ABSTRACT
Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens.
Subject(s)
Arenavirus , Hepatitis B , Mice , Animals , Hepatitis B Surface Antigens , Hepatitis B virus/genetics , Hepatitis B Vaccines , Hepatitis B Antibodies , Immunization , CD8-Positive T-Lymphocytes , Genotype , Antigens, SurfaceABSTRACT
BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.
Subject(s)
Antiviral Agents/therapeutic use , Hepatitis B Virus, Woodchuck/drug effects , Hepatitis B Virus, Woodchuck/genetics , Hepatitis B, Chronic/drug therapy , Hexanols/therapeutic use , Pyrimidines/therapeutic use , Toll-Like Receptor 8/agonists , Animals , Antiviral Agents/pharmacology , DNA, Viral/blood , Disease Models, Animal , Hepatitis Antibodies/blood , Hepatitis Antigens/blood , Hepatitis B Virus, Woodchuck/immunology , Hepatitis B, Chronic/complications , Hepatitis B, Chronic/immunology , Hexanols/pharmacology , Humans , Marmota , Pyrimidines/pharmacology , Virus Replication/drug effectsABSTRACT
The eastern woodchuck (Marmota monax) is a hibernating species extensively used as an in vivo efficacy model for chronic human hepatitis B virus infection. Under laboratory conditions, woodchucks develop a pseudohibernation condition; thus, the pharmacokinetics (PK) of small-molecule therapeutics may be affected by the seasonal change. The seasonal PK of four probe compounds were characterized over 12 months in seven male and nine female laboratory-maintained woodchucks. These compounds were selected to study changes in oxidative metabolism [antipyrine (AP)], glucuronidation [raltegravir (RTG)], renal clearance [lamivudine (3TC)], and hepatic function [indocyanine green (ICG)]. Seasonal changes in physiologic parameters and PK were determined. Seasonal body weight increases were ≥30%. Seasonal changes in body temperature and heart rate were <10%. The mean AP exposure remained unchanged from April to August 2017, followed by a significant increase (≥1.0-fold) from August to December and subsequent decrease to baseline at the end of study. A similar trend was observed in RTG and 3TC exposures. The ICG exposure remained unchanged. No significant sex difference in PK was observed, although female woodchucks appeared to be less susceptible to seasonal PK and body weight changes. Significant seasonal PK changes for AP, RTG, and 3TC indicate decreases in oxidative metabolism, phase II glucuronidation, and renal clearance during pseudohibernation. The lack of seasonal change in ICG exposure suggests there are no significant changes in hepatic function. This information can be used to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK. SIGNIFICANCE STATEMENT: Woodchuck is a hibernating species and is commonly used as a nonclinical model of hepatitis B infection. Investigation of seasonal PK changes is perhaps of greater interest to pharmaceutical industry scientists, who use the woodchuck model to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK and/or toxicity. This information is also valuable to drug metabolism and veterinary scientists in understanding woodchuck's seasonal metabolism and behavior under the pseudohibernation condition.
Subject(s)
Antiviral Agents/pharmacokinetics , Hepatitis B, Chronic/drug therapy , Hibernation/physiology , Marmota/physiology , Metabolic Clearance Rate/physiology , Animals , Antiviral Agents/therapeutic use , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Female , Humans , Male , SeasonsABSTRACT
BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Interferon Type I/immunology , Pteridines/pharmacology , Toll-Like Receptor 7/agonists , Animals , Antigen Presentation , Cells, Cultured , Cytokines/biosynthesis , DNA, Circular/genetics , DNA, Circular/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Hepatitis B Antigens/metabolism , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/virology , Hepatocytes/drug effects , Hepatocytes/immunology , Hepatocytes/virology , Humans , Immunity, Innate , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Toll-Like Receptor 7/geneticsABSTRACT
Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.
ABSTRACT
Background & Aims: Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Methods: Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n = 39) and HBeAg-negative (n = 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Results: Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Conclusions: Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). Impact and Implications: HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.
ABSTRACT
Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcgamma receptors (FcgammaR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcgammaR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.
Subject(s)
Antibodies, Viral/immunology , Antibody-Dependent Enhancement/immunology , Dengue/immunology , Immunoglobulin Fc Fragments/immunology , Animals , Antibodies, Neutralizing/immunology , Cell Separation , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunization, Passive , Immunohistochemistry , Mice , Neutralization Tests , Surface Plasmon Resonance , Viral LoadABSTRACT
Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation.
Subject(s)
Cell Differentiation/genetics , Cell Transformation, Neoplastic/genetics , Chromosomal Proteins, Non-Histone/biosynthesis , Epithelial Cells/cytology , Keratinocytes/cytology , Oncogene Proteins/biosynthesis , Animals , Blotting, Western , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/virology , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Epithelial Cells/pathology , Epithelial Cells/virology , Epithelium/metabolism , Epithelium/pathology , Fluorescent Antibody Technique , Foreskin/cytology , Gene Expression , Humans , Hyperplasia/genetics , Hyperplasia/metabolism , Hyperplasia/virology , Keratinocytes/pathology , Keratinocytes/virology , Male , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Papillomavirus E7 Proteins/genetics , Poly-ADP-Ribose Binding Proteins , Proto-Oncogene Mas , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.
Subject(s)
Antibodies, Monoclonal/therapeutic use , B7-H1 Antigen/immunology , Disease Models, Animal , Hepatitis B/therapy , Animals , Antibodies, Monoclonal/adverse effects , MarmotaABSTRACT
Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.
Subject(s)
Genes, Retinoblastoma , Oncogene Proteins, Viral/genetics , Animals , Base Sequence , Cell Cycle/radiation effects , Cell Differentiation , Cell Division , Centrosome/metabolism , DNA Primers/genetics , Epithelium/metabolism , Epithelium/pathology , Epithelium/virology , Female , Gene Deletion , Genes, Viral , Humans , Mice , Mice, Transgenic , Oncogenes , Papillomaviridae/genetics , Papillomaviridae/pathogenicity , Papillomavirus E7 Proteins , Phenotype , Retinoblastoma Protein/metabolism , Skin/metabolism , Skin/pathology , Skin/virology , Uterine Cervical Neoplasms/etiologyABSTRACT
This report describes the development and optimization of a quantitative real-time PCR assay for evaluating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assay measures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalization of CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability. The assay throughput was further enhanced by eliminating several wash steps and by lysing the cells directly in the presence of cell culture media, both of which had no impact on the assay metrics. The assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR) assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratory strains and clinical isolates.
Subject(s)
Cytomegalovirus/isolation & purification , Cytomegalovirus/physiology , Real-Time Polymerase Chain Reaction/methods , Virology/methods , Virus Replication , Cytomegalovirus/genetics , Cytosol/virology , DNA, Mitochondrial/analysis , DNA, Mitochondrial/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Humans , Real-Time Polymerase Chain Reaction/standards , Reference Standards , Reproducibility of Results , Time Factors , Virology/standardsABSTRACT
The E7 oncoprotein of the high-risk human papillomaviruses (HPV) is thought to contribute to cervical carcinogenesis at least in part by abrogating cell cycle regulation. E7 can dysregulate the cell cycle through its interaction with several cellular proteins including the retinoblastoma suppressor protein pRb, as well as the cyclin-dependent kinase inhibitor p21(Cip1). Inactivation of pRb in cervical epithelia is not sufficient to explain the ability of E7 to cause cervical cancers in transgenic mice. In the current study, we focused on the role of p21(Cip1) in cervical cancer. Cervical disease was significantly increased in p21(-/-) mice compared with p21(+/+) mice, showing that p21(Cip1) can function as a tumor suppressor in this tissue. Importantly, the ability of E7 to induce cervical cancers was not significantly enhanced on the p21-null background, consistent with the hypothesis that the ability of E7 to inhibit p21(Cip1) contributes to its carcinogenic properties. Further supportive of this hypothesis, cervical carcinogenesis in mice expressing a mutant form of HPV-16 E7, E7(CVQ), which fails to inactivate p21(Cip1), was significantly reduced compared with that in K14E7(WT) mice expressing wild-type HPV-16 E7. However, K14E7(CVQ) mice still displayed heightened levels of cervical carcinogenesis compared with that in nontransgenic mice, indicating that activities of E7 besides its capacity to inactivate p21(Cip1) also contribute to cervical carcinogenesis. Taken together, we conclude that p21(Cip1) functions as a tumor suppressor in cervical carcinogenesis and that p21(Cip1) inactivation by HPV-16 E7 partially contributes to the contribution of E7 to cervical carcinogenesis.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Oncogene Proteins, Viral/metabolism , Tumor Suppressor Proteins/metabolism , Uterine Cervical Neoplasms/pathology , Animals , Blotting, Western , Cell Cycle Proteins/metabolism , Cervix Uteri/drug effects , Cervix Uteri/metabolism , Cervix Uteri/pathology , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA-Binding Proteins/metabolism , Epithelium/metabolism , Epithelium/pathology , Estrogens/toxicity , Female , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Minichromosome Maintenance Complex Component 7 , Mutation , Nuclear Proteins/metabolism , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Phenotype , Phosphoproteins/metabolism , Retinoblastoma Protein/metabolism , Tumor Suppressor Proteins/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolismABSTRACT
Previous attempts to define dengue virus (DENV) tropism in human autopsy tissues have detected DENV antigens that are abundant in circulation during severe dengue, and thus may be present in uninfected cells. To better define DENV tropism, we performed immunostaining for the DENV2 nonstructural protein 3 (NS3) in humans and in a mouse model of DENV infection. In mice, NS3 was detected in phagocytes of the spleen and lymph node, hepatocytes in liver, and myeloid cells in bone marrow. In human autopsy tissues, NS3 was present in phagocytes in lymph node and spleen, alveolar macrophages in lung, and perivascular cells in brain. This protein was also found in hepatocytes in liver and endothelial cells in spleen, although NS3 was not present in endothelium in any other tissue. Thus, NS3-specific immunostaining supports roles for infected phagocytes, hepatocytes, and, to a limited degree, endothelial cells in the pathogenesis of severe dengue.
Subject(s)
Dengue Virus/metabolism , Dengue/virology , Tropism , Viral Nonstructural Proteins/metabolism , Adolescent , Adult , Animals , Antibodies, Monoclonal , Child, Preschool , Dengue Virus/classification , Disease Models, Animal , Endothelial Cells/virology , Hepatocytes/virology , Humans , Mice , Mice, Inbred BALB C , Middle Aged , Phagocytes/virology , Spleen/cytology , Spleen/virology , Staining and Labeling , Viral Nonstructural Proteins/immunologyABSTRACT
The four serotypes of dengue virus (DENV1-4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-alpha/beta and -gamma receptors (AG129) were used to model secondary infection using both DENV1-DENV2 and DENV2-DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.
Subject(s)
Antibodies, Viral/immunology , Dengue Virus/immunology , Severe Dengue/immunology , Severe Dengue/prevention & control , Adoptive Transfer , Animals , Immunization, Passive , Mice , Mice, Knockout , Receptors, Interferon/deficiency , Viral Plaque AssayABSTRACT
High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage-induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis.
Subject(s)
Carcinoma, Squamous Cell/genetics , Cell Transformation, Viral/genetics , Human papillomavirus 16/physiology , Oncogene Proteins, Viral/physiology , Papillomavirus Infections/genetics , Uterine Cervical Neoplasms/genetics , Alleles , Animals , Carcinoma, Squamous Cell/virology , Cell Cycle , Cell Cycle Proteins/physiology , Cell Differentiation , Cyclin E/physiology , DNA Damage , DNA-Binding Proteins/physiology , Estrogens/toxicity , Female , Gene Targeting , Genes, Retinoblastoma , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Minichromosome Maintenance Complex Component 7 , Nuclear Proteins/physiology , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Uterine Cervical Neoplasms/virology , Uterine Cervical Dysplasia/geneticsABSTRACT
High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRb(DeltaLXCXE)) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb(DeltaLXCXE) mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19(ARF). These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.
Subject(s)
Adenovirus E1A Proteins/physiology , Oncogene Proteins, Viral/physiology , Papillomaviridae/pathogenicity , Animals , Cell Cycle Proteins/metabolism , Cell Differentiation , Cyclin-Dependent Kinase Inhibitor p21 , Epidermal Cells , Hyperplasia/pathology , Hyperplasia/virology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasms, Glandular and Epithelial/pathology , Neoplasms, Glandular and Epithelial/virology , Nuclear Proteins , Papillomavirus E7 Proteins , Repressor Proteins , Tumor Suppressor Protein p53/metabolismABSTRACT
Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.