Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.

The IFN-alpha-inducing capacity of Sendai Virus in human leukocytes is independent from the hemagglutinating and hemolytic activities and from the viral proteins of the Sendai Virus preparations.

About 30 Sendai Virus (SV) preparations, examined for their capacity to induce natural human interferon alpha from fresh human leukocytes (Le-IFN-alpha) of healthy donors, were characterized for hemagglutinating (HA) and hemolytic (HemA) activities and for SDS-PAGE proteic pattern. The SV preparations were produced by a single passage or by serial propagations through eggs in different conditions of multiplicity of infection (m.o.i.). The produced SV subpopulations showed variable IFN-inducing capacity, the values of which are distributed over a 6-fold range resembling a Gaussian distribution. No detectable difference was observed comparing the SV preparation obtained by serial propagations with those obtained by a single passage. The variability of the measured HA activity and HemA activity and as well as the SDS-PAGE proteic pattern of the SV preparations did not correlate with the induced amount of IFN per cell. In the same experimental conditions to produce Le-IFN-alpha, u.v.-treated SV preparations were unable to induce interferon depending on the u.v.-treatment. So it can be concluded that the distinct nHu-IFN-alpha-inducing capacity of SV subpopulation could be mainly associated with divergent compositions of the viral RNAs rather than with a different contents of viral proteins, among those detectable in SDS-PAGE and those responsible for HA activity and for HemA activity.

Variations in the interferon-inducing capacity of Sendai virus subpopulations.

Several Sendai virus (SV) preparations, propagated through eggs from the same viral seed, exhibited significantly different capacities to induce interferon (IFN) in human leukocytes (nHu-IFN-alpha). The amount of induced IFN and the numbers of SV IFN-inducing particles (IFP) per cell were determined in dose (SV concentration)-response (IFN yield) curves, kinetics of IFN production, and coinfection experiments with SV preparations that differed in IFN-inducing capacities. The possible role of leukocyte sources and the quality of the SV preparations and of allantoic fluids in affecting the IFN-inducing capacity of SV populations also were tested. The data indicate that different SV preparations induced different amounts of IFN per leukocyte and contained approximately the same concentrations of IFN-inducing particles. There was no apparent correlation between the IFN induced and the apparent quality of the SV preparations examined (EID50, HAU, and EID50/HAU). The leukocyte source and the allantoic impurities of SV preparations did not have any influence on the magnitude of the IFN yield. Similar shapes of the dose-response curves, the absence of any lag in the kinetics of IFN production, and the ability of a viral preparation that induced low yields of IFN to suppress partially a high-yielding inducer suggest that a common mechanism of induction is always present. Hence, propagation of SV in eggs from low multiplicity produced virus stocks that differed significantly in their inducing capacity, suggesting that genetic bottlenecks may be operative.

Structure-activity of type I interferons.

Type I IFNs constitute a family of proteins exhibiting high homology in primary, secondary, and tertiary structures. They interact with the same receptor and transmit signals to cellular nucleus through a similar mechanism, eliciting roughly homogeneous biological activity. Nevertheless, the members of that family, IFN alpha species, IFN beta and IFN omega, due to local differences in the structure sometime show distinct properties. From the reported data it results that even minute changes or differences in the primary sequences could be responsible for a significant variety of biological actions, thus inducing to the hypothesis that Type I IFNs, rather than to be the result of a redundant replication during the evolution play definite roles in the defense of living organisms to foreign agents.

Capillary electrophoresis of heparin and dermatan sulfate unsaturated disaccharides with triethylamine and acetonitrile as electrolyte additives.

Capillary electrophoresis at constant voltage with the addition of triethylamine as electrolyte to a running buffer containing borate using fused-silica capillaries permits the complete resolution in less than 30 min of 11 standard heparin and 8 standard dermatan sulfate disaccharides, which represent degradation products of heparin and dermatan sulfate by specific lyases. Triethylamine influences the migration time of disaccharides by reducing both their electrophoretic mobility towards the anode and the electroosmotic flow towards the cathode. A modulated combination of these effects together with borate-disaccharide complex formation is responsible for separation, especially in the case of isomers which differ in the position of the sulfate groups. The addition of acetonitrile did not introduce any favourable effect in the separation of disaccharide mixtures. Under these conditions different dermatan sulfates were analysed to assess the source of the preparations.

Purification of recombinant human interferon-beta by immobilized antisense peptides.

Synthetic antisense peptides encoded in the antisense strands of DNA corresponding to the 1-14, 42-54 and 103-115 fragments of the human interferon-beta sequence were applied in the purification of recombinant human interferon-beta from a mammalian cell culture. The protein fragments were selected on the basis of their computer-predicted exposure on the surface of the protein. The antisense peptides were synthetized by the solid-phase method directly on the resin used as the stationary phase in affinity chromatography. All the tested antisense peptides showed a selective affinity for human interferon-beta, permitting a ten-fold purification of the protein.

Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

Large-scale purification of the synthetic peptide fragment 163-171 of human interleukin-beta by multi-dimensional displacement chromatography.

Multi-dimensional chromatography has been used successfully in the displacement mode for the purification of the synthetic peptide H-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the fragment 163-171 of human interleukin-beta. This peptide can mimic several of the in vivo and in vitro immunostimulatory activities of the entire protein, except for the inflammatory effect. A large-scale procedure has been developed to purify the synthetic peptide by reversed-phase (RP) and ion-exchange (IE) displacement chromatography (DC) in a single run without any pretreatment. Masses from 100 mg to about 35 g of the unpurified compounds synthesized by a solid-phase technique on a Merrifield-type resin and obtained by acidolytic cleavage from the solid support, can be purified in this way. In the RP-DC mode the carrier and the displacer were aqueous solutions of 0.1% trifluoroacetic acid and 50 mM benzyltributylammonium chloride, respectively, whereas in the IE-DC mode the carrier was water and the displacer 50 mM ammonium citrate solution. RP-DC and IE-DC were also performed in series by directing the effluent of the RP column onto the IE column. Peptide purities and recoveries greater than 96 and 90%, respectively, were obtained.

Preparative purification of Plasmodium falciparum circumsporozoite protein synthetic polypeptides by displacement chromatography.

Displacement chromatography was used for the preparative purification of a synthetic polypeptide that is a promising malaria vaccine. It was prepared by solid-phase synthesis and contains two important epitopes of circumsporozoite (CS) protein of Plasmodium falciparum sporozoite. With apparatus typically employed in analytical high-performance liquid chromatography (HPLC) and on a 250 x 4.6 mm I.D. reversed-phase column, up to 50 mg of crude polypeptide were purified in a single run and with a yield higher than 95%. The results demonstrate that displacement chromatography is suitable for the isolation of several milligrams of a pure polypeptide from a complex mixture that is difficult to separate even by analytical HPLC. In such a preparative application, displacement appears to be superior to elution chromatography as used traditionally.

Fast atom bombardment mass spectrometry and selective acid hydrolysis for the analysis of partially modified retro-inverso peptide analogues.

Fast atom bombardment (FAB) mass spectrometry has been successfully applied to the analysis of partially modified retro-inverso peptide isomers. The spectra are characterized by abundant protonated molecular ions and also by sequence ions due to fragmentation of the inverted bonds. Unambiguous information, as to the nature and the position in the backbone of the amino acids involved in the partial modification of the structure, are given by using a combination of FAB mass spectrometry and partial, selective acid hydrolysis, without separation of the resulting peptide mixtures.