ABSTRACT
BACKGROUND: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. AIM: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. METHODS: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl(4)), lipopolysaccaride (LPS), tumour necrosis factor alpha (TNFalpha), and anti-Fas antibody. RESULTS: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl(4), LPS, TNFalpha, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFalpha, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). CONCLUSIONS: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFalpha induced liver damage in vivo. The TNFalpha resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.
Subject(s)
Hepacivirus/immunology , Liver Diseases/immunology , Tumor Necrosis Factor-alpha/immunology , Animals , Hepatocytes/metabolism , Immunohistochemistry , Lymphocyte Subsets/immunology , Mice , Mice, Transgenic , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
OBJECTIVES: To investigate the prevalence of GB virus C (GBV-C) viraemia and GBV-C antibodies in a cohort of HIV-infected mothers and their infants between 1987 and 1994. METHODS: GBV-C viraemia and antibodies were determined by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in 52 HIV-infected mothers and their 53 infants, who were born before antiretroviral prophylaxis for reduction of HIV transmission was introduced at the end of 1994. Ten of these children acquired HIV. RESULTS: Mothers of three children had GBV-C viraemia and mothers of another 14 children carried antibodies against GBV-C. No mother had GBV-C antibodies and GBV-C viraemia simultaneously. GBV-C viraemia was detected in only one infant. This child was delivered by the vaginal route to a mother with GBV-C viraemia, and was not HIV-infected. No vertical transmission of GBV-C occurred from mothers with GBV-C antibodies. However, four of 10 children who were infected with HIV had a mother with past or ongoing GBV-C infection. CONCLUSION: Our findings suggest that the risk of vertical transmission of GBV-C is not elevated in HIV-infected mothers. Furthermore, although the number of HIV-1-infected children was low, we saw no evidence that the presence of ongoing or past GBV-C infection influenced the probability of vertical HIV transmission.
Subject(s)
Anti-Retroviral Agents , Flaviviridae Infections/transmission , GB virus C , HIV Infections/transmission , Pregnancy Complications, Infectious/immunology , Adolescent , Adult , Antibodies, Viral/blood , Female , Flaviviridae Infections/complications , Flaviviridae Infections/immunology , HIV Infections/complications , HIV Infections/immunology , Humans , Infant , Infectious Disease Transmission, Vertical , Pregnancy , Prognosis , Viremia/immunologyABSTRACT
Immune responses to two recombinant envelope 2 (E2) proteins, representing genotypes 1 and 2 of the GB virus C, or the hepatitis G virus (GBV-C/HGV), were studied in mice and in 48 individuals with, or without, chronic, or past GBV-C/HGV infection. Immunised mice developed E2-specific antibodies (mean titres, 1:1,167 to 1:9,360), recognising linear antigenic regions and proliferative and IL-2, IL-6 and gammaIFN cytokine responses regardless of the viral genotype. Individuals with past GBV-C infection had E2 antibody titres from 1:1,500 to 1:7,500 that did not recognise the E. coli derived E2 protein or linear antigenic regions. Proliferative E2-specific responses were detected in peripheral blood mononuclear cells from 6/22 (27%) persons with, and in none without GBV-C markers (P<0.05). Thus, E2-specific immune responses are mainly crossreactive between different variants of GBV-C/HGV, although proliferative responses appear to be rare.
Subject(s)
Antigens, Viral/immunology , Flaviviridae/immunology , Hepatitis, Viral, Human/immunology , Viral Envelope Proteins/immunology , Amino Acid Sequence , Animals , Antibody Formation , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Cytokines/analysis , Dose-Response Relationship, Immunologic , Genotype , Hepatitis Antibodies/blood , Humans , Immunity, Cellular , Immunization , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Mice , Molecular Sequence Data , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/geneticsSubject(s)
2-Aminopurine/analogs & derivatives , Antiviral Agents/therapeutic use , Hepatitis B e Antigens/drug effects , Hepatitis B, Chronic/drug therapy , Immunity, Cellular/drug effects , Lamivudine/therapeutic use , 2-Aminopurine/therapeutic use , Aged , DNA, Viral/blood , DNA, Viral/drug effects , Drug Therapy, Combination , Famciclovir , Hepatitis B e Antigens/blood , Hepatitis B virus/drug effects , Hepatitis B virus/genetics , Hepatitis B virus/growth & development , Humans , Male , Treatment Outcome , Virus ReplicationABSTRACT
GB virus C (GBV-C) markers were analyzed in two to three generations in three families with documented vertical transmission of GBV-C. None of the maternal grandparents had GBV-C markers, whereas the male spouses had GBV-C envelope 2 antibodies. Evidence was found for intrafamilial transmission but not for GBV-C transmission over three generations.
Subject(s)
Flaviviridae/genetics , Flaviviridae/isolation & purification , Hepatitis, Viral, Human/transmission , Adult , Aged , Biomarkers , Child , Family , Female , Flaviviridae/immunology , Hepatitis, Viral, Human/virology , Humans , Infectious Disease Transmission, Vertical , Male , Middle Aged , RNA, Viral/analysis , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain Reaction , Saliva/virology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolismABSTRACT
BACKGROUND: The role of the recently discovered GB virus C (GBV-C)/hepatitis G virus in fulminant hepatic failure (FHF) has been debated. Although GBV-C RNA has been detected in many cases of FHF, recent data suggest that the relation between GBV-C and FHF may be accidental. AIMS: To retrospectively investigate the possible relation between the presence of GBV-C markers (RNA or antibodies to the GBV-C envelope 2 (E2) glycoprotein) and FHF. METHODS: The presence of GBV-C RNA was determined in serum samples from 58 patients diagnosed with FHF using a reverse transcriptase polymerase chain reaction. Amplified genetic fragments were directly sequenced by the dideoxy chain termination method. Antibodies to GBV-C in serum samples were detected by enzyme immunoassay based on a recombinant GBV-C E2 protein. RESULTS: Nine (16%) patients with FHF had GBV-C RNA and 13 (22%) [corrected] had GBV-C E2 antibodies, which are higher frequencies than in healthy subjects (p<0.01 and p<0.05 respectively). Six of nine [corrected] patients with GBV-C markers during FHF tested negative for these markers before therapy with blood and/or blood products. Sequence analysis of the GBV-C NS3 region fragments of six FHF patients showed no common sequence pattern or motif. CONCLUSIONS: The frequencies of both GBV-C RNA and antibodies are higher in patients with FHF than in healthy subjects. However, these increased frequencies may in many cases be explained by the use of contaminated blood and/or blood products given as therapy.
Subject(s)
Flaviviridae/isolation & purification , Hepatic Encephalopathy/virology , Hepatitis, Viral, Human/transmission , Transfusion Reaction , Adolescent , Adult , Aged , Antibodies, Viral/blood , Blood Component Transfusion/adverse effects , Child , Child, Preschool , Flaviviridae/immunology , Hepatitis, Viral, Human/complications , Humans , Middle Aged , Polymerase Chain Reaction , RNA, Viral/blood , Retrospective StudiesABSTRACT
Ribavirin is effective in combination therapies against chronic hepatitis C virus (HCV) infection, although its direct antiviral properties are unclear. We therefore studied the immune-modulatory effects of ribavirin on hepatitis B virus (HBV)- and HCV-specific immune responses. During a 24 week placebo-controlled ribavirin trial in ten patients with chronic HCV infection, HCV antibodies and alanine aminotransferase (ALT) levels decreased transiently whereas the serum levels of HCV RNA remained stable. Effects of ribavirin on human and murine phytohaemagglutinin (PHA)-activated T cells included inhibition of in vitro proliferation and modulation of IL-2, IL-4, IFN-gamma and TNF-alpha levels. HBcAg- and HBeAg-specific IL-2 and IFN-gamma levels were > or = 25-fold higher in mice immunized with HBV core- or e-antigens (HBcAg, HBeAg) while receiving ribavirin compared to untreated mice, but IL-4 and IL-6 remained constant. Concordantly, a slight shift was observed in the IgG subclass distribution of the humoral responses of ribavirin-treated mice to HBeAg and HCV NS3 protein. Ribavirin treatment of HBeAg-transgenic (HBeAg-Tg) mice induced a dose-dependent down-regulation of T helper (Th)2-mediated antibody production to HBeAg. In ribavirin-treated HBeAg-Tg mice anti-HBe IgG1 (positively regulated by Th2 cytokines) decreased simultaneously as both anti-HBe IgG2a (positively regulated by Th1 cytokines) levels and in vitro T-cell IFN-gamma production increased, indicating a change in the Th1/Th2 balance. Thus, the present data suggest that ribavirin is not strictly an antiviral compound, but rather it alters the T-cell balance in the immune system.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/immunology , Hepatitis B virus/immunology , Ribavirin/pharmacology , Th1 Cells/drug effects , Th2 Cells/drug effects , Animals , Cytokines/biosynthesis , Hepatitis B Antibodies/blood , Hepatitis B e Antigens/immunology , Hepatitis C Antibodies/blood , Humans , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , RNA, Viral/analysis , Th1 Cells/immunology , Th2 Cells/immunologyABSTRACT
Cytotoxic T lymphocyte (CTL) activity and CD4+ helper T cell responses to the hepatitis B virus (HBV) core antigen (HBcAg) have been implicated in clearance of acute and chronic HBV infections. We showed that intramuscular injections of a novel recombinant retroviral vector expressing an HBcAg-neomycin phosphotransferase II (HBc-NEO) fusion protein induces HBc/eAg-specific antibodies and CD4+ and CD8+ T cell responses in mice and rhesus monkeys. We have now immunized three chronically infected chimpanzees, each with 10(10) CFU of nonreplicating retroviral vector particles expressing the HBc-NEO fusion protein. Of two immunized chimpanzees examined for CTL responses, one developed HBcAg-specific CTLs and showed marginal, transient elevations of alanine aminotransferase (ALT) levels following injection. However, both chimpanzees remained positive for serum HBeAg, negative for anti-HBe antibody by conventional assays, and displayed no change in HBV viral load throughout the study. In contrast, the third chimpanzee exhibited a traditional seroconversion evidenced by a loss of serum HBeAg and the subsequent emergence of anti-HBe antibodies within 24 weeks after the first injection. Simultaneously, two transient ALT flares and a significant decrease in the serum HBV DNA levels were noted. Despite its limitations, the present study demonstrates (1) the safety of treatment with high titers of retroviral vector in chimpanzees, (2) the capability of a retroviral vector expressing HBcAg to stimulate immune responses in HBV chronic carrier chimpanzees, and (3) that retroviral vector immunization may be therapeutically beneficial in the treatment of chronic HBV infection.
Subject(s)
Genetic Therapy , Genetic Vectors , Hepatitis B Core Antigens/genetics , Hepatitis B, Chronic/therapy , Retroviridae/genetics , Amino Acid Sequence , Animals , Base Sequence , Carrier State , DNA Primers , DNA, Viral/blood , Disease Models, Animal , Hepatitis B Antibodies/biosynthesis , Hepatitis B virus/genetics , Hepatitis B virus/isolation & purification , Hepatitis B, Chronic/immunology , Humans , Molecular Sequence Data , Pan troglodytes , Recombination, Genetic , Sequence Homology, Amino Acid , T-Lymphocytes, Cytotoxic/immunology , Viral LoadABSTRACT
The correlation among the presence of a 32-bp deletion in the CC-chemokine receptor 5 (CCR5) gene, disease progression, and human immunodeficiency virus type 1 (HIV-1)-specific immune responses was analyzed for a cohort of 79 Caucasian HIV-1-infected patients. The CCR5 genotype (CCR5/CCR5 = wild type/wild type or delta32CCR5/CCR5 = 32-bp deletion/wild type) in peripheral blood mononuclear cells was determined by PCR, followed by sequencing of both wild-type and delta32CCR5 gene fragments. HIV-1-specific humoral responses to gp41 and V3MN peptides were determined by enzyme immunoassays. The prevalence of the delta32CCR5 allele was lower among 37 patients with rapid progression (progression to AIDS or to a CD4 cell count of <200 x 10(6)/liter in less than 9 years; P < 0.01) compared to that for 42 patients with slow progression (no AIDS and CD4 cell count of >200 x 10(6)/liter after at least 9 years from infection) or to that for 25 non-HIV-1-infected Swedish blood donors (P < 0.05). No differences were observed in the wild-type CCR5 sequences between the different groups of patients. For three analyzed patients, the 32-bp delta32CCR5 gene deletions were identical. The antibody titers against gp41 and a V3MN peptide in patients with the delta32CCR5/CCR5 genotype were not significantly different from those in pair-matched CCR5/CCR5 controls. However, in 13 analyzed patients, a stronger serum neutralizing activity was associated with the delta32CCR5/CCR5 genotype. Thus, a CCR5/CCR5 genotype correlates with a shortened AIDS-free HIV-1 infection period and possibly with a worse neutralizing activity, without an evident influence on the antibody response to two major antigenic regions of HIV-1 envelope.
Subject(s)
HIV Infections/etiology , HIV-1 , Receptors, CCR5/genetics , Amino Acid Sequence , Antibody Specificity , Base Sequence , Cohort Studies , DNA Primers/genetics , Genotype , HIV Antibodies/blood , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/immunology , HIV Infections/genetics , HIV Infections/immunology , HIV-1/immunology , Humans , Molecular Sequence Data , Neutralization Tests , Peptide Fragments/genetics , Peptide Fragments/immunology , Polymerase Chain Reaction , Sequence Deletion , SurvivorsSubject(s)
B-Lymphocytes/immunology , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/immunology , Hepatitis B/genetics , Hepatitis B/immunology , Epitopes/immunology , Hepatitis B Antibodies/immunology , Hepatitis B e Antigens/genetics , Hepatitis B e Antigens/immunology , Humans , Mutation , Sequence Deletion , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.
Subject(s)
Antibodies, Viral/immunology , Hepacivirus/genetics , Hepacivirus/immunology , Major Histocompatibility Complex/immunology , Viral Nonstructural Proteins/immunology , Amino Acid Sequence , Animals , Antigens, Viral/genetics , Antigens, Viral/immunology , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Humans , Immunity , Major Histocompatibility Complex/genetics , Mice , Molecular Sequence DataABSTRACT
Production of antibody to hepatitis B e antigen (HBeAg); i.e., anti-HBe antibody,) in HBeAg-transgenic mice is believed to be mediated by T-helper 2 (Th2) cells. Injection of an HBeAg-specific Th2 clone into HBeAg-transgenic H-2k mice induced anti-HBe antibody production, confirming the function of Th2 cells in this model system.
Subject(s)
Adoptive Transfer , Hepatitis Antibodies/biosynthesis , Hepatitis B e Antigens/immunology , Th2 Cells/immunology , Animals , Clone Cells , Hepatitis Antibodies/immunology , Hepatitis B Core Antigens/immunology , Immunoenzyme Techniques , Mice , Mice, TransgenicABSTRACT
The susceptibility of monocyte-derived cultured dendritic cells (DCs) to human immunodeficiency virus (HIV) infection and their role in viral transmission in the immune response were studied in detail. We observed that highly purified cultured DCs were infected with the T-tropic Lai strain of HIV type 1 (HIV-1Lai) via the CD4 receptor, and this was followed by formation of the complete provirus as detected by PCR. HIV mRNAs were transcribed at only low levels, and virus production was undectable; however, the addition of the purified protein derivative antigen of tuberculin and of autologous resting T cells to HIV-1Lai-infected DCs but not to HIV-1Lai-infected macrophages led to massive HIV transmission and production. These data suggest that the interaction of infected DCs with T cells during the normal immune response could play an important role in the activation and expansion of HIV.
Subject(s)
Antigen Presentation , Dendritic Cells/virology , HIV-1/physiology , Monocytes/virology , T-Lymphocytes/virology , Base Sequence , Cell Communication , Cells, Cultured , HIV Core Protein p24/biosynthesis , Humans , Molecular Sequence Data , RNA, Messenger/analysisABSTRACT
An in vitro culture system was developed that facilitates detailed studies of the interaction of Human Immunodeficiency Virus (HIV) with dendritic cells (DC). Cultured immature DC were generated from adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. These cells were non-adherent, non-phagocytic and had a veiled surface appearance. They expressed high levels of MHC class I and II proteins, CD1a, B7/BB1 and low levels of CD4, and were known to possess a potent soluble antigen presenting capacity. Upon infection with the HIV-1 strains Lai (lymphocytotropic) and BaL (monocytotropic), the viral RNA was reverse transcribed to complete DNA provirus. However the infection was non-productive as judged from measuring the activity of the virus encoded reverse transcriptase in the culture supernatant. Thus HIV infection was restricted at a step post entry.
Subject(s)
Dendritic Cells/virology , HIV-1/pathogenicity , Antigen Presentation , Antigens, Surface/metabolism , Cell Differentiation , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , HIV Infections/etiology , HIV-1/genetics , HIV-1/physiology , Humans , In Vitro Techniques , Proviruses/genetics , Proviruses/physiology , Virus ReplicationABSTRACT
Human immunodeficiency virus type 1 (HIV-1) replication is shown to be sensitive to the intracellular concentration of deoxynucleoside triphosphate substrates. Addition of thymidine to established cell lines resulted in a dramatic reduction of virus production. The effect could be substantially alleviated by addition of deoxycytidine, which, alone, enhanced viral titers by a factor of 2 to 3. Hydroxyurea treatment abolished HIV-1 replication in peripheral blood mononuclear cells and could be reversed by deoxyadenosine. These data show that HIV-1 replication occurs under suboptimal DNA precursor conditions.