Genetic basis for variation in plasma IL-18 levels in persons with chronic hepatitis C virus and human immunodeficiency virus-1 infections.

Inflammasomes are multi-protein complexes integrating pathogen-triggered signaling leading to the generation of pro-inflammatory cytokines including interleukin-18 (IL-18). Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections are associated with elevated IL-18, suggesting inflammasome activation. However, there is marked person-to-person variation in the inflammasome response to HCV and HIV. We hypothesized that host genetics may explain this variation. To test this, we analyzed the associations of plasma IL-18 levels and polymorphisms in 10 genes in the inflammasome cascade. About 1538 participants with active HIV and/or HCV infection in three ancestry groups are included. Samples were genotyped using the Illumina Omni 1-quad and Omni 2.5 arrays. Linear regression analyses were performed to test the association of variants with log IL-18 including HCV and HIV infection status, and HIV RNA in each ancestry group and then meta-analyzed. Eleven highly correlated single-nucleotide polymorphisms (r2=0.98-1) in the IL-18-BCO2 region were significantly associated with log IL-18; each T allele of rs80011693 confers a decrease of 0.06 log pg ml-1 of IL-18 after adjusting for covariates (rs80011693; rs111311302 ß=-0.06, P-value=2.7 × 10-4). In conclusion, genetic variation in IL-18 is associated with IL-18 production in response to HIV and HCV infection, and may explain variability in the inflammatory outcomes of chronic viral infections.

Defective response to Toll-like receptor 3 and 4 ligands by activated monocytes in chronic hepatitis C virus infection.

Toll-like receptors (TLR) have a critical role in innate immunity against pathogens. We investigated the cytokine response to TLR stimulation in peripheral blood cells of subjects infected with hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) in the Women Interagency HIV Study (WIHS) cohort. Interleukin (IL)-6 in response to TLR3 and TLR4 ligands such as polyinosinic-polycytidylic acid and lipopolysaccharide was significantly compromised in HCV-infected women. High spontaneous secretion of IL-6 suggested pre-existing cell activation as a factor mediating reduced responses to TLR3 and TLR4 stimulation. To a lesser extent, tumour necrosis factor-alpha and IL-1beta responses to TLR stimulation were also compromised. Monocytes, but not B cells or NK cells, were identified as the cell population spontaneously secreting cytokines and also as the cells responding to TLR stimulation. These results highlight a functional defect in antigen-presenting cells of women with HCV infection or co-infection. In women with existing HIV co-infection, decreased cytokine function of antigen-presenting cells suggests another mechanism contributing to immune dysfunction in addition to the HIV-associated CD4 defect.

Human immunodeficiency virus-infected patients receiving highly active antiretroviral therapy maintain activated CD8+ T cell subsets as a strong adaptive immune response to cytomegalovirus.

CD8(+) T lymphocyte function specific for human cytomegalovirus (CMV) was evaluated in 14 patients infected with human immunodeficiency virus (HIV) receiving highly active antiretroviral therapy (HAART) and 26 CMV-seropositive donors without HIV infection. Fifty-seven percent of the HIV-infected group had CMV-specific cytolytic activity in freshly isolated peripheral blood mononuclear cells (PBMC) against targets expressing CMV pp65. Both interferon (IFN)-gamma secretion by CD8(+) T cells and the frequency of human leukocyte antigen (HLA)-tetramer-positive T cells in HLA-A*0201-positive HIV-infected subjects correlated with CMV-specific cytolysis. In contrast, PBMC from healthy CMV-seropositive donors did not have either measurable CMV-specific cytolysis or secretion of IFN-gamma without in vitro stimulation. The T helper response to CMV antigens was vigorous in healthy CMV-seropositive donors but low in the cohort of HIV-infected patients. Potent CD8(+) cytotoxic T lymphocyte responses to CMV in HIV-infected patients receiving HAART is the converse of what is found in healthy CMV-seropositive subjects and may be the predominant adaptive immune response against CMV in HIV-infected patients.

Maintenance of CD8(+) T-cell memory following infection with recombinant sindbis and vaccinia viruses.

CD8(+) T-cell memory is critical for protection against pathogens poorly controlled by humoral immunity. To characterize two distinct vaccine vectors, the acute and memory CD8(+) T-cell responses to an HIV-1 epitope (p18) expressed by recombinant vaccinia (vp18) and Sindbis (SINp18) viruses were compared. Whereas 9 to 13% of CD8(+) splenocytes were p18 specific during the acute response to vp18, 4% were induced by SINp18 as revealed by class I tetramer staining. Increased T-cell activation by vp18 was confirmed by higher numbers of both p18-specific IFN-gamma-secreting splenocytes and activated CD8(+) and CD4(+) T cells. Although higher frequencies of p18-specific CD8(+) T cells during primary responses correlated with higher frequencies during memory, the overall decline was only two- to threefold during the transition to memory, demonstrating equally efficient maintenance of memory in SINp18- as in vp18-immune mice. Despite modest in vivo activation, SINp18-induced CD4(+) T cells secreted substantial amounts of IFN-gamma and IL-2, potentially contributing to sustained CD8(+) memory. Collectively the data indicate that Sindbis virus recombinants provide effective vaccines for inducing protective memory CD8(+) T cells in the absence of the extensive inflammation and replication associated with vaccinia virus.

Status of Cytomegalovirus Prevention and Treatment in 2000.

Cytomegalovirus (CMV) infection continues to be a problem in selected populations following hematopoietic stem cell transplantation (SCT). Although there have been no new antiviral agents for management of this infection in recent years, the methods for using the existing agents have improved with newer assays for detection of virus. In addition, our understanding of immunity to CMV has undergone considerable expansion. This paper will address these new aspects relating to CMV infection in the setting of SCT. In Section I Dr. Zaia reviews the pathogenesis of CMV and the current epidemiology of CMV disease following marrow or blood allo-SCT with emphasis on late-onset disease. The current lab tests available for preemptive management are summarized including the role for conventional shell vial cultures, and a comparison of the CMV antigenemia assay with the new nucleic acid-based assays, including the hybrid capture assay, the NASBA assay, and "real-time" PCR assays. Use of antiviral agents with these tests in the preemptive management of CMV infection is discussed. Ultimately, what is necessary is restoration of adequate CMV immunity, and that requires understanding the basics of the CMV-specific immune response. In Section II, Dr. Sissons traces the evolution of the CTL response from primary infection into memory and reviews recent advances in the understanding of cytotoxic T cell based immunity to CMV, based on the use of T cell clonotypic analysis and markers of T cell memory and activation, with conventional CTL functional assays. In Section III Dr. Riddell presents approaches to correction of the problem of CMV pathogenesis, namely direct restoration of the CMV-specific cellular immune deficiency. Attempts at passive therapies will be reviewed with the focus on current problems and approaches to these problems. In Section IV, Dr. Diamond presents work on the identification of multiple HLA-allele specific cytotoxic T cell epitopes specific for CMV-pp65 and - pp150. Specific epitopes are recognized by CMV-seropositive individuals including healthy donors, SCT recipients, and AIDS patients, indicating their potential usefulness as vaccines. One of these epitopes is recognized by most individuals who express the HLA A(*)0201 Class I allele. Pre-clinical evaluation in HLA2.1 transgenic mice of vaccine structures utilizing this epitope, and alternative delivery systems are described. Possible methods for vaccination of donor and/or recipient of a SCT as well as their limitations, utilizing synthetic or viral vaccines, are discusseed.

Immune response to green fluorescent protein: implications for gene therapy.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Enhanced cytotoxic T cell activity in IL-4-deficient mice.

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-gamma by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.

Internalization of iscom-borne antigens and presentation under MHC class I or class II restriction.

Exogenous nonreplicating antigens (Ag) incorporated into immunostimulating complexes (iscoms) induce CTL responses under MHC class I restriction. A requirement for inducing CTL responses is that the Ag is delivered to the cytosol of antigen-presenting cells (APC), a route restricted to endogenously produced Ag. To investigate the mechanisms by which iscoms elicit MHC class I-restricted responses, the intracellular distribution of influenza virus envelope proteins incorporated in iscoms (flu-iscoms) or in micelles (flumicelles) was studied in vitro using murine peritoneal cells (PEC). Ultrathin sections of cells pulsed with biotinylated flu-iscoms or flu-micelles were analyzed by electron microscopy after detection of the biotin label by reaction with streptavidin-gold. PEC pulsed with flu-iscoms showed a pattern of scattered gold particles distributed in clear and dense vesicles as well as in the intracellular space but not associated with organelles. In cells pulsed with flu-micelles, Ag was also detected in most cellular compartments but at a considerably lower concentration. The intracellular distribution of particulate Ag in iscom or micelle form was confirmed by lysis and differential centrifugation of Ag-pulsed APC. Furthermore, P815 cells pulsed with flu-iscoms were lysed by specific immune effectors showing that the iscom-Ag was processed and presented by class I-expressing APC. Flu-iscoms were internalized about 50-fold more efficiently than ovalbumin iscoms (ovaiscoms) suggesting that the nature of the protein and/or the presence of cellular receptors are important factors influencing the capacity of APC to take up iscom-borne proteins. PEC accounted for the most active internalization of iscom-borne Ag, although splenic dendritic cells and B cells also took up fluiscoms with remarkable efficiency.

Characterization of immunostimulating complexes (ISCOMS) of HIV-1.

HIV-1, strain HTLV-III, propagated in H9 cells and purified by sucrose gradient centrifugation, was used as native antigen source for the preparation of immunostimulating complexes, HIV-iscoms. The major antigen detected in the iscom was the cell-derived HLA-DR, which readily could be removed from the virus lysate by immunosorbent. In the iscoms the HIV structural proteins MA p17, p55 and TM gp41 were identified; SU gp120 was present in only minute amounts in the virus lysate. The iscom particles appeared well preserved after freeze drying with a round shape, approximately 35 nm in diameter, comprising morphological subunits, assembled with icosahedral symmetry. Immunization experiments in mice reflected the antigen content of the iscoms. High antibody response was induced to HLA-DR in non-depleted iscoms. Major humoral responses were observed to the viral structural proteins MA p17, CA p24, p55, and also to TM gp41. A low or negligible antibody response to SU gp120 was induced by the HIV-iscoms. The negligible response was, however, overcome by the addition of recombinant gp160 to the virus lysate prior to formation of iscoms, resulting in a preparation evoking a clear serum antibody to gp160.