The clearance of hepatitis C virus infection in chimpanzees may not necessarily correlate with the appearance of acquired immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-gamma)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.     

Loss of CD4+ T Cells in Human Immunodeficiency Virus Type 1-Infected Chimpanzees Is Associated with Increased Lymphocyte Apoptosis

Supportive evidence that apoptosis contributes to loss of CD4⁺ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4⁺ T cells which correlated with persistently high viral burdens and increased levels of CD4⁺ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4⁺ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4⁺ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

The quality of chimpanzee T-cell activation and simian immunodeficiency virus/human immunodeficiency virus susceptibility achieved via antibody-mediated T-cell receptor/CD3 stimulation is a function of the anti-CD3 antibody isotype

While human immunodeficiency virus type 1 (HIV-1) infection is associated with hyperimmune activation and systemic depletion of CD4⁺ T cells, simian immunodeficiency virus (SIV) infection in sooty mangabeys or chimpanzees does not exhibit these hallmarks. Control of immune activation is thought to be one of the major components that govern species-dependent differences in the disease pathogenesis. A previous study introduced the idea that the resistance of chimpanzees to SIVcpz infection-induced hyperimmune activation could be the result of the expression of select sialic acid-recognizing immunoglobulin (Ig)-like lectin (Siglec) superfamily members by chimpanzee T cells. Siglecs, which are absent on human T cells, were thought to control levels of T-cell activation in chimpanzees and were thus suggested as a cause for the pathogenic differences in the course of SIVcpz or HIV-1 infection. As in human models of T-cell activation, stimulation had been attempted using an anti-CD3 monoclonal antibody (MAb) (UCHT1; isotype IgG1), but despite efficient binding, UCHT1 failed to activate chimpanzee T cells, an activation block that could be partially overcome by MAb-induced Siglec-5 internalization. We herein demonstrate that anti-CD3 MAb-mediated chimpanzee T-cell activation is a function of the anti-CD3 MAb isotype and is not governed by Siglec expression. While IgG1 anti-CD3 MAbs fail to stimulate chimpanzee T cells, IgG2a anti-CD3 MAbs activate chimpanzee T cells in the absence of Siglec manipulations. Our results thus imply that prior to studying possible differences between human and chimpanzee T-cell activation, a relevant model of chimpanzee T cell activation needs to be established.     

The Vancouver Lymphadenopathy-AIDS Study: 4. Effects of exposure factors,cofactors and HTLV-III seropositivity on number of helper T cells.

Results of testing for antibody to human T-lymphotropic virus (HTLV-III) and absolute numbers of helper T cells in 219 participants in the Vancouver Lymphadenopathy-AIDS (acquired immune deficiency syndrome) Study were analysed. The mean absolute helper T-cell counts in the 141 HTLV-III seronegative and the 78 seropositive men were 897/mL and 659/mL respectively (p less than 0.001). Established AIDS risk factors such as elevated lifetime number of male sexual partners and frequent receptive anal intercourse did not appear to have any significant effect on number of helper T cells that was independent of HTLV-III antibody status. Seropositive men with less than 100, 100 to 500 or more than 500 male sexual partners in their lifetime had mean absolute helper T-cell counts of 667/mL, 651/mL and 662/mL respectively. Most other risk factors, as well, did not appear to exert any effect on absolute number of helper T cells that was independent of the effect of HTLV-III antibody status. However, independent effects of a history of mononucleosis or hepatitis and of cigarette smoking were noted. The data support the hypothesis that no immune dysfunction beyond that due to the initial infection alone arises from repeated exposure to HTLV-III. Most risk factors appear to act as exposure factors, exerting their effect on the immune system merely by increasing the probability of contact with the agent. The independent effects of a history of mononucleosis or hepatitis suggest that viral agents may be cofactors in the production of immune dysfunction.     

Isolation of a novel simian T-cell lymphotropic virus from Pan paniscus that is distantly related to the human T-cell leukemia/lymphotropic virus types I and II.

An unusual serological profile against human T-cell leukemia/lymphotropic virus type I and II (HTLV-I and -II) proteins was reported in several human Pygmy tribes in Zaire and Cameroon with serum antibodies reactive with gp21 and p24. Here we describe a similar pattern of serum antibodies in a colony of captive pygmy chimpanzees and the isolation of a novel retrovirus, simian T-cell lymphotropic virus from Pan paniscus (STLVpan-p), from the peripheral blood mononuclear cells of several seropositive animals. Cocultures of peripheral blood mononuclear cells from three seropositive pygmy chimpanzees with human cord blood mononuclear cells led to the expression of an HTLV-I- and HTLV-II-related virus initially demonstrated by electron microscopy. Furthermore, several of these cocultures became immortalized T-cell lines expressing the CD4+ CD8+ DR+ phenotype of mature activated T cells. Southern blotting and DNA sequencing of a PCR fragment of viral DNA from these cell cultures demonstrated a distant evolutionary relationship of these viruses to HTLV-I and -II and distinct from the known STLV isolates. We designated this virus STLVpan-p. A genealogical analysis of the captive pygmy chimpanzees colony, originated from wild-caught animals, revealed a prevalence of seropositive offspring from infected mothers, as also observed with HTLVs. The presence in this old African Great Ape species of a virus which is genetically quite distinct from HTLV-I and -II could provide new insights in the phylogenesis of STLVs and HTLVs and be instrumental in the discovery of related human viruses.     

Upregulation of indoleamine 2,3-dioxygenase in hepatitis C virus infection

Indoleamine 2,3-dioxygenase (IDO) is induced by proinflammatory cytokines and by CTLA-4-expressing T cells and constitutes an important mediator of peripheral immune tolerance. In chronic hepatitis C, we found upregulation of IDO expression in the liver and an increased serum kynurenine/tryptophan ratio (a reflection of IDO activity). Huh7 cells supporting hepatitis C virus (HCV) replication expressed higher levels of IDO mRNA than noninfected cells when stimulated with gamma interferon or when cocultured with activated T cells. In infected chimpanzees, hepatic IDO expression decreased in animals that cured the infection, while it remained high in those that progressed to chronicity. For both patients and chimpanzees, hepatic expression of IDO and CTLA-4 correlated directly. Induction of IDO may dampen T-cell reactivity to viral antigens in chronic HCV infection.     

Serological differences between human T-lymphotropic virus type-I (HTLV-I) and HTLV-III/LAV

Sera from each of five preselected groups of patients with acquired immune deficiency syndrome (AIDS), AIDS-related complex (ARC), hemophilia, adult T-cell leukemia (ATL), and healthy controls were examined for antibodies to human T-cell leukemia (T-lymphotropic) virus type-I (HTLV-I) and HTLV-III by indirect immunofluorescence (IF) and radioimmunoprecipitation (RIP) methods. All sera from five patients with AIDS, ARC, and hemophilia reacted at titers from 1 : 512 to 1 : 5,120 with fixed H9/HTLV-III cells by IF but not with fixed MT-1 cells carrying HTLV-I. Similarly, sera from patients with AIDS, ARC, and hemophilia precipitated HTLV-III-specific polypeptides of 120K, 46K, and 24K. In contrast, sera from five patients with ATL did not react with fixed H9/HTLV-III cells, but reacted with fixed MT-1 cells. Moreover, HTLV-I-specific polypeptides of 68K, 28K, and 24K were precipitated with sera from ATL-patients but not with anti-HTLV-III-positive sera. Recently, we infected HTLV-I-carrying MT-4 cells with HTLV-III and provoked strong cytopathic effects. This system enabled testing for neutralizing antibodies to HTLV-III. Neutralizing titers to HTLV-III of five anti-HTLV-III-positive sera ranged from 1 : 720 to 1 : 9,000. In contrast, all five seronegative controls showed no or only low reactivity to HTLV-III envelope (1 : 80 and 100). However, three out of five anti-HTLV-I-positive sera exhibited weak cross-reactivities with HTLV-III. The reactivities were expressed as less than 1 : 160, except for one case (1 : 720). They were considered to be nonspecific since they were negative for HTLV-III antibodies in the radioimmunoprecipitation studies.     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

The resistance of HIV-infected chimpanzees to progression to AIDS correlates with absence of HIV-related T-cell dysfunction

Differences in the in vivo and in vitro responses of T lymphocytes from chimpanzees and human subjects were compared for evidence of HIV-1 related T-cell dysfunction. There was no increased level of programmed cell death (PCD) in HIV-1 infected chimpanzees in contrast to asymptomatic individuals. Anergy could be induced with HIV-1 gp120 in human but not chimpanzee T_H lymphocytes, however in vitro infection of chimpanzee T_H cultures with HIV-1 resulted in complete lysis of cells within three weeks. These findings suggest that the resistance of HIV-1 infected chimpanzees to progression to AIDS is due to their relative resistance to the systemic effects of HIV-1 on T-cell dysfunction.     

Escape mutations alter proteasome processing of major histocompatibility complex class I-restricted epitopes in persistent hepatitis C virus infection

Mutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+ T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+ T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.     

Rapid CD4(+) T-cell loss induced by human immunodeficiency virus type 1(NC) in uninfected and previously infected chimpanzees

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1(NC) (HIV-1(NC)). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4(+) T-cell loss to fewer than 26 cells/microl by 14 weeks after infection. CD4(+) T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1(LAV), experienced a more protracted course of peripheral CD4(+) T-cell loss after HIV-1(NC) inoculation, resulting in fewer than 200 cells/microl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1(NC) but were significantly and persistently increased after superinfection, with HIV-1(NC) representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.     

Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.     

Hepatic transcriptome analysis of hepatitis C virus infection in chimpanzees defines unique gene expression patterns associated with viral clearance

Hepatitis C virus infection leads to a high rate of chronicity. Mechanisms of viral clearance and persistence are still poorly understood. In this study, hepatic gene expression analysis was performed to identify any molecular signature associated with the outcome of hepatitis C virus (HCV) infection in chimpanzees. Acutely HCV-infected chimpanzees with self-limited infection or progression to chronicity were studied. Interferon stimulated genes were induced irrespective of the outcome of infection. Early induction of a set of genes associated with cell proliferation and immune activation was associated with subsequent viral clearance. Specifically, two of the genes: interleukin binding factor 3 (ILF3) and cytotoxic granule-associated RNA binding protein (TIA1), associated with robust T-cell response, were highly induced early in chimpanzees with self-limited infection. Up-regulation of genes associated with CD8+ T cell response was evident only during the clearance phase of the acute self-limited infection. The induction of these genes may represent an initial response of cellular injury and proliferation that successfully translates to a "danger signal" leading to induction of adaptive immunity to control viral infection. This primary difference in hepatic gene expression between self-limited and chronic infections supports the concept that successful activation of HCV-specific T-cell response is critical in clearance of acute HCV infection.     

Inducer T-cell-mediated killing of antigen-presenting cells

L3T4+ inducer/helper T-cell clones, once activated by antigen-presenting cells (APC) expressing the appropriate Ia allele and antigen, autonomously kill their target APC. All 13 L3T4+ inducer T-cell clones tested demonstrated this cytolytic activity. In addition, 11 different target cells representing the three major APC types, namely, macrophages, B cells, and dendritic cells, were all sensitive to this cytolytic activity. Moreover, normal macrophages which were treated with interferon-gamma to increase Ia expression were also killed. These observations convincingly demonstrate that the cytolytic activity of L3T4+ inducer T-cell clones is a general phenomenon. In contrast to other reports, lysis of target APC could not be detected following 4-6 hr of incubation. Marginal lysis was observed after 9 hr and a 20-hr incubation period was required to achieve maximal killing. The kinetics of killing paralleled other parameters of T-cell activation such as IL-2 release and cell proliferation. Activation of T cells for cytolysis of APC requires the interaction of T-cell receptors with Ia and antigen. Monoclonal antibody to Ia, L3T4 and the T-cell receptor inhibited the cytolysis of APC. The ability to mediate nonspecific bystander killing was variable depending on both the T-cell clone and the target. The implications of these findings to immune regulation and autoimmunity are discussed.     

Kinetic analysis by real-time PCR of hepatitis C virus (HCV)-specific T cells in peripheral blood and liver after challenge with HCV

Intrahepatic virus-specific CD8(+) T cells are thought to be important for the control of hepatitis C virus (HCV) infection, yet the precise kinetics for the expansion of epitope-specific T cells over the course of infection are difficult to determine with currently available methods. We used a real-time PCR assay to measure the frequency of clonotypic HCV-specific CD8(+) T cells in peripheral blood and snap-frozen liver biopsy specimens of two chimpanzees (Pan troglodytes) with previously resolved HCV infection who were rechallenged with HCV. In response to rechallenge, the magnitude of each clonotypic response was 10-fold higher in the liver than in the blood, and the peak clonotype frequency was concurrent with the peak viral load. The higher frequency of HCV-specific clonotypes in the liver than in peripheral blood was maintained for at least 3 months after the clearance of viremia. After antibody-mediated CD8(+) T-cell depletion and another viral challenge, the rebound of these clonotypes was seen prior to an appreciable reconstitution of CD8(+) T-cell values and, again, at higher frequencies in the liver than in peripheral blood. These data demonstrate the importance of intrahepatic virus-specific CD8(+) T cells for the clearance of infection and the rapid kinetics of expansion after virus challenge.     

Proliferative T-cell response to HIV envelope glycoprotein in immunized and infected primates and human beings

Human immunodeficiency virus (HIV)-specific helper T-cell response was studied in human subjects and nonhuman primates either infected with HIV or immunized with different HIV protein preparations. A strong group-specific T-cell response involving T-cell proliferation and lymphokine secretion was observed in immunized chimpanzees and rhesus monkeys as well as HIV-infected chimpanzees and gibbons. HIV-infected people demonstrated a low or no HIV-specific T-cell response. In contrast, five of 14 HIV antibody-negative sexual partners of HIV-infected men recognized one or more T-cell epitopes in the envelope glycoprotein of HIV.