Production of monospecific immunoglobulin against human leukocyte interferon

Donkeys were regularly, at seven-day intervals, immunized subcutaneously with human leukocyte interferon (IF) with the activity of 1.6 X 103 U/10 ml. IF-neutralizing antibodies in the titre of 1:128-1:256 were detected in the animals' sera after 38-40 injections. As a result of continuing injections the titre of these antibodies increased considerably. The antibodies to the components of the system in which the IF was obtained were revealed in parallel. Donkey antiinterferon plasma was prepared by plasmapheresis and antiinterferon immunoglobulin (AIFIG) was released from the plasma by centrifugation using ammonium sulphate at 50% saturation. Contamination antibodies were removed from AIFIG by affin chromatography on combined immunosorbent.     

Toxicity of interferon.

The effects of partially purified human leucocyte interferon (PIF) and of a preparation purified by passage twice through a monoclonal antibody affinity chromatography column (NK21F) were compared with those of a control solution in healhty volunteers. After intramuscular injections both interferon preparations caused rises in pulse rate and body temperature, changes in circulating white cell counts, and various unpleasant symptoms, the most common of which were headache, malaise, and fever. Slightly lower doses of NK21F were given, and this was reflected in lower peak serum concentrations. Mean symptom scores, however, were not lower after NK21F than after PIF. Local inflammatory reactions eight hours after intradermal inoculations of these interferons were similar. Purification of interferon using a monoclonal antibody does not reduce the facets of its activity considered in this study. They are therefore inherent in the leucocyte interferon type selected by the antibody.     

Production of human leukocyte interferon for clinical use under immunoelectrophoretic control.

Human leukocyte interferon (HLIF) can be contaminated by several antigens which may include also potentially pathogenic agents. For this reason, gel filtration as a separation method was included into the routine procedure of preparation and purification of HLIF for clinical use. Since production of HLIF requires the cultivation of leukocytes in the presence of serum (or albumin), serum proteins represent then the majority of proteins contaminating IF preparations. Measuring the total protein content, as a control of various antigens present in HLIF preparations during purification, becomes ineffective because, essentially, it indicates only the decrease of the amount of proteins derived from cultivation medium. For a better visualization of dissociation of different antigens from molecules with IF activity during the purification procedure, the method of quantitative immunoelectrophoresis was applied utilizing a sheep antiinterferon serum in assay. Our results indicate that this method represents a valuable control test.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Pooled human immunoglobulin inhibits IL-4 but not IFN-gamma or TNF-alpha secretion following in vitro stimulation of mononuclear cells with Staphylococcal superantigen.

Intravenous immunoglobulin preparations have been successfully used in many disorders, where immunomodulation rather than immunoglobulin replacement has been the goal of therapy. The exact mechanisms by which immunoglobulin exerts its immunomodulatory effects are unclear. Proposed mechanisms include modification of T cell activation and alteration to cytokine production. As intravenous immunoglobulin therapy has been used in a number of disorders where superantigens are proposed to play a role in the disease pathogenesis, we have examined the effect of in vitro human pooled immunoglobulin on cytokine production from peripheral blood mononuclear cells in response to activation with the Staphylococcal superantigen Staphylococcal enterotoxin B. The authors found inhibition of secretion of interleukin 4 (IL-4) (P<0.001) but not interferon gamma (IFN-gamma) (P=0.13) or tumour necrosis factor alpha (TNF-alpha) (P=0.66) by pooled immunoglobulin at concentrations (6 g/l) which approximate the rise in serum immunoglobulin following in vivo IVIG therapy. Mononuclear cell proliferation was also inhibited by addition of pooled immunoglobulin to superantigen stimulated cultures. These effects do not relate to specific anti-staphylococcal enterotoxin B antibodies in the immunoglobulin preparation. The authors show that pooled human immunoglobulin can differentially modulate the secretion of IL-4 and IFN-gamma in response to superantigen stimulation.     

Dry erythrocytic diagnostic agent for the determination of antiglobulins]

Dry erythrocytic diagnostic agents were obtained under experimental conditions for determination of antiglobulins forming in the organism of man and animals under the effect of serum preparations from the blood of horses and homologoum immunoglobulins. A study was made of the sera of 100 patients with tick-borne encephalitis treated with heterologous and homologous immunoglobulins of directed action; in response to the administration of horse gamma-globulin antiglobulins (in titres below 1 : 10000) appeared in the serum; they circulated in the blood for long periods and inhibited the accumulation of hormonal antibodies to the causative agent; in the majority of cases a high level of antiglobulins to the foreign protein correlated with the presence of remote side-reactions of the serum sickness type. In patients treated with immunoglobulin of human origin antiglobulins were determined in low titres, disappeared from the blood in 15--20 days and did not hinder the accumulation of antihemmagglutinins to the tick-borne encephalitis virus.     

Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.     

Purified interferon as protection against rhinovirus infection.

In a double-blind placebo-controlled study a preparation of human leucocyte interferon purified by affinity chromatography using a monoclonal antibody and applied by repeated nasal sprays reduced the incidence and severity of colds in volunteers challenged with human rhinovirus 9. Although interferon itself caused some symptoms, these were minor compared with the clinical colds. Interferon activity was still detectable in nasal washings as long as 26 hours after the last dose in about half the volunteers on active treatment.     

Immunologic effects of interferon-alpha in man: treatment with human recombinant interferon-alpha suppresses in vitro immunoglobulin production in patients with chronic type B hepatitis

The effect of human recombinant interferon-alpha on lymphocyte proliferation and differentiation was studied in 18 patients with chronic type B hepatitis who were participating in a randomized controlled trial of interferon-alpha therapy. Peripheral blood mononuclear cells (PBMC) were obtained by lymphopheresis before and during a 4 mo course of interferon. Pokeweed mitogen-induced immunoglobulin synthesis by PBMC obtained from patients before therapy was similar to that of PBMC from normal individuals. However, after 2 wk treatment with human recombinant interferon-alpha mitogen-induced immunoglobulin production was decreased by an average of 50%. Staining for cytoplasmic immunoglobulin revealed decreases that paralleled secreted immunoglobulin, indicating that interferon-alpha treatment inhibited immunoglobulin synthesis. Mixing autologous T and B cell enriched populations from before and during interferon treatment revealed that the decrease in immunoglobulin synthesis involved a defect in the B cell-enriched population. In contrast to immunoglobulin synthesis, pokeweed mitogen-induced lymphocyte proliferation was not significantly affected by in vivo administration of interferon-alpha. Thus a major effect of in vivo interferon-alpha on immunoregulation in patients with chronic type B hepatitis appears to be an inhibition of the late stages of B cell differentiation into immunoglobulin producing and secreting plasma cells.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Measurement of Rubella Antibody in Immunoglobulin: its Disappearance from the Blood after Injection: Report of the Public Health Laboratory Service Working Party on Rubella

Rubella antibody titrations were done on samples of human immunoglobulin by neutralization and haemagglutination-inhibition methods. No significant variation was found in the antibody content of different batches. The specificity of the methods was confirmed by tests on a batch of human globulin specially prepared from plasma samples lacking rubella antibody.Divided doses of immunoglobulin were given to volunteers who had no rubella antibody. Low titres were then detected in the blood for a limited period and the disappearance of this antibody was followed.     

Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase

Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.     

Expression of human leukocyte interferon type A in Saccharomyces cerevisiae under the control of regulatory elements of the yeast gene URA3

Expression of a chromosomal gene for human leucocyte interferon A was obtained due to interaction of 5'-nontranslating region of a cloned interferon gene with the regulatory sequences from UNA3 yeast gene. The sequence of 5'-nontranslating region from interferon gene essential for its expression in yeast is localized within 130 b p. from the initiating codon. Due to increasing of plasmid stability and copy number a 60-fold increase. in interferon yield was obtained in yeast transformants reaching the level of 6 X 10(7) u X 1(-1). The data are presented supposing the existence of functional polycistronic mRNA in yeast.     

Immune-type interferon-induced transfer of viral resistance.

Mouse immune-type interferon (type II), a lymphokine, caused the transfer of viral resistance from mouse L cells to human WISH cells. The interferon was incapable of protecting WISH cells in the absence of L cells. The transfer of viral resistance occurred with interferon preparations of various specific activities, and was in proportion to the interferon concentration in the preparations. The transferred resistance had the characteristics of an interferon-induced antiviral state in that it was blocked by actinomycin D, effective against different types of viruses, and resulted from an action on the cell rather than on the virus. Mouse immune-type interferon was more efficient than virus-type (type I) at eliciting the transfer of protection. The transfer phenomenon may represent a mechanism for amplification of the interferon system as a host defense against viral infection. Further, it serves as a model for studying the mechanism of lymphokine-induced transfer of information between cells.     

Interferon Studies with Japanese and U.S. Rubella Virus Strains

Japanese strains of rubella virus have been claimed not to be teratogenic, and tests on three Japanese strains showed that they induced high levels of interferon in human placental cell cultures obtained from conceptuses ranging from 13 to 24 weeks'' gestational age, whereas two strains derived from the U.S.A. induced low levels. Both Japanese and U.S. strains induced similar but low levels in fetal lung cell cultures and leucocyte preparations. A representative Japanese strain and a U.S. strain were both interferon-sensitive. If indeed a strain can be shown to be non-teratogenic it could lead to an alternative, safer rubella vaccine.     

Antibodies against Pseudomonas aeruginosa toxin in preparations of normal human immunoglobulin

Some lots of commercial normal human immunoglobulin have been found to contain antibodies neutralizing the action of P. aeruginosa exotoxin. The content of antibodies in human immunoglobulin preparations correlates to a certain degree with their protective activity determined in the passive protection test in white mice. Certain lots of normal human immunoglobulin have been found to possess protective activity, but contain no specific antitoxins. The clinical testing of these immunoglobulin preparations used for treating patients with Pseudomonas infections has yielded promising results.     

Evaluation of prekallikrein activator (PKA) activity in plasma-derived preparations

The study was carried out on 111 series of human immunoglobulin preparations (81 series for intravenous use, 30 series for intramuscular use) produced abroad and in Poland. Kallikrein (KK) and prekallikrein activator (PKA) activities were identified using chromogenic substrate. S-2302 and proteinase inhibitors were tested by double immunodiffusion method. Several biochemical, biological and serological methods were also applied which are valid in the control of intravenous and intramuscular immunoglobulin preparations. It was found that trace amounts of PKA and KK are present in human immunoglobulin preparations for intravenous use such as Bioglobulina and Gamma-Venina (PKA 0.0092-0.0505 mu kat/L, and KK 0,-0.0093 mu kat/L). On the other hand normal human immunoglobulin preparations for intramuscular use had somewhat higher level of KK and PKA and anti-HBs Immunoglobulin preparations (5.50 mu kat/L and 153.91 mu kat/L) and Tetabulina preparations had very high levels of these factors as compared to remaining preparations (4.50 mu kat/L and 316.5 mu kat/L).     

The potency determination of human varicella-zoster immunoglobulin by enzyme-linked immunosorbent assay, complement-fixation test and indirect fluorescent antibody tests

Traditionally, plasma for the production of the human varicella-zoster immunoglobulin (VZIG) has been selected on the basis of the complement-fixing antibody (CFA) titre. Since immune individuals may lack CFA to varicella-zoster virus (VZV), non-CFA may be of importance in protection. In a search for a simple and reliable method for potency determination, 24 VZIG preparations were quantified by enzyme-linked immunosorbent assay (ELISA), the complement-fixation test (CFT), the indirect fluorescent antibody test to acetone-fixed (IF) and viable (FAMA) VZV-infected cells, respectively. The antibody titres obtained by the various methods were compared. Arranged in order of decreasing agreement, the correlation coefficients (r) of the regression equations between the variables were 0.62 for CFT and FAMA, 0.50 for CFT and ELISA and 0.26 for CFT and IF in a log2 plot. There was complete agreement between the titres obtained by the commercially available Enzygnost Varicella/Zoster kits (Behring Institute, Marburg, F.R. Germany) and the ELISA microtitre plates produced at our institute (r = 1). The regression equation lines for ELISA/CFT and FAMA/CFT titres tended to be parallel to each other, while the line for IF/CFT titres had a less steep slope. Similar titration curves were obtained for VZIGs fractionated by two different methods. Furthermore, the titration curves of serum pools from varicella and zoster convalescents, respectively, had a similar shape below delta OD = 0.4. Generally, a steeper slope was observed above delta OD = 0.4. As antibody detectable by ELISA seems to correlate with protection and the method is sensitive, specific, reproduceable, simple to carry out and easily automated, it may be suitable for the potency determination of VZIGs.     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

Nonspecificity of antiviral protein action]

The authors studied the antiviral activity of the informative RNA of antiviral protein (M-RNA AVP) isolated from the cells following superinduction of interferon in them for the purpose of ascertaining the action specificity of the product of their translation--AVP. Following the administration of M-RAN AVP a marked (from 1 to 5 lg PFU/mil) reduction of the infectious titres was observed in the homologous and heterologous cells. RNA preparations from control (noninduced) cells possessed a weak (0.4--0.1 lg PFU/mil) antiviral activity.