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Suzanne U Emerson

Publications and source records attributed to Suzanne U Emerson.

At least 19 recordsLinked to original sources
PubMed2006

Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous.

Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this caveat should be kept in mind while characterizing them.

Antibodies, Monoclonal↗
PubMed2006

ORF3 protein of hepatitis E virus is not required for replication, virion assembly, or infection of hepatoma cells in vitro.

A subclone of Huh-7 cells that could be relatively efficiently transfected and infected with hepatitis E virus was identified. Following transfection, infectious virus was produced but remained predominantly cell associated. Intracellular virus, recovered by lysis of transfected cells, infected naïve cells. This in vitro-produced virus appeared to be antigenically identical to virus isolated from clinical samples. Lysates from cells transfected with mutant viral genomes unable to synthesize ORF3 protein contained infectious virions that were similar in number, thermostability, and sedimentation characteristics to those in lysates transfected with wild-type viral genomes. Therefore, in contrast to its requirement in vivo, ORF3 protein is not required for infection of Huh-7 cells or production of infectious virus in vitro.

Carcinoma, Hepatocellular↗
PubMed2006

Functional analyses of GB virus B p13 protein: development of a recombinant GB virus B hepatitis virus with a p7 protein.

GB virus B (GBV-B), which infects tamarins, is the virus most closely related to hepatitis C virus (HCV). HCV has a protein (p7) that is believed to form an ion channel. It is critical for viability. In vitro studies suggest that GBV-B has an analogous but larger protein (p13). We found that substitutions of the -1 and/or -3 residues of the putative cleavage sites (amino acid 613/614 and 732/733) abolished processing in vitro and rendered an infectious GBV-B clone nonviable in tamarins. Internal cleavage was predicted at two sites (amino acid 669/670 and 681/682), and in vitro analysis indicated processing at both sites, suggesting that p13 is processed into two components (p6 and p7). Mutants with substitution at amino acid 669 or 681 were viable in vivo, but the recovered viruses had changes at amino acid 669 and 681, respectively, which restored cleavage. A mutant lacking amino acid 614-681 (p6 plus part of p7) was nonviable. However, a mutant lacking amino acid 614-669 (p6) produced high titer viremia and acute resolving hepatitis; viruses recovered from both animals lacked the deleted sequence and had no other mutations. Thus, p6 was dispensable but p7 was essential for infectivity. The availability of a recombinant GBV-B virus containing a p7 protein with similarities to the HCV p7 will enhance the relevance of this model and will be of importance for identifying compounds that inhibit p7 function as additional therapeutic agents.

Animals↗
PubMed2006

Active surveillance for acute viral hepatitis in rural villages in the Nile Delta.

BACKGROUND: Acute viral hepatitis is less frequent in Egypt than serum antibody levels suggest. Because acute viral hepatitis has a wide clinical spectrum, we tested the hypothesis that many cases are undetected because of mild illness caused by initial, early-childhood exposure to hepatitis viruses. METHODS: During active case detection among 20,000 inhabitants of rural villages in Egypt, we screened 1715 symptomatic patients for serum alanine aminotransferase (ALT) levels. Viral hepatitis markers were tested in 47 subjects who had ALT levels that were least twice the normal level. RESULTS: Of the 47 individuals tested, 4 children aged 3-5 years had immunoglobulin M (IgM) antibodies to hepatitis A virus (anti-HAV IgM). One also had a possible false-positive result to a test for IgM antibodies to hepatitis E virus. None had serological evidence of acute hepatitis B virus (HBV) infection or hepatitis C virus (HCV) infection. However, 33 of the remaining 43 had active HCV infection, having both antibodies to HCV (anti-HCV) and HCV RNA. Four others anti-HCV without HCV RNA, and 2 others had seroconversion to anti-HCV during follow-up. Two patients who were positive for hepatitis B surface antigen had chronic HBV infection. Only 3 with elevated ALT levels had no evidence of acute or chronic infections with known hepatitis viruses. Immunoglobulin G antibodies to hepatitis E virus was detected in 40 patients. CONCLUSION: Active surveillance covering approximately 50,000 person-years detected only 4 cases of acute HAV infection. Almost all persons with mild symptoms and elevated ALT levels had serological evidence of chronic viral hepatitis, most often associated with HCV. Many of these cases were probably "flare-ups" of HCV infection or incidental illness in patients with chronic HCV infection, but some could have been caused by difficult-to-confirm initial HCV infections. Although serological evidence for exposures was highly prevalent, hepatitis viruses seldom caused acute viral hepatitis in these communities.

Adolescent↗
PubMed2006

Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay.

Monolayers of Hep G2/C3A cells were inoculated with genotype 1 Hepatitis E virus (HEV) mixed with either anti-HEV or an appropriate control. After 5 or 6 days, cell monolayers were stained with anti-HEV and infected cells were identified by immunofluorescence microscopy and counted. Anti-HEV from vaccinated or infected rhesus monkeys neutralized the virus, as did mAbs that recognized epitopes on the C terminus of a recombinant vaccine protein. Antibodies were broadly cross-reactive, since convalescent serum from animals infected with any one of the four mammalian genotypes all neutralized the genotype 1 virus.

Animals↗
PubMed2006

A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus.

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.

Capsid Proteins↗
PubMed2005

High prevalence of hepatitis E antibodies in pregnant Egyptian women.

The epidemiology of hepatitis E virus (HEV), an enterically-transmitted cause of acute viral hepatitis (AVH), is not fully understood. During outbreaks on the Indian subcontinent and elsewhere, HEV causes severe AVH with mortality rates around 20% during pregnancy. In Egypt, where prevalence of HEV antibodies (anti-HEV) in rural communities is very high, severe HEV-caused AVH in pregnant women has not been reported. This study examined a cohort of 2,428 pregnant women in the Nile Delta to assess prevalence of, and risk factors for, anti-HEV and correlated these with history of liver disease. Anti-HEV prevalence was 84.3%. Several risk factors associated with anti-HEV included older age, many siblings, not using soap to wash produce and frequent contact with cats. History of jaundice and liver disease was rare and not increased in those having anti-HEV. Our results confirm Egypt's high HEV endemicity and show that almost all women of childbearing age in these communities had prior HEV exposures without a history of liver disease. Reasons for the lack of clinical hepatitis remain unclear but could be the result of early childhood HEV exposures, producing long-lasting immunity and/or modify subsequent responses to exposure. Alternatively, the predominant HEV strain(s) in Egypt are less virulent than those in South Asia.

Adolescent↗
PubMed2005

Hepatitis E antibody seroconversion without disease in highly endemic rural Egyptian communities.

Hepatitis E virus (HEV) is enterically transmitted and causes self-limiting acute viral hepatitis (AVH) primarily in less developed countries. A prospective cohort study to assess incidence of, and risk factors for, seroconversion to HEV (anti-HEV) was conducted in two Egyptian villages with a 67.7% anti-HEV prevalence. Nine hundred and nineteen villagers who were initially anti-HEV-negative were followed for 10.7 months. Thirty-four (3.7%) had strong anti-HEV serologic responses at follow-up giving an estimated anti-HEV incidence of 41.6/1,000 person-years. No significant associations were found between anti-HEV seroincidence and demographic and socioeconomic factors, source of water, household plumbing or sanitation, hand and vegetable washing, ownership of animals, jaundice and many other variables. None of the seroconverting subjects gave a history compatible with AVH during the interval. We hypothesize that both zoonotic and anthroponotic transmission of avirulent (possibly genotype-3) HEV is occurring extensively in these rural villages. An alternative explanation for the lack of morbidity among anti-HEV incident cases could be initial asymptomatic infections occur during early childhood with subsequent antibody titer boosting without illness upon re-exposure to the virus.

Adolescent↗
PubMed2005

Thermal stability of hepatitis E virus.

The thermal stability of virulent hepatitis E virus (HEV) and hepatitis A virus (HAV) was compared. Fecal suspensions of virus were heated to temperatures between 45 degrees C and 70 degrees C, and residual infectivity was determined in a cell culture system that was permissive for both viruses. Although HEV was less stable than was HAV, some HEV would most likely survive the internal temperatures of rare-cooked meat.

Cell Line↗
PubMed2005

A truncated ORF2 protein contains the most immunogenic site on ORF2: antibody responses to non-vaccine sequences following challenge of vaccinated and non-vaccinated macaques with hepatitis E virus.

A candidate hepatitis E vaccine is composed of amino acids (aa) 112-607 of the 660-aa protein encoded by open reading frame 2 (ORF2) of hepatitis E virus (HEV). We have studied the antibody response to vaccine-associated epitopes and to epitopes excluded from the vaccine to determine if important epitopes were omitted from the vaccine and if antibody responses to these regions could be used to differentiate between infection and vaccination. ELISAs were developed based on genotype 1 ORF2 peptides, containing aa 112-607 (vaccine), 458-607 (minimum neutralization site), 1-111 (N-terminus) and 607-660 (C-terminus), as well as on ORF3 peptides, containing aa 1-123 (complete) and 91-123 (C-terminus). All naive macaques infected with HEV genotype 1, 2, 3 or 4 produced antibodies to all ORF2 peptides. Anti-ORF3 was detected in both monkeys infected with genotype 1 virus and in one of two infected with genotype 2 virus. These antibody responses were considerably weaker than those directed against the neutralization site. In contrast, vaccinated animals that were challenged with HEV had a diminished or absent immune response to the peptides not included in the vaccine. Thus, only minor epitopes were excluded from the vaccine; they had limited utility for distinguishing between vaccination and infection.

Animals↗
PubMed2005

Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.

The lack of a cell culture system to support hepatitis C virus (HCV) replication has hampered studies of this frequent cause of chronic liver disease. However, pseudotyped retroviral particles (pp) bearing the HCV envelope glycoproteins have provided a different approach to HCV studies. We used genotype 1a pp to detect neutralizing antibodies (NtAb) in eight chimpanzees and four humans infected with 1a strains, and developed pp of genotypes 2a, 3a, 4a, 5a, and 6a to study crossreactivity. NtAb was detected in one of four chimpanzees and none of three humans with acute resolving infection, suggesting that NtAb is not required for HCV clearance. NtAb were detected at high titer in two of four chimpanzees and, in Patient H, all with persistent infection; responses paralleled humoral responses to envelope 1 and 2 proteins and, in some cases, correlate also with antibodies to the hypervariable region 1, previously thought to be the primary site of neutralization. NtAb raised during 1a infections could neutralize HCVpp of genotypes 4a, 5a, and 6a but had only limited reactivity against 2a and 3a. The detection of high-titer NtAb with cross-genotype reactivity has important implications for the development of active and passive immune-prophylaxis strategies against HCV. Finally, we found that HCVpp infectivity was enhanced by human or chimpanzee sera; apolipoprotein C1 alone or as a component of high-density lipoproteins caused this enhancement. Future studies of the in vivo role of apolipoprotein C1 might provide additional insights into the infection process of HCV.

Animals↗
PubMed2005

Human monoclonal antibodies that react with the E2 glycoprotein of hepatitis C virus and possess neutralizing activity.

Active and/or passive immunoprophylaxis against hepatitis C virus (HCV) remain unachieved goals. Monoclonal antibodies might provide one approach to protection. We derived human monoclonal antibodies from the bone marrow of a patient with a well-controlled HCV infection of 22 years duration. Five distinct antibodies reactive with the E2 glycoprotein of the homologous 1a strain of HCV were recovered as antigen-binding fragments (FAbs). They demonstrated affinity constants as high as 2 nanomolar. "Neutralization of binding" titers paralleled the affinity constants. All five FAbs reacted with soluble E2 protein only in nonreducing gels, indicating that the relevant epitopes were conformational. The FAbs could be divided into two groups, based on competition analysis. Three of the FAbs neutralized the infectivity of pseudotyped virus particles (pp) bearing the envelope glycoproteins of the homologous HCV strain (genotype 1a). The three FAbs also neutralized genotype 1b pp and one also neutralized genotype 2a pp. In conclusion, one or more of these monoclonal antibodies may be useful in preventing infections by HCV belonging to genotype 1 or 2, the most medically important genotypes worldwide.

Amino Acid Sequence↗
PubMed2005

The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques.

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.

Amino Acid Sequence↗
PubMed2005

In vitro and in vivo mutational analysis of the 3'-terminal regions of hepatitis e virus genomes and replicons.

Hepatitis E virus (HEV) replication is not well understood, mainly because the virus does not infect cultured cells efficiently. However, Huh-7 cells transfected with full-length genomes produce open reading frame 2 protein, indicative of genome replication (6). To investigate the role of 3'-terminal sequences in RNA replication, we constructed chimeric full-length genomes with divergent 3'-terminal sequences of genotypes 2 and 3 replacing that of genotype 1 and transfected them into Huh-7 cells. The production of viral proteins by these full-length chimeras was indistinguishable from that of the wild type, suggesting that replication was not impaired. In order to better quantify HEV replication in cell culture, we constructed an HEV replicon with a reporter (luciferase). Luciferase production was cap dependent and RNA-dependent RNA polymerase dependent and increased following transfection of Huh-7 cells. Replicons harboring the 3'-terminal intergenotypic chimera sequences were also assayed for luciferase production. In spite of the large sequence differences among the 3' termini of the viruses, replication of the chimeric replicons was surprisingly similar to that of the parental replicon. However, a single unique nucleotide change within a predicted stem structure at the 3' terminus substantially reduced the efficiency of replication: RNA replication was partially restored by a covariant mutation. Similar patterns of replication were obtained when full-length genomes were inoculated into rhesus macaques, suggesting that the in vitro system could be used to predict the effect of 3'-terminal mutations in vivo. Incorporation of the 3'-terminal sequences of the swine strain of HEV into the genotype 1 human strain did not enable the human strain to infect swine.

Animals↗
PubMed2004

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↗
PubMed2004

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↗
PubMed2004

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↗