Efficacy of a hepatitis C virus core antigen enzyme-linked immunosorbent assay for the identification of 'window-phase' blood donations.

BACKGROUND AND OBJECTIVES: Recent studies have suggested that potentially infectious donations provided during the antibody-negative 'window' phase of hepatitis C virus (HCV) infection may be identified by testing for viral RNA or HCV core protein. We therefore evaluated the performance of an HCV antigen enzyme-linked immunosorbent assay (ELISA) for identification of window-phase donations and for prospective screening of blood donors. MATERIALS AND METHODS: One-hundred and twenty-eight archived plasma donations containing HCV RNA, but lacking antibody to HCV (anti-HCV), were tested by using the HCV antigen ELISA, together with 9951 freshly collected serum and plasma specimens from blood donors. RESULTS: HCV core antigen was detected in 94% (120/128) of window-phase plasma donations. Overall specificity in freshly collected blood donor specimens was 99.74%. Two putative window-phase donations containing HCV core protein and viral RNA were identified from paid plasma donors by prospective testing with the HCV antigen ELISA. CONCLUSION: These results indicate that an HCV antigen ELISA can identify almost all (94%) of viraemic donations given during the seronegative window phase of infection. The performance of the HCV antigen ELISA appears to be suitable for large-scale screening of blood donations.

Lymphoproliferative responses to hepatitis C virus core, E1, E2, and NS3 in patients with chronic hepatitis C infection treated with interferon alfa.

The quality of the hepatitis C virus (HCV)-specific T-cell response may greatly determine the course of an HCV infection. An adequate T-cell response may contribute to a successful clearance of the virus and a rapid recovery from the disease. An inadequate response may lead to viral persistence and may eventually contribute to the pathogenesis of hepatocellular damage in chronic disease. The effect of interferon alfa (IFN-alpha), presently the most popular therapeutic agent for chronic HCV infections, on HCV-specific T-cell responses is completely unknown. To demonstrate the presence of HCV-specific T lymphocytes during chronic HCV infections, to know their antigenic specificities, and to examine possible effects of IFN-alpha treatment on their presence and antigen recognition patterns, we have stimulated peripheral blood mononuclear cells (PBMC) from 35 chronic HCV patients with nine pools of synthetic peptides representing the HCV Core, E1, and E2 proteins as well as with a recombinant NS3 protein. The proliferative responses of PBMC from 16 healthy control subjects toward these antigens were measured for comparison. Lymphoproliferative responses of patients with chronic HCV infections were assayed either before (in 10 patients), during (in 13 patients), or after (in 21 patients) treatment with IFN-alpha. The analysis showed that PBMC from most HCV patients consistently recognized the COOH-terminal part of the core protein. E1, E2, and NS3 were recognized less frequently. This recognition pattern was not related to the therapy with IFN-alpha nor to the clinical response of the patient toward this therapy. The response to the Core protein could be fine-mapped to the COOH-terminal region encompassing amino acids (aa) 73 to 92, 121 to 140, 145 to 164, and 157 to 176.

Follow-up study of clinical and immunological findings in patients presenting with acute parvovirus B19 infection.

This study was undertaken to examine the natural history of parvovirus B19 infection in persons without a known immune defect in terms of both clinical symptoms and immune responsiveness to the virus. Fifty-three patients with acute B19 infection (positive for serum anti-B19 IgM) were studied; symptoms at acute infection were rash and arthralgia (n = 26), rash (n = 7), arthralgia (n = 16), aplastic crisis (n = 3), and intrauterine fetal death (n = 1). Patients were followed for 26-85 months (mean 57 months) and reassessed for persistent symptoms, anti-B19 antibodies, and antibodies to the unique region of B19 VP1. There were 23 cases of arthralgia persisting for longer than 1 year after acute infection. One of these patients, a 48-year-old woman at follow-up, had had persistent arthralgia for 4 years following acute B19 infection, had rheumatoid factor at a titre of 1920 IU/ml detected at follow-up, and had been independently diagnosed as having rheumatoid arthritis at the time of follow-up. All 53 patients were positive for serum anti-B19 IgG compared to 45 of 53 age- and sex-matched control patients, a significant difference (two-tailed P value = 0.008). All test patients at follow-up and control patients were negative for serum anti-B19 IgM and antibodies to the unique region of B19 VP1. Serum from acute infection from 33 of 53 test patients was tested for antibodies to the unique region of VP1, and 16 of these were positive. The presence of this antibody did not correlate with subsequent duration of symptoms but did correlate with a short interval between symptom onset and blood sampling. The unique region of B19 VP1 is known to be crucial for a successful humoral response to the virus, and it seems that the antigenic role played by this region is important only during the acute phase of B19 infection.

Design and production of a target-specific monoclonal antibody to parvovirus B19 capsid proteins.

Native parvovirus B19 was used as antigen to produce a mouse monoclonal antibody, R92F6, which reacted with B19 VP1 and VP2, neutralised the virus in bone marrow culture, and labelled infected cells in paraffin-embedded tissues from cases of B19-related fetal hydrops. The B19 epitope recognised by R92F6 (amino acids 328-344 from the amino terminal region of B19 VP2) appears to be highly conserved, since these tissue specimens were obtained over a 13 year period from widely spaced locations in the UK. This epitope was synthesised as a peptide (S7b) which was used as antigen to produce a mouse monoclonal antibody, 3H8, which specifically reacted with the B19 capsid proteins VP1 and VP2 in immunofluorescence and immunoblot assays. 3H8 was also capable of labelling formalin-fixed, paraffin-embedded, B19-infected fetal tissue and was shown to be of the same isotype as R92F6 (IgG1). Highly conserved epitopes derived from conserved amino acid sequences are valuable in the diagnosis of infectious disease. If these can be recognised and accurately synthesised, the production of specific mouse monoclonal antibodies may be possible for many human pathogens. Considering the vast amount of sequence data available in the literature, this approach seems to be both feasible and of wide potential.

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.

Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans.

We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.

T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

Synthetic model peptides for apolipoproteins. I. Design and properties of synthetic model peptides for the amphipathic helices of the plasma apolipoproteins.

Amphipathic helical peptides are the lipid-binding motives of the plasma apolipoproteins, and synthetic peptide analogs have been used to unravel the mechanism of lipid association within this class of proteins. Hydrophobic interactions between the apolar amino acid residues belonging to the hydrophobic face of the amphipathic helices and the lipids are the major driving forces in the peptide-lipid association to form discoidal complexes. Ionic interactions and salt bridge formation between contiguous peptide chains in the complex can, however, contribute to the overall stability of the lipid-protein particle. This was studied by designing peptide analogs to the helical repeats of the apolipoproteins with variable degrees of salt bridge formation between adjacent peptide chains. The most stable conformation for pairs of synthetic peptides was calculated by energy minimisation together with the energy of interaction between peptides. The sequence of the peptides was derived from that of the 18A peptide synthesized by Segrest et al., and the theoretical calculations confirmed that ionic interactions between residues close to each other, along the edge of two adjacent anti-parallel peptides, can significantly contribute towards the stability of a peptide-phospholipid complex.

Synthetic model peptides for apolipoproteins. II. Characterization of the discoidal complexes generated between phospholipids and synthetic model peptides for apolipoproteins.

The structure, composition and physico-chemical properties of complexes generated between phospholipids and synthetic model peptides for the amphipathic helices of the plasma apolipoproteins were studied. The sequences of the peptides were derived from that of the 18A peptide and designed to either enhance or decrease ionic interactions between pairs of peptides, as described in the accompanying paper. Complexes were prepared with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), or with DPPC and cholesterol, and isolated on a Superose 6HR column. Association kinetics for the DMPC-peptides complexes were followed by measuring the turbidity as a function of the temperature. The diameters of the DPPC-peptide complexes, measured by gradient gel electrophoresis (GGE), were about 120 A. Fluorescence polarization measurements after labeling with diphenyl hexatriene (DPH) yielded transition temperatures of, respectively, 40.6, 41.5 and 41.8 degrees C for the DPPC/18AM1-, DPPC/18AM4- and DPPC/18A-peptide complexes. These values were confirmed by differential scanning calorimetry. Circular dichroism and infrared spectroscopy revealed that the peptides adopt an alpha-helical structure in solution and this percentage increased from 30-40% in the free peptides up to 50-60% in the complexes. Attenuated total reflection (ATR) infrared measurements of the complexes indicated that the peptides are oriented parallel to the acyl chains of the phospholipid bilayer. Denaturation of the peptides and of the peptide-lipid complexes was monitored by Trp fluorescence under addition of increasing amounts of GdmCl. The mid-points of the denaturation curves lie at, respectively, 0.05, 0.25 and 0.35 M GdmCl for the 18AM4, 18A and 18AM1 peptide and are shifted towards higher GdmCl concentrations after peptide-lipid binding. GdmCl denaturation decreased the alpha-helical content of the peptides and of the complexes, as monitored by circular dichroism measurement. The helix to random coil structure transition occurred at, respectively, 2.1, 2.2, and 2.0 M GdmCl for 18A, 18AM1 and 18AM4, compared to 5.1, 5.0, and 5.3 M in the corresponding complexes. These data suggest altogether that the structural properties, the mode of lipid-protein association and the stability of the phospholipid-peptide complexes are similar to those of native plasma apolipoproteins. The 18A and 18AM4 peptides which contain charged residues along the edge of the helix, leading to salt bridge formation between peptides were shown to mimic the amphipathic helices of the plasma apolipoproteins.

Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes.

OBJECTIVE: To determine the extent of genetic variation among internationally collected HIV-1 isolates, to analyse phylogenetic relationships and the geographic distribution of different variants. DESIGN: Phylogenetic comparison of 70 HIV-1 isolates collected in 15 countries on four continents. METHODS: To sequence the complete gag genome of HIV-1 isolates, build multiple sequence alignments and construct phylogenetic trees using distance matrix methods and maximum parsimony algorithms. RESULTS: Phylogenetic tree analysis identified seven distinct genotypes. The seven genotypes were evident by both distance matrix methods and maximum parsimony analysis, and were strongly supported by bootstrap resampling of the data. The intra-genotypic gag distances averaged 7%, whereas the inter-genotypic distances averaged 14%. The geographic distribution of variants was complex. Some genotypes have apparently migrated to several continents and many areas harbor a mixture of genotypes. Related variants may cluster in certain areas, particularly isolates from a single city collected over a short time. CONCLUSIONS: The genetic variation among HIV-1 isolates is more extensive than previously appreciated. At least seven distinct HIV-1 genotypes can be identified. Diversification, migration and establishment of local, temporal 'blooms' of particular variants may all occur concomitantly.

Redistribution of a murine humoral immune response following removal of an immunodominant B cell epitope from a recombinant fusion protein.

Immunization of different mice strains with a recombinant fusion protein composed of the vector-encoded N-terminal leader peptide CroLac (containing lambda Cro and LacI fragments) and a part of the transmembrane protein of HIV-1 (gp41) led to a high anti-CroLac humoral immune response. A detailed analysis of this response revealed the presence of an immunodominant, linear B cell epitope localized near the C-terminus of the CroLac fragment. The immune response seemed to be biased towards this epitope since few or no monoclonal antibodies (mAb) could be generated against the remaining part of CroLac and the gp41 fragment. Upon removal of the immunodominant region from the fusion protein the immune response was redirected and spread over the previously non-immunogenic regions. Consequently, we report a model system in which an immunodominant B cell epitope biases the immune response away from less immunogenic epitopes on the same molecule.

Analysis of the putative E1 envelope and NS4a epitope regions of HCV type 3.

Using a Line Probe Assay, type 3 HCV genotype-infected sera were selected from Brazilian blood donors. The partial nucleotide sequences of the core/E1 and NS4a epitope-containing regions and the NS5b typing region were determined. The E1 region had a nucleic acid homology of only 61 to 65% with the type 1 prototype genomes, and 56 to 58% homology with the type 2 prototype HCV genomes. Similar homologies were also found for the NS4a epitope region and for NS5. Furthermore, the deduced amino acid sequence of type 3 NS4a was used to generate synthetic peptides which were strongly reactive with human HCV-infected sera which were previously determined as anti-NS4 negative, indicating that a type-specific antibody response to the NS4a protein may exist.

Identification and use of a neutralising epitope of parvovirus B19 for the rapid detection of virus infection.

A conserved neutralising epitope was confirmed as the site of specific activity for the monoclonal antibody R92F6. This monoclonal antibody was used to detect B19 viral antigen in serum samples after SDS-PAGE and Western blotting. Twenty samples from the United Kingdom, Ireland, Japan, and the United States were positive with this technique. Serum samples from various control groups were negative.

HIV antigen detection in circulating immune complexes.

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.

Unusual effects of penicillin G and chloramphenicol on the growth of Moraxella osloensis.

Growth of exponential-phase liquid cultures of Moraxella osloensis was inhibited by 0.5 U of penicillin G per ml. For this organism, low concentrations of penicillin acted primarily in a bacteriostatic rather than in a bactericidal manner. At higher concentrations of penicillin some killing did take place, but the rate of killing was rather slow and appeared to be independent of penicillin concentration. Microscopic observation of cells from penicillin-treated cultures showed little or no cellular swelling or lysis. The total cell count did not decrease significantly during 6 h of incubation in 5,000 U of penicillin per ml. The rates of respiration, nucleic acid synthesis, and protein synthesis were not affected by the presence of penicillin. Attempts to counteract the bactericidal action of high concentrations of penicillin with growth inhibitory concentrations of chloramphenicol were unsuccessful, since chloramphenicol itself was more bactericidal than penicillin for M. osloensis.

Electrophoretic analysis of covalently closed SV40 DNA: Boltzmann distributions of DNA species.

Covalently closed relaxed SV40 DNA [SV40(I')] generated by polynucleotide ligase closure of nicked circular SV40 DNA was analyzed by agarose gel electrophoresis. The DNA can be resolved into a series of bands differing in superhelical density whose intensities are approximately symmetrical about a central most intense band. Densitometric analysis of the gel pattern has revealed that the distribution of DNA species conforms to a Boltzmann distribution and has enabled us to derive an equation for the free energy of superhelix formation for SV40 DNA. We believe the observed bands reflect the time-averaged distribution of thermally induced fluctuations in DNA chain conformation in solution at the time of ligase catalyzed phosphodiester bond formation. Densitometric analysis of native supercoiled SV40 DNA, partially unwound in the presence of ethidium bromide, demonstrates that the separation between adjacent bands is approximately half that seen with SV40(I'). Agarose gel electrophoresis was also used to measure the change in average base rotation angle as a function of temperature by a procedure independent of ethidium dye binding.