Recent advances in the bioanalytical applications of dried matrix spotting for the analysis of drugs and their metabolites.

DBS techniques for the bioanalysis of drugs and metabolites from whole blood have been demonstrated to be a useful tool in drug development. The term dried matrix spot (DMS) has been used to indicate that the DBS technique has been applied to nonblood matrices. DMS methods often employ a color-indicating process that enhances the ability to analyze these mostly transparent fluids when spotted onto collection paper. The color-indicating dye allows the analyst to visually confirm the location of the dried sample spot. Other benefits of using a color-indicating dye include improved method accuracy and precision, because the process of adding the dye allows for the concurrent addition of the IS prior to sample addition and extraction. To date, matrices that have been analyzed using DMS include cerebrospinal fluid, synovial fluid, saliva, tears, urine and plasma.

The advantages of microflow LC-MS/MS compared with conventional HPLC-MS/MS for the analysis of methotrexate from human plasma.

BACKGROUND: In support of bioanalysis, there has always been a desire to improve detection limits and reduce scale. Microflow LC (MFLC) coupled with MS accomplishes both of these goals. RESULTS: As such, MFLC coupled with an MS system was used to generate bioanalytical validation data that met US FDA criteria. The MFLC-MS/MS data was compared with the same method with the use of conventional HPLC-MS/MS and a more than 14× S/N improvement was found with the MFLC-MS/MS method. Methotrexate was used as a model molecule to demonstrate the validation of the method from human plasma. The MFLC-MS/MS method was demonstrated to be accurate (±7%) and precise (12.9% at the LLOQ and a maximum of 11.6% at all other concentrations) across the dynamic range of the assay (1-1000 ng/ml) and compared well with the HPLC-MS/MS method. The MFLC bioanalytical validation was performed at a flow rate of 35 µl/min on a 0.5-mm inner diameter (I.D.) column, whereas, for the same linear velocities on the 2.0-mm I.D. column, the conventional HPLC bioanalytical validation was performed at 700 µl/min. Since the flow rate of the MFLC system is 20-times less than the HPLC system, the consumable solvent and disposal cost to perform the MFLC validation was significantly less. CONCLUSION: MFLC-MS/MS can be used to perform bioanalytical method validations with increased MS signal, reduced source contamination and reduced solvent consumption.

Peptides specific for Mycobacterium avium subspecies paratuberculosis infection: diagnostic potential.

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease (JD). Current serological diagnostic tests for JD are limited by their sensitivity when used in sub-clinical stages of the disease. Our objective was to identify peptides that mimic diagnostically important Map epitopes that might be incorporated into a new-generation JD diagnostic. Four peptides were isolated from a phage-displayed random peptide library by screening on antibodies derived from Map-infected goats. The peptides were recognised by antibodies from Map-infected goats but not by antibodies from uninfected goats. The peptides elicited immune responses in rabbits, which reacted strongly with bona fide Map antigens proving the peptides were true epitope mimics. To assess the diagnostic value a panel of goat sera was screened for reactivity's with peptides. The peptides were recognised by antibodies from a proportion of goats infected with Map compared with control animals with a diagnostic specificity of 100% and the sensitivity ranged from 50 to 75%. Combinations of any two peptides improved sensitivity 62.5-87.5% and 100% sensitivity was achieved with three of the four peptides in combination. These data suggest peptides representing diagnostically important Map epitopes could be incorporated into a sensitive diagnostic test.

Isolation of antibodies specific to a single conformation-dependant antigenic determinant on the EG95 hydatid vaccine.

EG95 is a recombinant vaccine protein that elicits protection against hydatid disease in sheep. Previous studies have shown that the host-protective epitopes on EG95 depend on correct conformation and cannot be represented by simple "linear" peptides. By screening random peptide phage display libraries with polyclonal antibodies directed against conformation-dependant epitopes of EG95, we have selected a number of peptides that mimic these epitopes. The selected peptides did not show sequence homology to EG95. Antigen binding assays involving these peptides have provided evidence of at least four conformationally-dependant epitope regions on EG95. One of the selected peptides, E100, has been used to purify antibodies from anti-sera raised in sheep vaccinated with EG95. This yielded monospecific antibodies capable of recognizing recombinant EG95 in ELISA and native EG95 in Western blot assays. This antibody was demonstrated to be effective in antibody-dependant complement-mediated in vitro killing of Echinococcus granulosus oncospheres. Peptide E100 may represent the basis for a quality control assay for EG95 production, and has the potential to become a component of a synthetic peptide-based vaccine against E. granulosus.

Peptide mimics selected from immune sera using phage display technology can replace native antigens in the diagnosis of Epstein-Barr virus infection.

There is an expanding area of small molecule discovery, especially in the area of peptide mimetics. Peptide sequences can be used to substitute for the entire native antigen for use in diagnostic assays. Our approach is to select peptides that mimic epitopes of the natural immune response to Epstein-Barr virus (EBV) that may be recognised by antibodies typically produced after infection with EBV. We screened a random peptide library on sera from rabbits immunised with a crude preparation of EBV and serum antibodies from a patient with a high titer of EBV antibodies. We selected four peptides (Eb1-4) with the highest relative binding affinity with immune rabbit sera and a single peptide with high affinity to human serum antibodies. The peptides were coupled to the carrier molecule BSA and the recognition of the peptides by IgM antibodies in clinical samples after infection with EBV was measured. The sensitivities were Eb1 94%, Eb2, 3, 4 88%, H1 81% and all had 100% specificity. This study illustrates that the phage display approach to select epitope mimics can be applied to polyclonal antibodies and peptides that represent several diagnostically important epitopes can be selected simultaneously. This panel of EBV peptides representing a wide coverage of immunodominant epitopes could replace crude antigen preparations currently used for capture in commercial diagnostic tests for EBV.

RNA editing in hepatitis delta virus.

Hepatitis delta virus (HDV) relies heavily on host functions and on structural features of the viral RNA. A good example of this reliance is found in the process known as HDV RNA editing, which requires particular structural features in the HDV antigenome, and a host RNA editing enzyme, ADAR1. During replication, the adenosine at the amber/W site in the HDV antigenome is edited to inosine. As a result, the amber stop codon in the hepatitis delta antigen (HDAg) open reading frame is changed to a tryptophan codon and the reading frame is extended by 19 or 20 codons. Because these extra amino acids alter the functional properties of HDAg, this change serves a critical purpose in the HDV replication cycle. Analysis of the RNA secondary structures and regulation of editing in HDV genotypes I and III has indicated that although editing is essential for both genotypes, there are substantial differences. This review covers the mechanisms of RNA editing in the HDV replication cycle and the regulatory mechanisms by which HDV controls editing.

The woodchuck model of HDV infection.

The Eastern woodchuck, Marmota monax, has been a useful model system for the study of the natural history of hepadnavirus infection and for the development and preclinical testing of antiviral therapies. The model has also been used for hepatitis delta virus (HDV). In this chapter several new applications of the woodchuck model of HDV infection are presented and discussed. The development of a woodchuck HDV inoculum derived from a molecular clone has facilitated the analysis of viral genetic changes occurring during acute and chronic infection. This analysis has provided insights into one of the more important aspects of the natural history of HDV infection-whether a superinfection becomes chronic. These results could renew interest in further vaccine development. An effective therapy for chronic HDV infection remains an important clinical goal for this agent, particularly because of the severity of the disease and the inability of current hepadnaviral therapies to ameliorate it. The recent application of the woodchuck model of chronic HDV infection to therapeutic development has yielded promising results which indicate that targeting the hepadnavirus surface protein may be a successful therapeutic strategy for HDV.

The most polymorphic residue on Plasmodium falciparum apical membrane antigen 1 determines binding of an invasion-inhibitory antibody.

Apical membrane antigen 1 (AMA1) is currently one of the leading malarial vaccine candidates. Anti-AMA1 antibodies can inhibit the invasion of erythrocytes by Plasmodium merozoites and prevent the multiplication of blood-stage parasites. Here we describe an anti-AMA1 monoclonal antibody (MAb 1F9) that inhibits the invasion of Plasmodium falciparum parasites in vitro. We show that both reactivity of MAb 1F9 with AMA1 and MAb 1F9-mediated invasion inhibition were strain specific. Site-directed mutagenesis of a fragment of AMA1 displayed on M13 bacteriophage identified a single polymorphic residue in domain I of AMA1 that is critical for MAb 1F9 binding. The identities of all other polymorphic residues investigated in this domain had little effect on the binding of the antibody. Examination of the P. falciparum AMA1 crystal structure localized this residue to a surface-exposed alpha-helix at the apex of the polypeptide. This description of a polymorphic inhibitory epitope on AMA1 adds supporting evidence to the hypothesis that immune pressure is responsible for the polymorphisms seen in this molecule.

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study.

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab' cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab' maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab')(2). Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab' maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab' maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab')(2) used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab' maleimide over murine versions of this antibody suggesting that humanised divalent-Fab' maleimide should be a useful vehicle for repeated therapies.

Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.

Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally deficient for the expression of interferon gamma and tumor necrosis factor-alpha messenger RNAs.

Surgical biopsies of the liver were obtained from woodchuck hepatitis virus (WHV)-infected neonatal woodchucks at 2 time points before the self-limited or chronic outcomes became obvious by serologic criteria. Following segregation of outcomes, livers were analyzed for intrahepatic type 1 cytokine messenger RNAs (mRNAs) (interleukin 2 [IL-2], interferon gamma [IFN-gamma], tumor necrosis factor-alpha [TNF-alpha]) and leukocyte inflammatory phenotype (IgG+ plasma cells, lysozyme+ macrophages, CD3+ T cells). Baselines were assessed using age-matched uninfected control livers. At week 8 (early acute phase), intrahepatic type 1 cytokine mRNAs were similarly low in both outcome settings and no different from age-matched uninfected controls. This was consistent with the minimal initial viral loads and lack of histologic inflammation at this time. At week 14 (mid-acute phase), changes in viral load between outcome groups related inversely to the intrahepatic inflammatory responses. Animals that eventually became resolved had increased intrahepatic expression of IFN-gamma and TNF-alpha mRNAs and robust inflammation by CD3+ T cells, plasma cells, and macrophages. At the same time point of infection, animals that eventually became chronic carriers had an acute hepatitis involving the same cell types, but at diminished levels, and markedly deficient intrahepatic expression of IFN-gamma and TNF-alpha mRNAs. IL-2 mRNA remained at baseline control levels in both outcome groups. These cotemporal comparisons map a critical deviation in host response to the acute stage of an evolving chronic infection. They strongly suggest that increasing viral load and chronicity as an outcome of neonatal WHV infection result from a temporal deficiency in the acute intrahepatic effector mechanisms mediated by IFN-gamma and TNF-alpha.

Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium.

We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors.

Green fluorescent antibodies: novel in vitro tools.

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

An outbreak of fulminant hepatitis delta in the Waorani, an indigenous people of the Amazon basin of Ecuador.

An outbreak of delta hepatitis occurred during 1998 among the Waorani of the Amazon basin of Ecuador. Among 58 people identified with jaundice, 79% lived in four of 22 Waorani communities. Serum hepatitis B surface antigen (HBsAg) was found in the sera of 54% of the jaundiced persons, and 14% of asymptomatic persons. Ninety-five percent of 105 asymptomatic Waorani had hepatitis B core (HBc) IgG antibody, versus 98% of 51 with jaundice. These data confirm that hepatitis B virus (HBV) infection is highly endemic among the Waorani. Sixteen of 23 (70%) HBsAg carriers identified at the onset of the epidemic had serologic markers for hepatitis D virus (HDV) infection. All 16 were jaundiced, where as only two of seven (29%) with negative HDV serology were jaundiced (P = .0006). The delta cases clustered in families, 69% were children and most involved superinfection of people chronically infected with HBV. The data suggest that HDV spread rapidly by a horizontal mode of transmission other than by the sexual route.

Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab' fragments.

We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab')2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab')2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397-1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab')2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM.

Intrafamilial transmission of hepatitis delta virus: molecular evidence.

BACKGROUND/AIMS: Epidemiologic studies have suggested that transmission of hepatitis delta virus (HDV) occurs by intrafamilial routes in some populations in southern Italy, where HDV infection is endemic. To further evaluate intrafamilial transmission of HDV, we obtained the partial sequence of the viral genome from HDV-RNA positive members of families in which two or more immediate family members were positive for HDV-RNA. METHODS: The region analyzed was the semi-conserved region from nucleotides 908 to 1265. Sequences obtained from family members were compared with those obtained from a control group of 20 unrelated patients. RESULTS: The mean genetic divergence among HDV isolates was 2.8 +/- 1.7% within the 9 families analyzed, and 7.6 +/- 2.2% among the control group of unrelated individuals (p < 0.0001). A Receiver Operating Characteristic curve and Youden Index were used to define a cut-off value of 3.5% to discriminate sequence variations calculated within families and in the control group. CONCLUSIONS: The data indicate that in most family units, HDV-infected members harbored nearly identical strains of HDV, and provide molecular support that HDV infection can be transmitted within the family. Such spreading among family members highlights the role of inapparent transmission through personal contacts.

A novel technique, using radioluminography, for the measurement of uniformity of radiolabelled antibody distribution in a colorectal cancer xenograft model.

PURPOSE: Radioimmunotherapy of cancer employs an antitumour antibody to carry a radionuclide selectively to deposits of cancer. Conventional dose estimates, based on the Medical Internal Radiation Dose (MIRD) formulation, assume uniform distribution of radiolabelled antitumour antibody within tissue source regions. This assumption has been tested by using a statistical model to predict the pixel value distribution obtained from the digitised radioluminographs of a known radioactive source. The model uses the statistical nature of the detection of radiation where any uniform source distribution can be expected to have a detected histogram of pixel counts that is normal or Gaussian. Therefore, any test for the degree of normality in the detected distribution is also a measure of the degree of uniformity in the source. METHODS AND MATERIALS: Three statistical techniques have been used to test the normality of the histogram of pixel values produced from the antibody distribution in a tissue section. Kurtosis, skew, and Lilliefor's are tests for normality and have statistically defined critical values for a normal distribution. After administration of 125I-labelled F(ab)2 antibody to nude mice bearing the LS174T colorectal cancer xenograft, the uniformity of antibody distribution in tumour and healthy tissues is measured using the radioluminographs of formalin-fixed paraffin sections. The test statistic for kurtosis, skew, and Lilliefor's is calculated for each tissue and is compared to critical values from statistical tables. RESULTS: The radiolabelled antibody is distributed uniformly in liver, spleen, muscle, lung, and colon and, therefore, conforms to conventional use of the MIRD formulation. The study showed that the kidney cortex and medulla should be considered separately in macroscopic absorbed-dose calculations, as should bone marrow and hard bone. Antibody heterogeneity in the tumour necessitates the incorporation of a microdosimetric tumour model into a macrodosimetry model for the accurate calculation of absorbed dose in all tissues.

Clearance of yttrium-90-labelled anti-tumour antibodies with antibodies raised against the 12N4 DOTA macrocycle.

Radioimmunotherapy (RIT) is currently limited by toxicity to normal tissues as a result of prolonged circulating radioantibody in the blood. In this study, the use of a clearing antibody was investigated (second antibody) in an attempt to reduce blood background levels of [90Y]A5B7 immunoglobulin G (IgG) activity, and, therefore, improve the therapeutic tumour-blood ratio in nude mice bearing human colorectal tumour xenografts. The second antibody was raised against the 12N4 macrocycle group used for chelation of 90Y, and is, thus, applicable to any anti-tumour antibody labelled with this methodology. Second antibody was administered 18, 24 or 48 h after radiolabelled antibody injection and produced up to a tenfold reduction in blood levels and a tenfold improvement in tumour-blood ratios. This has the advantage of reducing the risk of myelotoxicity caused by prolonged retention of activity in the blood. For all normal tissues, there was a similar or slightly lower uptake of [90Y]IgG with second antibody clearance, apart from a transient rise in liver activity due to complexes of primary and secondary antibody clearing via the liver. As a result of clearance of [90Y]IgG from the blood pool, there was an associated fall in the amount of antibody at the tumour site (up to 3.3-fold) at later time points for mice injected with second antibody. However, despite this, tumour-blood ratios remained superior to the control group at these later time points. Estimated dosimetry evaluation revealed that total dose to normal tissues, blood and tumour was lower than for the non-clearance group. Surprisingly, however, there was little improvement in total estimated tumour-blood dose ratio over the time period studied. This was probably because the majority of the dose was delivered to both the blood and tumour within the first 24 h after administration of [90Y]IgG, so that giving the clearing agent after this time did not produce a large difference in total estimated dose. The anti-macrocycle second antibody proved to be a successful clearing agent and could potentially be applied to any anti-tumour antibody coupled with the 12N4 macrocycle. In the light of the estimated dosimetry results described here, it would probably be most useful given at earlier time points (i.e. before 18 h after injection of primary antibody) to produce an improved tumour-blood ratio of total dose. Development of this strategy may allow higher levels of activity to be administered for RIT, and repeated dosing regimens.

Hepatitis D viremia following orthotopic liver transplantation involves a typical HDV virion with a hepatitis B surface antigen envelope.

Patients receiving orthotopic liver transplantation (OLT) because of type D hepatitis frequently exhibit what appears to be an autonomous, or "isolated," hepatitis D virus (HDV) infection following the transplantation, with no evidence of hepatitis B virus (HBV) in the graft or in the serum. These observations have led to the hypothesis that HBV might not always be required for HDV infection, or that HDV could exist as a latent infection until rescued by HBV. Alternatively, an apparently autonomous HDV infection could be explained by coinfection of a small number of hepatocytes with both viruses following transplantation, with a very low level of HBV expression that supports low-level HDV propagation. Our results are consistent with the latter hypothesis. Sensitive polymerase chain reaction (PCR)-based analysis of HBV and HDV viremia in transplantation patients with HDV infection previously characterized as isolated showed that HDV viremia was not independent of HBV viremia. Additional analyses, including PCR amplification, buoyant density analysis in a CsCl gradient, and immunoprecipitation with monoclonal hepatitis B surface antigen antibodies (anti-HBs), indicated that the posttransplant HDV particle is typical: it contains full-length HDV RNA and an envelope of hepatitis B surface antigen (HBsAg) and is not different from that found during the acute and chronic stages of HDV superinfection or coinfection. Moreover, an experimental test of the first hypothesis in chimpanzees did not support the idea that HDV can persist for several weeks as an isolated, latent infection that can be rescued subsequently by HBV. The data indicate, therefore, that latent HDV infection is not a factor in OLT recipients. We conclude that the HDV virion in the posttransplantation setting is typical, and that HDV viremia following OLT requires the helper function of HBV infection.