Running like water--the omnipresence of hepatitis E.
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Disease Outbreaks↗
Biomedical subjects
Robert H Purcell
Publications and source records attributed to Robert H Purcell.
An ELISA for putative neutralizing antibodies to hepatitis E virus detects antibodies to genotypes 1, 2, 3, and 4.
Two monoclonal antibodies that neutralize hepatitis E virus (HEV) were used to identify a subregion of ORF2 capsid protein spanning amino acids 459-607 as the shortest peptide to form the corresponding neutralization epitopes. An enzyme-linked immunosorbent assay (ELISA) based on a purified recombinant protein covering amino acids 458-607 in ORF2 of the Sar-55 strain (genotype 1) efficiently detected anti-HEV in non-human primates which had been experimentally infected with the four known mammalian genotypes of HEV, respectively. However, anti-HEV in these animals did not react with a shorter ORF2 peptide spanning amino acids 475-607. The ELISA was highly specific and sensitive when human or non-human primate sera were tested in parallel with a previously established ELISA based on amino acids 112-607 in ORF2. The antibody titer to peptides 458-607 in two ORF2-vaccinated rhesus monkeys which had different HEV challenge outcomes differed at the time of challenge. Since the ELISA appeared to be specific for neutralizing antibodies against HEV, it should be especially useful for quantifying the humoral immune response in hepatitis E vaccine trials.
Animals↗
Neutralizing antibodies to hepatitis C virus (HCV) in immune globulins derived from anti-HCV-positive plasma.
The role of humoral immunity in hepatitis C virus (HCV) infections is uncertain. Nevertheless, there is increasing evidence for neutralizing antibodies to HCV in the serum or plasma of chronically infected individuals. Immune globulins prepared by ethanol fractionation of plasma had long been considered safe until a commercial immune globulin product, Gammagard, prepared from plasma from which units containing anti-HCV had been excluded, transmitted HCV to recipients. Studies suggested that the exclusion might have removed neutralizing antibodies from the plasma and hence compromised the safety of the resulting immune globulins. In the present study, by using chimpanzees and a recently validated in vitro system based on neutralization of infectious HCV pseudoparticles, we found broadly reactive neutralizing and protective antibodies in experimental immune globulin preparations made from anti-HCV-positive donations. Neutralizing antibodies were also found in Gammagard lots made from unscreened plasma that did not transmit hepatitis C but not in Gammagard lots, which were prepared from anti-HCV-screened plasma, that did transmit hepatitis C. The results provide an explanation for the mechanism by which the safety of this product was compromised. Immune globulins made from anti-HCV-positive plasma and containing broadly reactive neutralizing antibodies may provide a method of preventing HCV infection.
Animals↗
Genomic analysis of the host response to hepatitis B virus infection.
Previous studies in hepatitis B virus (HBV)-infected humans and chimpanzees suggest that control of HBV infection involves the cells, effector functions, and molecular mediators of the immune response. The objective of the current study was to identify, in the liver of acutely HBV-infected chimpanzees, the spectrum of virus-induced and immune response-related genes that regulate the infection. The results demonstrate that HBV does not induce any genes during entry and expansion, suggesting it is a stealth virus early in the infection. In contrast, a large number of T cell-derived IFN-gamma-regulated genes are induced in the liver during viral clearance, reflecting the impact of an adaptive T cell response that inhibits viral replication and kills infected cells, thereby terminating the infection.
Animals↗
Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees.
We have previously shown that hepatitis B virus (HBV) replication is controlled by noncytolytic mechanisms that depend primarily on the effector functions of the CD8(+) T cell response, especially the production of IFN-gamma in the liver. The mechanisms that control the nuclear pool of viral covalently closed circular DNA (cccDNA) transcriptional template of HBV, which must be eliminated to eradicate infection, have been difficult to resolve. To examine those mechanisms, we quantitated intrahepatic HBV cccDNA levels in acutely infected chimpanzees whose virological, immunological, and pathological features were previously described. Our results demonstrate that the elimination kinetics of the cccDNA are more rapid than the elimination of HBV antigen-positive hepatocytes during the early phase of viral clearance, and they coincide with the influx of small numbers of IFN-gamma producing CD8(+) T cells into the liver. In contrast, terminal clearance of the cccDNA is associated with the peak of liver disease and hepatocellular turnover and with a surge of IFN-gamma producing CD8(+) T cells in the liver. Collectively, these results suggest that cccDNA clearance is a two-step process mediated by the cellular immune response. The first step reduces the pool of cccDNA molecules noncytolytically, probably by eliminating their relaxed circular DNA precursors and perhaps by destabilizing them. The second step enhances this process by destroying infected hepatocytes and triggering their turnover. Surprisingly, despite this multipronged response, traces of cccDNA persist indefinitely in the liver, likely providing a continuous antigenic stimulus that confers lifelong immunity.
Acute Disease↗
Long-term benefit of interferon alpha therapy of chronic hepatitis D: regression of advanced hepatic fibrosis.
BACKGROUND & AIMS: Little is known about the long-term effects of interferon alpha on clinical outcome and survival of patients with chronic hepatitis D. METHODS: Thirty-six patients with chronic hepatitis D who participated in a randomized controlled trial of a 48-week course of high (9 million units) or low (3 million units) doses of interferon alpha or no treatment were followed for an additional 2 to 14 years. RESULTS: Long-term survival was significantly longer in the high-dose group than in untreated controls (P = 0.003) or in the low-dose group (P = 0.019) but did not differ between patients treated with 3 million units and controls. Among surviving patients at 12 years of follow-up, a biochemical response was present in 7 of 12 treated with 9 million units, in 2 of 4 who received 3 million units, and in none of 3 controls. Long-term alanine aminotransferase (ALT) normalization correlated with improved hepatic function and loss of IgM antibody to hepatitis delta antigen (anti-HD). Patients in the high-dose group had a sustained decrease in HDV replication (P = 0.008), leading to clearance of HDV RNA and, eventually, hepatitis B virus (HBV) in some patients, as well as a dramatic improvement in liver histology with respect to activity grade (P = 0.0004) and fibrosis stage (P = 0.007). Strikingly, we documented an absence of fibrosis in the final biopsy of 4 patients with a long-term biochemical response and an initial diagnosis of active cirrhosis. CONCLUSIONS: High doses of interferon alpha-2a significantly improved the long-term clinical outcome and survival of patients with chronic hepatitis D, even though the majority had active cirrhosis before the onset of therapy.
Adult↗
Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses.
Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.
Amino Acid Sequence↗
Epitope determinants of a chimpanzee Fab antibody that efficiently cross-neutralizes dengue type 1 and type 2 viruses map to inside and in close proximity to fusion loop of the dengue type 2 virus envelope glycoprotein.
The epitope determinants of chimpanzee Fab antibody 1A5, which have been shown to be broadly reactive to flaviviruses and efficient for cross-neutralization of dengue virus type 1 and type 2 (DENV-1 and DENV-2), were studied by analysis of DENV-2 antigenic variants. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of the envelope protein (E), and another variant contained a His-to-Gln substitution at position 317 in E. Substitution of Gly(106)Val in DENV-2 E reduced the binding affinity of Fab 1A5 by approximately 80-fold, whereas substitution of His(317)Gln had little or no effect on antibody binding compared to the parental virus. Treatment of DENV-2 with beta-mercaptoethanol abolished binding of Fab 1A5, indicating that disulfide bridges were required for the structural integrity of the Fab 1A5 epitope. Binding of Fab 1A5 to DENV-2 was competed by an oligopeptide containing the fusion peptide sequence as shown by competition enzyme-linked immunosorbent assay. Both DENV-2 antigenic variants were shown to be attenuated, or at least similar to the parental virus, when evaluated for growth in cultured cells or for neurovirulence in mice. Fab 1A5 inhibited low pH-induced membrane fusion of mosquito C6/36 cells infected with DENV-1 or DENV-2, as detected by reduced syncytium formation. Both substitutions in DENV-2 E lowered the pH threshold for membrane fusion, as measured in a fusion-from-within assay. In the three-dimensional structure of E, Gly(106) in domain II and His(317) in domain III of the opposite E monomer were spatially close. From the locations of these amino acids, Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody.
Amino Acid Sequence↗
Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus.
A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.
Amino Acid Sequence↗
In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein.
Hepatitis E virus (HEV) RNA replication occurred in seven of nine primate cell cultures transfected with in vitro transcripts of an infectious cDNA clone. Cell-to-cell spread did not occur in cell cultures, but rhesus monkeys inoculated with lysates of HEV-transfected PLC/PRF/5 and Huh-7 cells became infected with HEV. A replicon with the ORF2 and ORF3 genes deleted and replaced with the green fluorescent protein gene also replicated in the same primate cells that supported the replication of the full-length genome. Fluorescence-activated cell sorter analysis confirmed that the 7mG cap structure was critical for efficient infectivity, although replication could be initiated at a very low level in its absence. HEV virions were also able to infect a limited number of cells of certain lines.
Animals↗
Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine.
In order to obtain monoclonal antibodies that might have prophylactic applications and to understand better the immune response to hepatitis E virus (HEV), we used phage display to isolate chimpanzee antibodies to HEV. The panning antigen was an two open reading frame (ORF2) recombinant protein that elicits a broadly protective immune response in vaccinated monkeys. Two major antigenic sites were identified on the ORF2 protein: one site was not accessible on the surface of infectious virions but the other site was accessible to antibodies and was recognized specifically by antibodies that neutralize the virus.
Animals↗
In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes.
Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.
Amino Acid Sequence↗
The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences.
The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous in vitro data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the amino- and carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent in vitro studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7 in vivo. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype 1a cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and 1a TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and 1a tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino- and/or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function.
Animals↗
Pre-clinical immunogenicity and efficacy trial of a recombinant hepatitis E vaccine.
We have demonstrated that recombinant hepatitis E vaccine suitable for clinical evaluation was highly immunogenic and efficacious in preventing hepatitis E and even infection in rhesus macaques following intravenous challenge with three different genotypes of hepatitis E virus (HEV). Two doses of vaccine were essential for optimal protection; the two-dose regimen was more important than the formulation of the vaccine for achieving efficacy. The titers of anti-HEV that were protective in this study were quantified against a World Health Organization (WHO) standard. This permits direct comparison of the results with other studies. The results of this pre-clinical trial of a candidate hepatitis E vaccine strongly suggest that it will be highly efficacious for preventing hepatitis E in the field trial of this vaccine that is currently in progress in Nepal.
Animals↗
CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection.
Although the CD4(+)- and CD8(+)-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8(+) cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8(+)-T-cell response.
Acute Disease↗
Long PCR amplification of large fragments of viral genomes: a technical overview.
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Genome, Viral↗
Genomic analysis of the host response to hepatitis C virus infection.
We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-alpha response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-gamma-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.
Animals↗