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Biomedical subjects

Jack T Stapleton

Publications and source records attributed to Jack T Stapleton.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of an immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in cell binding.

GB virus type C (GBV-C) is a human flavivirus that may cause persistent infection, although most infected individuals clear viremia and develop antibodies to the envelope glycoprotein E2. To study GBV-C E2 antigenicity and cell binding, murine anti-E2 monoclonal antibodies (MAbs) were evaluated to topologically map immunogenic sites on GBV-C E2 and for the ability to detect or block recombinant E2 binding to various cell lines. Five competition groups of MAbs were identified. Groups I and II did not compete with each other. Group III competed with both groups I and II. Group IV did not compete with group I, II, or III. One MAb competed with all of the other MAbs, suggesting that the epitopes bound by these MAbs are intimately related. Individually, none of the MAbs competed extensively with polyclonal human convalescent antibody (PcAb); however, combinations of all five MAb groups completely blocked PcAb binding to E2, suggesting that the epitopes bound by these MAbs form a single, immunodominant antigenic site. Only group I and III MAbs detected purified recombinant E2 bound to cells in binding assays. In contrast, group II MAbs neutralized the binding of E2 to cells. Both PcAb and MAbs were conformation dependent, with the exception of one group II MAb (M6). M6 bound to a five-amino-acid sequence on E2 if the peptide included four C-terminal or eight N-terminal residues, suggesting that the GBV-C E2 protein contains a single immunodominant antigenic site which includes a complex epitope that is involved in specific cellular binding.

Amino Acid Sequence↗
PubMed2006

An 85-aa segment of the GB virus type C NS5A phosphoprotein inhibits HIV-1 replication in CD4+ Jurkat T cells.

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.

CD4-Positive T-Lymphocytes↗
PubMed2006

In vitro interaction between hepatitis C virus (HCV) envelope glycoprotein E2 and serum lipoproteins (LPs) results in enhanced cellular binding of both HCV E2 and LPs.

Hepatitis C virus (HCV) particles in serum associate with lipoproteins (LPs), and the low-density lipoprotein receptor (LDLr) has been implicated in virus attachment and entry into cells. To clarify the basis of interactions between virus and LPs, we determined whether HCV interacts with human LPs via its envelope glycoprotein E2. The binding of serum-derived virus-like particles, HCV E2, and HCV E2-LP complexes to CD81 and LDLr was studied. Incubation of HCV E2 protein with human and bovine LPs (very low density, low density, and high density) enhanced the binding of both HCV E2 and LPs to CD4+ lymphoblastoid (MOLT-4) cells, foreskin fibroblasts, and hepatocytes. The binding of HCV E2 to MOLT-4 cells was not enhanced when it was preincubated with lipid-free apoprotein B, which suggests that E2 interacts with the lipid moiety of human lipoproteins. The LP interaction was specific for HCV E2--incubation of HIV gp120 with LPs did not enhance gp120 binding to MOLT-4 cells. The enhanced HCV E2 binding required expression of both human CD81 and LDLr. These data suggest that HCV E2 associates with LDL and that the resulting complex enhances binding of both ligands to cells, which may contribute to the finding that HCV-infected individuals have significantly lower levels of LDL than control subjects.

Antigens, CD↗
PubMed2005

GB virus C replicates in primary T and B lymphocytes.

GB virus C (GBV-C) infection is common in humans and may persist for decades, although most infected persons clear the virus and subsequently develop antibodies to the envelope glycoprotein. GBV-C replicates in peripheral blood mononuclear cells (PBMCs) and CD4(+) T lymphocytes in vitro, and depletion of CD4(+) T lymphocytes has been proposed as the reason for clearance of GBV-C among persons positive for human immunodeficiency virus. We identified GBV-C RNA in purified CD4(+) and CD8(+) T lymphocytes and CD19(+) B lymphocytes removed ex vivo from infected donors and found that GBV-C replicated in vitro in these PBMC subsets, suggesting that GBV-C is a panlymphotropic virus.

Antigens, CD19↗
PubMed2005

South African GB virus C isolates: interactions between genotypes 1 and 5 isolates and HIV.

GB virus C (GBV-C) is a common, apparently nonpathogenic human flavivirus that has been associated in some studies with prolonged survival in human immunodeficiency virus (HIV)-positive persons. There are 5 distinct GBV-C genotypes localized to specific geographic regions, and genotype 2 has been previously shown to inhibit HIV replication in vitro in lymphocyte cultures. We identified GBV-C virus isolates representing genotypes 1, 2, and 5 in samples from South African blood donors. GBV-C genotype 1 and 5 isolates replicated in lymphocyte culture, inhibited X4 and R5 HIV-1 isolates, and induced RANTES and stromal-derived factor-1 chemokines in vitro. Thus, African GBV-C genotypes can inhibit HIV replication in vitro.

5' Untranslated Regions↗
PubMed2005

Effect of GB virus C viremia on HIV acquisition and HIV set-point.

We performed both a case-control study and a cohort study to determine whether GB virus C (GBV-C) viremia prevents the acquisition of HIV infection or alters the HIV set-point after infection. The prevalence of GBV-C viremia in HIV-uninfected individuals who did and did not acquire HIV were similar (odds ratio 1.32; 95% confidence interval 0.6-2.6). Pre-existing GBV-C viremia at the time of HIV acquisition was also not associated with lower plasma HIV-RNA levels.

Adult↗
PubMed2005

GB virus type C NS5A sequence polymorphisms: association with interferon susceptibility and inhibition of PKR-mediated eIF2alpha phosphorylation.

GB virus type C (GBV-C) causes persistent infection in humans, although the mechanism by which the virus avoids clearance by the host is unknown. To determine if amino acid polymorphisms in the GB virus type C (GBV-C) NS5A and E2 proteins alter response to interferon (IFN) therapy, we studied the sequence of GBVC NS5A and E2 obtained from people receiving IFN therapy. In addition, we expressed recombinant GBVC NS5A protein to determine if it interferes with RNA-activated protein kinase (PKR) function in vitro. GBVC NS5A amplified from a person whose virus was cleared by IFN therapy (IFN sensitive) demonstrated unique amino acid changes occurring in the region that aligns with the hepatitis C virus (HCV) IFN sensitivity-determining region (ISDR) compared with NS5A sequences from individuals who did not clear GBV-C (IFN resistant). There were no differences in the E2 sequences obtained from IFN-sensitive and IFN-resistant isolates. Using a yeast genetic system, IFN-resistant NS5A inhibited PKR-mediated phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) in yeast, whereas IFN-sensitive NS5A did not inhibit PKR function. GBV-C NS5A amino acid polymorphisms appear to be involved in response to IFN therapy, and IFN-resistant GBV-C NS5A inhibited PKR-mediated eIF2alpha phosphorylation in a yeast genetic system, suggesting a mechanism by which GBV-C may evade clearance by naturally occurring host antiviral responses.

Amino Acid Sequence↗
PubMed2005

GB virus C infection in children with perinatal human immunodeficiency virus infection.

BACKGROUND: GB virus C (GBV-C) infection occurs in 20-40% of human immunodeficiency virus (HIV)-infected adults, and coinfection is associated with improved HIV disease outcome. METHODS: To determine the prevalence of GBV-C infection in children who were perinatally infected with HIV, we conducted a cross-sectional prevalence survey in a cohort of perinatally infected HIV-positive children selected from a large, multicenter observational protocol. A blood specimen was obtained and tested for GBV-C viremia with the use of a qualitative GBV-C RNA assay and screened for past GBV-C infection with enzyme-linked immunosorbent assay to detect antibodies to the GBV-C envelope protein E2 (E2 Ab). RESULTS: The 354 children who participated in the substudy were relatively healthy, with a median CD4 of 784 cells/mm and median HIV-1 viral load of 1055 copies/mL. The prevalence of GBV-C viremia was 20 of 353 or 5.7% (95% confidence interval, 3.5-8.6%), and the prevalence of E2 Ab was 12 of 354 or 3.4% (95% confidence interval, 1.8-5.8%). GBV-C viremic patients were older than patients without past GBV-C infection (median age, 12.8 years versus 10.7 years). Median CD4 lymphocyte counts were highest in subjects without GBV-C infection and lowest in those with E2 Ab. CONCLUSIONS: GBV-C prevalence rates are lower in children with perinatal HIV infection than those reported for HIV-infected adults. With the exception of evidence that GBV-C viremic children had lower rates of Centers for Disease Control and Prevention HIV disease category C disease before GBV-C testing, we did not find evidence of improved HIV disease outcome in coinfected patients, but the number of HIV/GBV-C-coinfected children was small.

AIDS-Related Opportunistic Infections↗
PubMed2004

Vaccination success rate and reaction profile with diluted and undiluted smallpox vaccine: a randomized controlled trial.

CONTEXT: Additional smallpox vaccine doses are needed to augment current US national stockpile. Aventis Pasteur smallpox vaccine (APSV), initially manufactured in the 1950s from the New York Board of Health vaccinia strain in a frozen preparation, appears as effective as lyophilized vaccine but the effectiveness of diluted doses of APSV is unclear. OBJECTIVE: To compare the vaccination success rate and the reaction profile of various APSV dilutions. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized controlled trial of 340 healthy vaccinia-naive adults aged 18 to 32 years from 3 academic medical centers who were vaccinated with 1 of 3 strengths of APSV dilutions (undiluted, 1:5, and 1:10) between October 9, 2002, and February 24, 2003. Volunteers were followed up every 3 to 5 days until the vaccination site healed for bandage changes, vaccine response assessment, and adverse event evaluation, followed by 1- and 2-month clinic evaluations and 6-month telephone interview. MAIN OUTCOME MEASURES: Successful vaccination, defined by presence of a vesicle or pustule at the inoculation site 6 to 11 days postvaccination, and local and systemic reactions to vaccination. RESULTS: A total of 340 volunteers were vaccinated (vaccine dose: undiluted, n = 113; 1:5 dilution, n = 114; and 1:10 dilution, n = 113). Following vaccination, 99.4% (95% confidence interval [CI], 97.9%-99.9%) of all volunteers had successful vaccinations. Success rates did not differ between the dilution groups (undiluted, 100.0%; 95% CI, 96.8%-100.0%; 1:5 dilution, 98.2%; 95% CI, 93.8%-99.8%; 1:10 dilution, 100.0% 95% CI, 96.8%-100.0%; P =.33). Overall, 99.7% of volunteers reported at least 1 local symptom at the vaccination site, and 61.8% had axillary lymphadenopathy, 15.0% developed satellite lesions, and 7.6% developed a rash away from the vaccination site. Fever developed in 21.5%. No differences were noted in local or systemic reactions between the 3 dilution groups (P>.05 for each comparison). A total of 25% of volunteers missed scheduled duties due to vaccine-related symptoms. CONCLUSIONS: Even at diluted doses, APSV is an effective smallpox vaccine, allowing for expansion of the current stockpile. However, reactogenicity was not reduced with dilution of the vaccine and, as with other smallpox vaccines, may impair daily activities.

Adult↗
PubMed2004

Inhibition of HIV-1 replication by GB virus C infection through increases in RANTES, MIP-1alpha, MIP-1beta, and SDF-1.

Background People coinfected with HIV and GB virus C (GBV-C) have lower mortality than HIV-positive individuals without GBV-C infection. HIV uses either of the chemokine receptors CCR5 and CXCR4 for entry into CD4-positive cells. Longer survival in HIV-positive individuals is associated with high serum concentrations of ligands for CCR5 (RANTES [regulated on activation, normal T-cell expressed and secreted] and macrophage inflammatory proteins [MIP] 1alpha and 1beta) and CXCR4 (stromal-derived factor [SDF-1]), and with decreased expression of CCR5 on lymphocytes. Methods Peripheral-blood mononuclear cells were coinfected with GBV-C and HIV, and HIV replication was monitored by measuring infectivity and HIV p24 antigen production. Chemokine secretion was measured by ELISA, chemokine-receptor expression by flow cytometry, and cellular chemokine mRNA expression by differential hybridisation. Findings GBV-C infection of peripheral-blood mononuclear cells resulted in decreased replication of both clinical and laboratory HIV strains that use either CCR5 or CXCR4 as their coreceptor. Inhibition was related to the dose and timing of the GBV-C infection. Expression of mRNA for RANTES, MIP-1alpha, MIP-1beta, and SDF-1 and secretion of the chemokines into culture supernatants were higher in GBV-C-infected cells than in mock-infected cells. The inhibitory effect of GBV-C on HIV replication was blocked by incubation with neutralising antibodies against the relevant chemokines, and surface expression of CCR5 was significantly lower in GBV-C-infected cells than in mock-infected cells. Interpretation GBV-C induces HIV-inhibitory chemokines and reduces expression of the HIV coreceptor CCR5 in vitro. This study provides insight into the epidemiological association between GBV-C infection and longer survival in HIV-infected individuals.

Cells, Cultured↗
PubMed2004

Persistent GB virus C infection and survival in HIV-infected men.

BACKGROUND: GB virus C (GBV-C), which is not known to be pathogenic in humans, replicates in lymphocytes, inhibits the replication of human immunodeficiency virus (HIV) in vitro, and has been associated with a decreased risk of death among HIV-positive persons in some, but not all, studies. Previous studies did not control for differences in the duration of HIV or GBV-C infection. METHODS: We evaluated 271 men who were participants in the Multicenter Acquired Immunodeficiency Syndrome Cohort Study for GBV-C viremia (by means of a reverse-transcriptase-polymerase-chain-reaction assay) or E2 antibody (by means of an enzyme-linked immunosorbent assay) 12 to 18 months after seroconversion to positivity for HIV (the early visit); a subgroup of 138 patients was also evaluated 5 to 6 years after HIV seroconversion (the late visit). RESULTS: GBV-C infection was detected in 85 percent of men with HIV seroconversion on the basis of the presence of E2 antibody (46 percent) or GBV-C RNA (39 percent). Only one man acquired GBV-C viremia between the early and the late visit, but 9 percent of men had clearance of GBV-C RNA between these visits. GBV-C status 12 to 18 months after HIV seroconversion was not significantly associated with survival; however, men without GBV-C RNA 5 to 6 years after HIV seroconversion were 2.78 times as likely to die as men with persistent GBV-C viremia (95 percent confidence interval, 1.34 to 5.76; P=0.006). The poorest prognosis was associated with the loss of GBV-C RNA (relative hazard for death as compared with men with persistent GBV-C RNA, 5.87; P=0.003). CONCLUSIONS: GBV-C viremia was significantly associated with prolonged survival among HIV-positive men 5 to 6 years after HIV seroconversion, but not at 12 to 18 months, and the loss of GBV-C RNA by 5 to 6 years after HIV seroconversion was associated with the poorest prognosis. Understanding the mechanisms of interaction between GBV-C and HIV may provide insight into the progression of HIV disease.

Adult↗
PubMed2004

Active or prior GB virus C infection does not protect against vertical transmission of HIV in coinfected women from Tanzania.

To determine whether GB virus C (GBV-C) infection is associated with protection against vertical transmission of human immunodeficiency virus (HIV), we tested 186 HIV-positive pregnant women for GBV-C. Neither active nor prior GBV-C infection was associated with a lower rate of HIV acquisition among infants. Thus, GBV-C does not appear to protect against perinatal HIV acquisition.

Adult↗
PubMed2004

Neutralizing human monoclonal antibodies to hepatitis A virus recovered by phage display.

Four human monoclonal antibodies (MAbs) to hepatitis A virus (HAV) were isolated from a phage-displayed antibody library constructed from the peripheral blood lymphocytes (PBLs) of HAV-immune donors. The four MAbs showed differences in their affinity: two (HA6, HA9) of them were dominant after four rounds of panning, and showed higher affinity than the other two (HA1, HA12). All four MAbs showed HAV-neutralizing activity in radioimmunofocus inhibition assay and their neutralizing activity was positively correlated with their affinities. Analysis of their epitope specificity by cross-competition binding assays suggested that HA6 and HA9 recognize extensively overlapping epitopes, which overlap with those of HA1 and HA12, although HA1 and HA12 recognize distinct epitopes. In addition, competition assays with known neutralizing murine MAbs suggested that the epitopes of four human MAbs extensively overlap with those of B5B3 and K34C8 which are distinct but reside within the single, immunodominant neutralization site on the HAV capsid. The human MAbs (HA6 and HA9) with highest affinity may be useful in the immunoprophylaxis of HAV infection.

Amino Acid Sequence↗