Uncommon pathways of immune escape attenuate HIV-1 integrase replication capacity

An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF_114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

Paradoxical effects of cytokines in tumor immune surveillance and tumor immune escape

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity.     

Widespread impact of HLA restriction on immune control and escape pathways of HIV-1

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.     

Innate immune response to Mycobacterium tuberculosis Beijing and other genotypes

Background

As a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evoluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes.

Methodology/Principal Findings

We infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-α than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion.

Conclusions/Significance

Our results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity.     

DC-SIGN points the way to a novel mechanism for HIV-1 transmission

Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a recently discovered type II transmembrane protein on DCs with a C-type lectin extracellular domain, is capable of binding ICAM-3 on resting T cells in the secondary lymphoid organs, providing the initial contact between these cells during the establishment of cell-mediated immunity. DC-SIGN also binds the HIV-1 envelope glycoprotein gp120 but does not function as a receptor for viral entry into DCs. Instead, DC-SIGN allows DCs in the peripheral mucosa to carry HIV-1 through the lymphatics in a "Trojan horse" fashion, where it is eventually delivered to the T cells. Also, the period of infectivity of HIV-1 is increased by several days as a result of DC-SIGN-gp120 binding, allowing for efficient trans-infection of T cells on DC arrival. The discovery of a cluster of related genes colocalized with DC-SIGN on chromosome 19p13.2-3, all displaying complex alternative splicing patterns, has led to a reexamination of the mechanisms underlying both the interactions between antigen-presenting cells (APCs) and T cells and the pathogenesis of HIV-1 infection.     

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.     

Mycobacterium tuberculosis induces an atypical cell death mode to escape from infected macrophages

Background

Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds ∼20 per macrophage but the precise nature of this demise has not been defined.

Methodology/Principal Findings

We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.

Conclusions/Significance

Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis.     

Viruses and the nucleolus: The fatal attraction

Viruses are small obligatory parasites and as a consequence, they have developed sophisticated strategies to exploit the host cell's functions to create an environment that favors their own replication. A common feature of most – if not all – families of human and non-human viruses concerns their interaction with the nucleolus. The nucleolus is a multifunctional nuclear domain, which, in addition to its well-known role in ribosome biogenesis, plays several crucial other functions. Viral infection induces important nucleolar alterations. Indeed, during viral infection numerous viral components localize in nucleoli, while various host nucleolar proteins are redistributed in other cell compartments or are modified, and non-nucleolar cellular proteins reach the nucleolus. This review highlights the interactions reported between the nucleolus and some human or animal viral families able to establish a latent or productive infection, selected on the basis of their known interactions with the nucleolus and the nucleolar activities, and their links with virus replication and/or pathogenesis. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V-M. tb) and attenuated M. tb H37Ra (A-M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V-M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A-M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V-M. tb-infected adipocytes versus those infected with A-M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V-M. tb induced a high level of necrosis versus A-M. tb in conjunction with increased levels of LHD. These results suggest that V-M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.     

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27⁻ phenotype was associated with active TB in HIV⁻ (p = 0.0003) and HIV⁺ (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV⁻ subjects, MTB-specific CD4 T cell populations from HIV⁺ TB-asymptomatic subjects were often dominated by CD27⁻ cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression.     

Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor

HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.     

Antibodies to HIV-1: aiming at the right target

HIV-1 evades antibody-mediated neutralization in many cunning ways. The recent structural characterization of a conserved neutralization epitope on the envelope glycoprotein complex (Env) provides clues to the vulnerabilities of the virus. We discuss these observations and explain their relevance for HIV-1 vaccine design.     

A model to predict cell-mediated immune regulatory mechanisms during human infection with Mycobacterium tuberculosis

A key issue for the study of tuberculosis infection (TB) is to understand why individuals infected with Mycobacterium tuberculosis experience different clinical outcomes. Elaborating the immune mechanisms that determine whether an infected individual will suffer active TB or latent infection can aid in developing treatment and prevention strategies. To better understand the dynamics of M. tuberculosis infection and immunity, we have developed a virtual human model that qualitatively and quantitatively characterizes the cellular and cytokine control network operational during TB infection. Using this model, we identify key regulatory elements in the host response. In particular, factors affecting cell functions, such as macrophage activation and bactericidal capabilities, and effector T cell functions such as cytotoxicity and cytokine production can each be determinative. The model indicates, however, that even if latency is achieved, it may come at the expense of tissue damage if the response is not properly regulated. A balance in Th1 and Th2 immune responses governed by IFN-gamma, IL-10, and IL-4 facilitate this down-regulation. These results are further explored through virtual deletion and depletion experiments.     

The human immune response to Mycobacterium tuberculosis in lung and lymph node

A key issue for the study of tuberculosis is to understand why individuals infected with Mycobacterium tuberculosis (Mtb) experience different clinical outcomes. To better understand the dynamics of Mtb infection and immunity, we have previously developed a temporal mathematical model that qualitatively and quantitatively characterizes the cellular and cytokine control network during infection. In this work we extend that model to a two compartmental model to capture the important processes of cellular activation and priming that occur between the lung and the nearest draining lymph node. We are able to reproduce typical disease progression scenarios including primary infection, latency or clearance. Then we use the model to predict key processes determining these different disease trajectories (i.e. identify bifurcation parameters), suggesting directions for further basic science study and potential new treatment strategies.     

DC-SIGN on B lymphocytes is required for transmission of HIV-1 to T lymphocytes

Infection of T cells by HIV-1 can occur through binding of virus to dendritic cell (DC)-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on dendritic cells and transfer of virus to CD4+ T cells. Here we show that a subset of B cells in the blood and tonsils of normal donors expressed DC-SIGN, and that this increased after stimulation in vitro with interleukin 4 and CD40 ligand, with enhanced expression of activation and co-stimulatory molecules CD23, CD58, CD80, and CD86, and CD22. The activated B cells captured and internalized X4 and R5 tropic strains of HIV-1, and mediated trans infection of T cells. Pretreatment of the B cells with anti-DC-SIGN monoclonal antibody blocked trans infection of T cells by both strains of HIV-1. These results indicate that DC-SIGN serves as a portal on B cells for HIV-1 infection of T cells in trans. Transmission of HIV-1 from B cells to T cells through this DC-SIGN pathway could be important in the pathogenesis of HIV-1 infection.     

Mechanisms of cell recruitment in the immune response to Mycobacterium tuberculosis

Recent advances in understanding cell traffic, especially the roles of adhesion proteins, chemokines, and chemokine receptors, provide the opportunity for understanding mechanisms involved in the immune response to tuberculosis. This review concentrates on the roles of these molecules and the immune response in tuberculosis, based on studies of humans and mice infected with Mycobacterium tuberculosis.