Lentiviral hepatitis B pseudotype entry requires sodium taurocholate co-transporting polypeptide and additional hepatocyte-specific factors.

Hepatitis B virus (HBV) is one of the world's major unconquered infections, resulting in progressive liver disease, and current treatments rarely cure infection. A limitation to discovering new therapies is our limited knowledge of HBV entry and dissemination pathways that hinders the development of in vitro culture systems. To address this gap in our understanding we optimized the genesis of infectious lentiviral pseudoparticles (HBVpps). The recent discovery that the bile salt transporter sodium taurocholate co-transporting polypeptide (NTCP) acts as a receptor for HBV enabled us to assess the receptor dependency of HBVpp infection. HBVpps preferentially infect hepatoma cells expressing NTCP, whereas other non-liver cells engineered to express NTCP do not support infection, suggesting that additional hepatocyte-specific factors are required for HBVpp internalization. These results highlight the value of the HBVpp system to dissect the pathways of HBV entry and dissemination.

A surrogate hepatitis B virus antigenic epitope represented by a synthetic peptide and an internal image antiidiotype antibody.

The use of molecules that represent single, defined epitopes able to substitute for antigen (i.e. surrogate antigens) offers considerable advantages over the use of native antigen for the precise manipulation of the immune response. We have investigated the immunochemical characteristics of two types of surrogate hepatitis B surface antigen (HBsAg) epitopes: (a) linear and cyclical synthetic peptides representing amino acid residues 139-147, a hydrophilic region corresponding to part of the a determinant of the HBsAg, and (b) four monoclonal antiidiotypes raised against anti-HBs mAb, two of which behave as an internal image of an a determinant. Polyclonal anti-HBs antisera bound the monoclonal antiidiotypes with affinities of the order of 10(8)/M, and to the peptides with greater than 10-fold lower affinities. However, the levels of antibody in the polyclonal antisera for the peptides was greater than for the antiidiotypes. In inhibition RIA, the surrogate antigens show concordance in that the internal image antiidiotypes inhibit the binding of both monoclonal and polyclonal anti-HBs to the linear and cyclical 139-147 peptides. These results imply that surrogate antigens could indeed be useful as potential hepatitis vaccines, but while the antiidiotypes may stimulate B cells of higher affinity, they would react with a more restricted range of B cell reactivities than would the peptides. A future HBV vaccine may thus comprise a synthetic peptide such as cyclical 139-147 or a cluster of monoclonal internal image antiidiotypes.

A peptide mimotope of hepatitis C virus E2 protein is immunogenic in mice and block human anti-HCV sera.

Conformational B-cell epitopes on the HCV E2 protein recognized by human antibodies were characterized by the use of a peptide mimotope named K1. K1 was identified by two HCV anti-E2 monoclonal antibodies (mAbs) following selection and purification of phage clones containing a 15-mer random peptide insert. Murine antisera to the mimotope K1 recognized the E2 protein. Five of eight human sera from patients who had cleared HCV recognized the K1 mimotope. Binding to E2 in four individuals with the capacity to block E2-CD81 interaction was inhibited by the mimotope K1. The results demonstrate that anti-E2 antibodies in sera from patients who have cleared HCV infection are directed against a conformational B-cell epitope on E2 that can be mimicked with linear synthetic peptides. These findings could have implications for vaccine design by employing linear mimotopes to direct B-cell responses against those specific E2 epitopes that may correlate with immunity.

High level expression of genes cloned in phage lambda gt11.

Plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in Escherichia coli. They are based on the pEMBL and pUC vectors, with the genes transcribed from the lac promoter. The EcoRI site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11. Cloned proteins are expressed fused to a 2-kDa leader sequence containing a run of six Aparagine residues which considerably improves the stability of the recombinant proteins, but does not interfere with immunological assays. Using these vectors, the Mycobacterium leprae 18-kDa protein was expressed at 20 mg per litre of culture and constituted 15% of total cell protein.

An improved selection procedure for the screening of phage display peptide libraries.

Peptide sequences that bind to a wide range of ligands such as monoclonal antibodies, receptors and carbohydrates have been successfully identified after screening phage display peptide libraries. However, these procedures tend to select mainly medium to low affinity-binding clones. A modified screening procedure has been developed in order to improve the efficiency of this process such that high avidity/affinity binding clones are preferentially selected. Three different solid phase binding surfaces were evaluated for the attachment of antibody during the screening procedure and a stepwise decrease in the pH of the elution buffer introduced during the final round of biopanning. The monoclonal antibody MA 18/7 was used to screen a 15-mer peptide library. This antibody is well-characterised and its binding site has been mapped to residues 28-37 of the pre-S1 protein of the hepatitis B virus. The antibody was either biotinylated and attached to polystyrene plates via a streptavidin-biotin 'bridge', or bound directly to 1/4 in. polystyrene beads, or to 11 microm latex beads. A significant enrichment of binding clones was observed when the monoclonal antibody was attached directly to polystyrene or latex beads as compared to the biotinylated antibody. All mimotopes identified after biopanning with the antibody attached to the polystyrene beads possessed a central core motif, identical or similar to the sequence DPAF contained within the epitope binding site of MA 18/7 on the native pre-S molecule. However, this motif was only observed in 30% of clones isolated after biopanning using the 11 microm latex beads and in 2% of clones isolated after biopanning on the streptavidin-coated plates. Immunoblotting with the monoclonal antibody MA 18/7 confirmed binding to clones containing the DPAF sequence or a similar motif. A stepwise reduction in the pH of the elution buffer in the final round of biopanning resulted in the removal of clones that possessed low affinity binding motifs, thereby increasing the percentage of clones containing high affinity binding motifs in the final elution step at pH 2.0. Thus, the combined use of polystyrene beads and a stepwise decrease in the pH of the elution buffer in the final round of biopanning resulted in the elimination of non-binding clones and an increase in the efficiency in isolating high affinity binding clones.

Core antigen and circulating anti-core antibody in hepatitis B infection.

Core antigen was obtained from the sera of persistent chronic carriers of hepatitis B virus by centrifugation and treatment with Nonidet P40 and 2-mercaptoethanol. The separated core antigen was radiolabelled and identified as a nucleoprotein structure of buoyant density 1.36 g/cm3 and possessing an isoelectric point of 4.4. This material was employed in a radioimmunoassay procedure of high sensitivity for the detection of core antibody. In a series of sera from patients with acute type B hepatitis, core antibody was demonstrated 2 to 3 weeks after the onset of jaundice during the period of surface antigenaemia. The presence of core antibody may therefore provide an accurate serological marker for the detection of active or recent virus replication in future epidemiological studies of hepatitis B infection.

Production of anti-peptide specific antibody in mice following immunization with peptides conjugated to mannan.

In order to investigate the usefulness of polysaccharides as carriers for the induction of antibody to synthetic peptides, peptides representing residues 139-147 of the surface antigen of hepatitis B and residues 129-140 of the pre-S2 region of the protein were coupled to mannan and dextran via an aminocaproic spacer molecule. Of the two conjugates studied, only mannan was useful as a carrier for the efficient production of anti-peptide antibodies.

HBsAg: anti-HBs immune complexes. A method for separating the constituent components and assessment of the affinity of the antibody.

A number of chemical disruption agents were assessed for their ability to dissociate HBsAg:anti-HBs immune complexes and to release both the antibody and antigen component in immunologically active forms. The most appropriate reagent was 0.1 M diethylamine which could elute up to 81% of anti-HBs antibody bound to solid-phase HBsAg and retained 93% of its antigen-combining activity. Complexes formed at various degrees of antigen excess and pre-exposed to 0.1 M diethylamine at room temperature for 18 h before ultracentrifugation on sucrose density gradients were effectively dissociated. The released antibody and antigen banded at their expected densities. However, the affinity of the isolated antibody for the detergent-solubilized polypeptide complex from purified HBsAg (gp30/p25) and cyclical peptides representing amino acids 124-137 and 139-147 of HBsAg were found to be considerably lower than that of the original pooled anti-HBs immunoglobulin used to form the immune complexes. These results suggest that the highest affinity antibody subpopulation may not be completely dissociated from the complex. Care should thus be exercised in the interpretation of the significance of the observed affinity of the antibody isolated by this and other similar dissociation procedures.

Determination of the affinity of antibodies to hepatitis B surface antigen in human sera.

The measurement of the affinity of anti-HBs antibody in human sera using 3 HBsAg-related antigens is described. The antigens used were (i) a synthetic linear peptide corresponding to amino acids 139-147 of the major polypeptide of HBsAg, (ii) a cyclical form of this same peptide and (iii) a polypeptide complex of a 28,000 MW glycoprotein and a 23,000 MW protein from purified HBsAg. The method was established with a pooled human anti-HBs immunoglobulin preparation and a monoclonal anti-HBs antibody reactive to the 'a' determinant of HBsAg. The results indicate that both these antibody preparations effectively bind the 3 antigens with affinity values of between 2 X 10(6) to 9 X 10(7) litres/mole. However, the affinity of both antibody preparations for the cyclical form of the peptide was higher than for the linear form. The level of antibody (expressed as Abt, molar antigen binding sites) in the pooled human immunoglobulin for each of the 3 antigens was similar. Measurements of anti-HBs antibodies in the sera of recovered acute hepatitis B patients and from HBsAg negative chronic liver disease patients showed that the cyclical form of the antigen was bound with a higher affinity than the linear form. Affinity values of antibody in the sera of the latter group of patients was significantly lower (3 X 10(5) to 2.7 X 10(6) litres/mole) than those observed in sera from other individuals. The implication of these results in determining the importance of the measurement of affinity in the assessment of the efficacy of vaccines is discussed.

Acetaminophen analgesia in neonatal circumcision: the effect on pain.

OBJECTIVE: Recognizing the concerns about the use of local anesthesia in neonatal circumcision, a painful procedure usually performed without analgesia or anesthesia, we undertook a study of acetaminophen for pain management of this procedure. DESIGN: A prospective, randomized, double-blind, placebo-controlled, clinical trial of acetaminophen analgesia in 44 healthy full-term neonates undergoing circumcision was conducted. Beginning 2 hours before Gomco circumcision, neonates received either acetaminophen (15 mg/kg per dose, 0.15 mL/kg per dose) or placebo (0.15 mL/kg per dose) every 6 hours for 24 hours. Neonates were monitored intraoperatively for changes in heart rate, respiratory rate, and crying time. Postoperative pain was assessed at 30, 60, 90, 120, 360 minutes, and 24 hours using a standardized postoperative comfort scoring system. Feeding behavior was also assessed before and after circumcision by nursing observation. RESULTS: Neonates in both groups showed significant increases in heart rate, respiratory rate, and crying during circumcision with no clinically significant differences observed between the groups. Postoperative comfort scores showed no significant differences between the groups until the 360-minute postoperative assessment, at which time the acetaminophen group had significantly improved scores (P < .05). Feeding behavior deteriorated in breast- and bottle-fed neonates in both groups, and acetaminophen did not seem to influence this deterioration. CONCLUSIONS: This study confirms that circumcision of the newborn causes severe and persistent pain. Acetaminophen was not found to ameliorate either the intraoperative or the immediate postoperative pain of circumcision, although it seems that it may provide some benefit after the immediate postoperative period.

Neonatal circumcision and pain relief: current training practices.

OBJECTIVE: We conducted a national survey of pediatric, family practice, and obstetrics and gynecology residency program directors to determine the curriculum content and predominant practices in US training programs with regard to neonatal circumcision and anesthesia/analgesia for the procedure. METHODS: Residency directors of accredited programs were surveyed in two mailings of a forced response and short answer survey (response rate: 680/914, 74%; pediatrics 83%; family practice 72%; obstetrics 71%). RESULTS: Pediatric residents were less likely than family practice [odds ratio (OR), 0.04; 95% confidence interval (CI), 0.02-0.08] or obstetrical (OR, 0.14; 95% CI, 0.08-0.23) residents to be taught circumcision. Training and local custom were rated as important determinants of medical responsibility for neonatal circumcision. Pediatric residents training in programs in which community pediatricians perform circumcisions were more likely to learn circumcision (OR, 39.0; 95% CI, 14.3-110.6) as were obstetric residents (OR, 79.0; 95% CI, 22.4-306.4) training in programs in which community obstetricians perform circumcision. In programs that teach circumcision, pediatric (84%; OR, 3.4; 95% CI, 1.7-7.1) and family practice (80%; OR, 2.7; 95% CI, 1.7-4.2) programs were more likely than obstetric programs (60%) to teach analgesia/anesthesia techniques to relieve procedural pain. Overall, 26% of programs that taught circumcision failed to provide instruction in anesthesia/analgesia for the procedure. Significant regional variations in training in circumcision and analgesia/anesthesia techniques were noted within and across medical specialties. CONCLUSIONS: Residency training standards are not consistent for pediatric, family practice, and obstetrical residents with regard to neonatal circumcision or instruction in analgesia/anesthesia for the procedure. Training with regard to pain relief is clearly inadequate for what remains a common surgical procedure in the United States. Given the overwhelming evidence that neonatal circumcision is painful and the existence of safe and effective anesthesia/analgesia methods, residency training in neonatal circumcision should include instruction in pain relief techniques.

Physiologic stability of newborns during cup- and bottle-feeding.

BACKGROUND: To prevent breastfeeding problems, cup-feeding has been recommended as a method of providing medically necessary supplemental feedings to breastfed infants. OBJECTIVES: To compare amounts ingested, administration time, and infant physiologic stability during cup-, bottle-, and breastfeeding. DESIGN/METHODS: A total of 98 term, healthy newborns were randomized to either cup-feeding (n = 51) or bottle-feeding (n = 47). The heart (HR), respiratory (RR), and oxygen (O(2)) saturation rates were monitored on these infants and 25 breastfed newborns during 1 feeding. Differences in amounts ingested and administration times were evaluated with t tests and physiologic data with repeat measures analysis of variance. RESULTS: There were no significant differences in administration time, amounts ingested or overall HR, RR, and (O(2)) saturation rates, between cup and bottle groups. Breastfed infants had longer administration times and lower overall HR, RR, and higher O(2) saturation as compared with cup- and bottle-fed infants. CONCLUSIONS: Administration times, amounts ingested, and infant physiologic stability do not differ with cup- and bottle-feeding. Breastfeeding takes longer than cup- or bottle-feeding, but infants experience less physiologic variability. These data support cup-feeding as an alternative to bottle-feeding for supplying supplements to breastfed infants.

Analysis of the antigenic epitopes of hepatitis B surface antigen involved in the induction of a protective antibody response.

Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.

Naked DNA when co-administered intranasally with heat-labile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen.

In this study a novel prime-boost immunisation strategy was evaluated. Priming of BALB/c mice by the intranasal route with plasmid DNA encoding beta-galactosidase (LacZ) with or without heat-labile enterotoxin (LT) of Escherichia coli as a mucosal adjuvant, resulted in the induction of weak serum antibody and proliferative T-cell responses. However, following an intraperitoneal booster injection with the beta-galactosidase protein (beta-gal), strong antibody and proliferative T-cell responses were induced in all the mice. These responses were highest in mice primed intranasally with a mixture of LacZ+LT as compared to those mice primed with DNA (LacZ) or protein (beta-gal) alone. Moreover, LacZ+LT primed mice produced high avidity antibodies and the subclasses of serum antibodies were IgG1 and IgG2a, suggesting a mixed Th1/Th2-type response. Priming of mice with either protein (beta-gal) or DNA (LacZ) alone, produced predominantly IgG1 antibodies, suggesting a Th2-type response. These findings suggest that the use of a heterologous DNA-prime, protein-boost immunisation scheme combining different routes of administration, might be an advantageous strategy for the induction of accelerated immune responses.

Viral antigens and antibodies in hepatitis B infection.

Hepatitis B virus antigens and antibodies were detected in the sera of acute and persistently infected patients. Evidence of active virus replication was confined to immediately before or during the initial detection of hepatitis B surface antigen during acute hepatitis B. Hepatitis B core antibody appeared during the period of antigenaemia and preceded recovery. Hepatitis B e antigen was dound in a proportion of sera which contained significant levels of virus particles. In contrast, all sera containing hepatitis B virus particles from persistently infected patients treated by maintenance haemodialysis also contained the e antigen. Among a group of 50 persistent carriers of hepatitis B virus, significant levels of virus production occurred in the presence of antibody to e antigen. In addition, evidence of exposure to hepatitis B virus was found among 3% of blood donors in whose sera the surface antigen was not detected by radioimmunoassay. The significance of these findings is discussed in relation to the aetiology of hepatitis type B.

The physician as advertiser: the unintentional discouragement of breast-feeding.

To be consistent with national health goals and ACOG policies and recommendations, physicians providing prenatal care should encourage breast-feeding whenever possible. The parents' choice to breast- or formula-feed their infant is the consequence of a complex decision. The physician's role is to provide information objectively so that the parents' decision can be made on an informed and factual basis. Clearly, the physician must support parents' decisions. However, the distribution of formula or vouchers in the physician's office during the antepartum period places the physician in the position of advertising or promoting a specific product and of potentially contributing to the failure of some patients to nurse their infants. We urge physicians not to distribute formula or formula vouchers to their pregnant patients, and encourage local and national obstetrics organizations to consider devising and discussing a policy statement discouraging such practices.

The ligase chain reaction distinguishes hepatitis B virus S-gene variants.

A novel method for the identification of hepatitis B virus (HBV) variants was developed. The ligase chain reaction (LCR) distinguished the sequences of two isolates from the Gambia showing a change at nucleotide 421 within the region coding for the surface protein a antigenic determinant. One sequence was derived from a child previously immunized with HBV vaccine, while the other, reported here for the first time, was from a chronic carrier. Nucleotide variations within the target sequence at, or up to 3 bases from the point of ligation inhibited the reaction. The LCR recognised variations in as little as 0.9 fmol DNA and will permit the rapid detection of HBV variants in molecular epidemiological studies.

The use of markers in immune electron microscopy.

Immune electron microscopy (IEM) cannot be used successfully for structures that do not have recognisable morphology. However, at least some of these structures or components are related antigenically to recognisable antigens or viruses. We have therefore mixed unknown antigens with known markers and looked for the presence of mixed aggregates. The present study examined a low molecular weight subunit of rotavirus and a micellar form of hepatitis B surface antigen. In both cases mixed immune aggregates were found showing that the unknown components had antigens in common with the established virus or antigen.