Kathleen M. Brasky

DNA Microarray Analysis of Chimpanzee Liver during Acute Resolving Hepatitis C Virus Infection

ABSTRACT Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understood and are difficult to study. Here, we describe the changes in liver gene expression during the course of an acute-resolving HCV infection in a chimpanzee. Clearance of viremia in this animal occurred between weeks 6 and 8, while clearance of residual infected hepatocytes did not occur until 14 weeks postinfection. The most notable changes in gene expression occurred in numerous interferon response genes (including all three classical interferon antiviral pathways) that increased dramatically, some as early as day 2 postinfection. The data suggest a biphasic mechanism of viral clearance dependent on both the innate and adaptive immune responses and provide insight into the response of the liver to a hepatotropic viral infection.

Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity

The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant κ domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (Kd) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the Kd of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees

ABSTRACT Hepatitis C virus (HCV) infections represent a global health problem and are a major contributor to end-stage liver disease including cirrhosis and hepatocellular carcinoma. An improved understanding of the parameters involved in disease progression is needed to develop better therapies and diagnostic markers of disease manifestation. To better understand the dynamics of host gene expression resulting from persistent virus infection, DNA microarray analyses were conducted on livers from 10 chimpanzees persistently infected with HCV. A total of 162 genes were differentially regulated in chronically infected animals compared to uninfected controls. Many genes exhibited a remarkable consistency in changes in expression in the 10 chronically infected animals. A second method of analysis identified 971 genes altered in expression during chronic infection at a 99% confidence level. As with acute-resolving HCV infections, many interferon (IFN)-stimulated genes (ISGs) were transcriptionally elevated, suggesting an ongoing response to IFN and/or double-stranded RNA which is amplified in downstream ISG expression. Thus, persistent infection with HCV results in a complex and partially predictable pattern of gene expression, although the underlying mechanisms regulating the different pathways are not well defined. A single genotype 3-infected animal was available for analysis, and this animal exhibited reduced levels of ISG expression compared to levels of expression with genotype 1 infections and increased expression of a number of genes potentially involved in steatosis. Gene expression data in concert with other observations from HCV infections permit speculation on the regulation of specific aspects of HCV infection.

GS-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees

BACKGROUND & AIMS Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection. METHODS GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620. RESULTS Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets. CONCLUSIONS The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection.

Cross-Genotype Immunity to Hepatitis C Virus

ABSTRACT Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.

Ribavirin Induces Error-Prone Replication of GB Virus B in Primary Tamarin Hepatocytes

ABSTRACT GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV) and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.

Genomic response to interferon-α in chimpanzees: Implications of rapid downregulation for hepatitis C kinetics

The mechanism of the interferon‐alpha (IFN‐α)‐induced antiviral response during hepatitis C virus (HCV) therapy is not completely understood. In this study, we examined the transcriptional response to IFN‐α in uninfected chimpanzees after single doses of chimpanzee, human, or human‐pegylated IFN‐α. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours post‐inoculation, a time when high levels of circulating pegylated IFN‐α were still present. The rapid downregulation of the IFN‐α response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN‐α therapy in HCV‐infected patients. The response to all three forms of IFN‐α was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN‐α were not due to species specificity of IFN‐α. The response to IFN‐α was partially tissue‐specific. A total of 1,778 genes were altered in expression by twofold or more by IFN‐α, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN‐α and IFN‐γ responses in primary chimpanzee and human hepatocytes were compared as well. IFN‐α and IFN‐γ induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN‐α is largely tissue‐specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response. (HEPATOLOGY 2006;43:961–972.)

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

A live attenuated vaccine for Lassa fever made by reassortment of Lassa and Mopeia viruses.

ABSTRACT Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Old World arenaviruses that can exchange genomic segments (reassort) during coinfection. Clone ML29, selected from a library of MOPV/LASV (MOP/LAS) reassortants, encodes the major antigens (nucleocapsid and glycoprotein) of LASV and the RNA polymerase and zinc-binding protein of MOPV. Replication of ML29 was attenuated in guinea pigs and nonhuman primates. In murine adoptive-transfer experiments, as little as 150 PFU of ML29 induced protective cell-mediated immunity. All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions. Rhesus macaques inoculated with clone ML29 developed primary virus-specific T cells capable of secreting gamma interferon in response to homologous MOP/LAS and heterologous MOPV and lymphocytic choriomeningitis virus. Detailed examination of two rhesus macaques infected with this MOPV/LAS reassortant revealed no histological lesions or disease signs. Thus, ML29 is a promising attenuated vaccine candidate for Lassa fever.

Protection against lethal challenge by Ebola virus-like particles produced in insect cells

Ebola virus-like particles (VLPs) were produced in insect cells using a recombinant baculovirus expression system and their efficacy for protection against Ebola virus infection was investigated. Two immunizations with 50 microg Ebola VLPs (high dose) induced a high level of antibodies against Ebola GP that exhibited strong neutralizing activity against GP-mediated virus infection and conferred complete protection of vaccinated mice against lethal challenge by a high dose of mouse-adapted Ebola virus. In contrast, two immunizations with 10 microg Ebola VLPs (low dose) induced 5-fold lower levels of antibodies against GP and these mice were not protected against lethal Ebola virus challenge, similar to control mice that were immunized with 50 microg SIV Gag VLPs. However, the antibody responses against GP were boosted significantly after a third immunization with 10 microg Ebola VLPs to similar levels as those induced by two immunizations with 50 microg Ebola VLPs, and vaccinated mice were also effectively protected against lethal Ebola virus challenge. Furthermore, serum viremia levels in protected mice were either below the level of detection or significantly lower compared to the viremia levels in control mice. These results show that effective protection can be achieved by immunization with Ebola VLPs produced in insect cells, which give high production yields, and lend further support to their development as an effective vaccine strategy against Ebola virus.