control of infections and tumors. The key interaction required to initiate the process of T cell activation is the engagement of the T cell receptor (TCR) with a major histocompatibility complex (MHC) class I/peptide complex on an antigen presenting cell (APC). Depending on the affinity of the interaction between the TCR and MHC class I molecule, different arrays of signaling pathways can be activated in the T cell. Molecular alterations in the peptide bound to the MHC class I can lead to a lower affinity of the MHCTCRinteraction resulting in incomplete or qualitatively different T-cell responses. Altered peptide ligands (APLs) exhibiting such activity are referred to as partial agonists and often occur naturally through genetic instability, which affects T cell epitopes derived from rapidly mutating viruses or tumor-associated cellular antigens. We studied the molecular basis of partial agonism using MHC class I/peptide tetramer complexes. By using tetramers assembled with a fully agonist peptide or its synthetic variant we could study the relationship between tetramer staining, cytokine production and different pathways of activation induced cell death (AICD). We found that positive tetramer staining correlated with at least two different activation programs in CD8+ T lymphocytes: full scale activation associated with Fas-dependent AICD and an incomplete activation followed by Fasindependent AICD. Further, we used raft-disrupting agents to assess the role of lipid rafts in determining the agonistic potency of different peptide ligands. We showed that overall binding of specific tetramers to CTLs was reduced upon raft disruption, although the half-life of tetramer:TCR complexes formed under these conditions was not affected. These findings suggest that different TCR complexes on the surface of CTLs may have different requirements for cholesterol and CTLs may be heterogeneous in their raft structure. In addition we analyzed programs of negative regulation of CD8+ T lymphocytes and the capacity of APLs to activate and modulate such programs. Upon specific triggering CD8+ T-cells become refractory to a secondary stimulus; a condition referred to Activation induced Non-Responsiveness (AINR). We have shown that TCRtriggering results in a novel degradation pathway of Lck, a kinase which plays a critical role in the initiation of T cell activation. Down-regulation of Lck through degradation correlated with AINR in CTLs. By blocking Lck degradation we could prevent the development of AINR. We further investigated how activation of CTLs with APLs affected Lck expression. The capacity of different peptide variants to induce Lck degradation correlated with their agonistic potency. Inefficient recognition of APLs by specific T lymphocytes is believed to contribute to the failure of the immune system to control certain tumor types and progressive viral diseases. To better understand the regulation of APL, activity by immunologic help, we analyzed the capacity of exogenous IL-2 and IL-15 to influence different aspects of activation triggered in CTLs by either fully or partially agonistic peptide ligands. We showed that signals induced by the lymphokines synergize with weak TCR signaling induced by partially agonistic APL, converting many of these peptides from inhibitory to stimulatory ligands. We also demonstrated that IL-2 and IL-15 suppress induction of a death receptor-independent apoptotic program triggered by partially agonistic APL. In conclusion, we have analyzed the molecular basis of partial agonism in CTL recognition of peptide epitopes and characterized molecular changes associated with death and AINR in specific CTLs. We have shown that structural changes in the sequence of CTL peptide epitopes may decrease the affinity of MHC/TCR interactions and generate APLs, which not only trigger incomplete activation programs but also induce and modulate negative regulation programs in CTLs. This APL induced signaling of suppressive nature appears to be more prominent in the absence of immunological help, suggesting that under conditions of immune deregulation APLs may actively suppress CTL responses against infectious agents or tumors

JC polyomavirus (JCV) and monoclonal antibodies: friends or potential foes?

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis