Robert Deleys

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 1gM) 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 lgG anti‐19 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 lgM 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 B19VP1 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.

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.

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.

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.

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.

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.