Mathematical model of immune processes. II. Kinetic features of antigen-antibody interrelations

The dynamics of populations of self-replicating antigen and specific antibodies due to the antigen-stimulated antibody production and the antagonistic antibody-antigen interaction is treated in the framework of the mathematical model (Dibrov, Livshits &; Volkenstein, 1977). A special emphasis on the time lag in antibody production is made. The ability of the system to respond on the antigen challenge by antibody production is assumed to be constant during the reaction. The conditions of antigen elimination, of unlimited antigen multiplication, of self-sustaining oscillations and of stationary co-existence of antigen with specific antibodies are obtained. The discrete stochastic character of real birth and death processes enables the complete antigen elimination. A simple procedure for the evaluation of the probability of antigen extinction is proposed. The effect of therapy executed at different moments is examined.     

Factors Influencing the Production of H and M Antigens by Histoplasma capsulatum: Development and Evaluation of a Shake Culture Procedure

Studies were undertaken to improve the production of histoplasmin for use in complement-fixation tests and in the determination of H and M antibodies. A shake culture method performed at 25 C was developed with a yeast-phase inoculum. Eight strains of Histoplasma were tested in three synthetic media to evaluate the effects of strain and medium on H and M antigen production. Intrastrain variation was negligible, and antigen production was reproducible. All of the strains produced H antigen; six strains produced both H and M antigens, and two produced only H antigen. The time of H and M antigen appearance varied with the medium; M antigen appearance was dependent upon the strain and medium used. Titers of M antigen appeared to be greater in stagnant culture.     

Enhancement of lysophospholipase activity with Trichinella spiralis antigen: evidence for cell cooperation

Murine lymphocytes, neutrophils, macrophages and eosinophils were assayed for lysophospholipase in order to determine the cellular source of the enzyme. The eosinophil was the only cell that demonstrated a positive reaction for the enzyme. The role of other cells and/or antigen in production of the enzyme by the eosinophil was also investigated. Results demonstrated that eosinophils cultured with both Trichinella spiralis antigen and other leukocytes (lymphocytes and/or macrophages) yielded enzyme activities significantly greater than did eosinophils cultured alone or with only antigen. More specific experiments showed that T-lymphocytes were the cells responsible for influencing the eosinophils' production of lysophospholipase in the presence of antigen, and that their influence was enhanced by the presence of macrophages. These results suggest that increased lysophospholipase activity present in parasitized tissue is not only due to an increased number of eosinophils infiltrating parasitized tissues, but is also due to each eosinophil synthesizing more of the enzyme for release. The necessity for antigen and other cells suggests a need for cell cooperation in the production of the enzyme, specifically T-lymphocytes and macrophage interaction with the eosinophil.     

Influence of pH,nitrogen and phosphorus sources on the production of hepatitis B virus pre-S2 antigen by Hansenula polymorpha

Experimental design techniques were used to study the influence of the composition of the culture medium on the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. pH, phosphoric acid, ammonium chloride and yeast extract concentrations were selected as experimental factors and their influence was investigated using Central Composite design techniques. The results indicated that antigen yield was maximized at high pH and in a culture medium containing both ammonium chloride and yeast extract. Phosphoric acid was found to have a detrimental effect on antigen production. This study allowed a 50% increase in antigen production in a medium in which the yeast extract cocentration was decreased to 32 g/1. These optimal conditions have been confirmed with an octagonal design experiment. Moreover, it was shown that the antigen produced was very stable up to at least 8 days after induction and that the yeast extract concentration could be lowered to 22 g/1 without apprciable effect on antigen yield. The increase in antigen production was not due to an increase in cell biomass, since no correlation could be found between these two parameters. The newly defined culture medium should allow a greatly increased antigen production at the fermentor level, at a lower cost and with minimal operational problems.Correspondence to: D. Groleau     

Electron microscopic studies on the peritoneal exudate of the mouse to determine the effect of colloidal carbon on the primary immune response to heterologous erythrocytes]

Antibody production to sheep erythrocytes was markedly depressed in mice injected intraperitoneally with colloidal carbon. Peritoneal exudate cells were investigated electron microscopically with regard to mechanisms responsible for lower antibody production. Peritoneal macrophages were not disturbed in antigen phagocytosis and antigen digestion. Only in connection with immunosuppression was there an increase in antigen containing macrophages in the peritoneal cavity. The changing relation of antigen-stimulated macrophages and lymphocytes in favour of the macrophages and its consequence to decreased immune response is discussed.     

Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and ¹³C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.     

免疫反应动力学的几个数学模型

免疫系统对抗原刺激的应答过程非常复杂,由抗原刺激导致抗体产生的现象,可借助数学模型的研究获得有意义的结果。本文讨论有关抗体产生与免疫反应的动力学的问题,介绍有关的数学模型,并根据近斯免疫学研究的进展分析了若干模型。     

Factors Influencing the Production of H and M Antigens by Histoplasma capsulatum: Effect of Physical Factors and Composition of Medium

Stagnant culture methods have permitted only limited physiological studies of the production of H and M antigens by Histoplasma capsulatum because, with such methods, antigen production is uncontrolled. In this investigation, a shake culture method was used to convert yeast-phase inoculum to mycelialphase growth at 25 C. Results strongly suggest that the release of H and M antigens relates to autolysis of the cells. Among the factors influencing production of H and M antigens under shaking conditions, choice of strain was the most important. Alterations of carbon or nitrogen source or variations in amino acid to carbohydrate ratios had limited influence on antigen production. With a strain that produced both H and M antigens, however, proportions of titers of M to H antigens could be made to vary considerably by changes in the medium, the pH, and the temperature. Results suggest that the source of M antigen during autolysis is enzymatic dissolution of the cell wall. The source of H antigen is more obscure. Production of both antigens may be differentially controlled under conditions of good reproducibility by a correct choice of strain and manipulation of culture medium.     

Adjuvant activity of alum in inducing antigen specific IgE antibodies in BALB/c mice: a reevaluation

The IgE production was compared in the presence and absence of aluminum hydroxide gel (alum). Without alum, the IgE production was induced within a suitable range of the antigen dosage; however, alum enhanced it. Alum did not affect the minimum requirement for the antigen dosage, indicating that alum may not take part in the efficiency of antigen presentation.     

Expression of viral capsid protein antigen against Epstein-Barr virus in plastids of Nicotiana tabacum cv. SR1

Epstein-Barr virus (EBV) infects nearly 90% of adults worldwide and is the pathogenic source of a broad spectrum of malignancies originating from lymphoid and epithelial cells. Currently, no vaccine has been developed to immunologically inactivate this virus. In infected patients, anti-EBV viral capsid antigen (VCA) immunoglobins represent some of the useful diagnostic markers for carcinoma development. To demonstrate that the EBV VCA antigen can be produced in plants, the plastid genome of tobacco (Nicotiana tabacum cv. SR1) was transformed with a VCA-expressing cassette. The EBV VCA mRNA was actively transcribed in the transplastomic plants and antigen production was detected. This study indicates that plastid transformation could be a promising strategy in EBV VCA antigen production.     

In situ removal and purification of biosurfactants by automated surface enrichment

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64–69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.     

Effect of growth factors and antigen on hybridoma cell culture dynamics

The cell growth and monoclonal antibody production kinetics of hybridoma cell cultures continuously exposed to growth factors and the cognate antigen were investigated. The growth factors were the epidermal growth factor, fibroblast growth factor, and interleukin-2, whereas the antigen was the trinitrophenyl group conjugated to a carrier protein. The cultures were carried out in a protein-free medium in batch operation. During the entire cultivation period there was continuously available free, antibody-unbound antigen to interact with the cells. The produced antibody was measured with an ELISA after it was released from the antigen-protein conjugate by competitive elution with non-protein-conjugated antigen. Cultures with growth factors and without antigen increased the total antibody produced by up to 30%, whereas cell growth remained unaffacted. Soluble antigen-protein conjugates had no effect on the hybridoma cultures. In contrast, immobilized antigen-protein on sepharose beads in cultures with growth factors induced significant changes. Total antibody produced was higher by up to 40%. More importantly, the specific antibody production shifted from a growth-phase-independent to a growth-phase-dependent profile, with approximately twice as much specific antibody production during the late growth-early stationary phase relative to constant specific antibody production in the antigen-free, factor-free culture. The culture changes induced by the presence of immobilized antigen and growth factors were reversed when the antigen and the growth factors were removed from the cells' environment. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 357-364, 1997.     

The specificity of antisera raised by oestradiol-17beta-3-hemisuccinyl-bovine serum albumin.

The preparation of pure oestradiol-17beta-3-hemisuccinyl-bovine serum albumin conjugate is described. Contrary to previous findings this antigen raised reasonably specific antisera in rabbits which possessed a cross reaction of only 2.0% with oestrone, and 0.8% with oestriol. The production of this specific antisera is considered to be due to the high purity of the antigen. The role of the C-3 phenolic hemisuccinyl linkage of the antigen in raising this specific antisera is discussed.     

Isolation of fractions from Bordetella parapertussis extracts and their use for preparing an erythrocyte diagnostic preparation]

Soluble parapertusis antigen, serologically active, having hemosensitive properties and containing only 2 antigenic components was obtained by the method of ethanol fractionation of microbial extracts. This method is simple and convenient for production purposes. The antigen thus obtained was used for the production of a highly specific erythrocytic diagnostic preparation (formalinized, liquid). When tested in reaction with animal and human sera, the new diagnostic preparation proved to be sufficiently active and species specific.     

Role of IL-4 in aversion induced by food allergy in mice

To ascertain the role of IL-4 in aversion to antigen induced by food allergy, wild type and IL-4 deficient BALB/c mice were sensitized with ovalbumin and challenged orally with egg white. Sensitized wild type mice had increased production of IL-4 by spleen and mesenteric lymph node cells in vitro, higher levels of serum anti-ovalbumin IgE and IgG1, aversion to ingestion of the antigen and loss of body weight after continuous oral challenge. Intestinal changes in wild type sensitized mice included eosinophil infiltration and increased mucus production. The IL-4 deficiency impaired the development of food allergy and the aversion to antigen, suggesting the involvement of the antigen specific antibodies. When IL-4 deficient mice received serum from sensitized wild type donors, the aversion was restored. These results indicate that production of IL-4 and specific IgE/IgG1 antibodies correlate with aversion to antigen induced by food allergy in mice.     

IL-1 secretion and membrane IL-1 expression by neonatal spleen cells during soluble antigen presentation

Antigen presentation and IL-1 production by neonatal spleen cells were studied in a murine model. The T-helper-cell line (D10-G4.1) (D10), which is specific for soluble antigen presented on syngeneic antigen-presenting cells and dependent on IL-1 for its proliferation, was used as an indicator cell for the ability of syngeneic neonatal or adult spleen cells to present antigen and produce IL-1. The antigen-presenting capacity of neonatal spleen cells is low as attested by D10 proliferation. During antigen presentation there is an augmentation of IL-1 production by the antigen-presenting spleen cell population. However, neonatal spleen cells do not respond to the same levels as do adult spleen cells. These reduced levels of secreted IL-1 cannot be attributed to a low potential for producing IL-1 as attested by the high levels of IL-1 made by these cells after induction by a crude IL-1 inducer factor (IL-1-IF) and by the stimulus of the IL-1-IF produced by D10 cells during antigen presentation by paraformaldehyde-fixed adult cells. The spontaneous expression of membrane IL-1 by neonatal cells is low. Membrane IL-1 levels on neonatal cells can be brought to adult levels by induction with IL-1-IF. Neonatal spleen cells have an impaired capacity to process and/or present soluble antigen. This impairment leads to a decreased stimulus of the T helper cell to produce inducer factors and thus a reduced level of IL-1 production by the neonatal cells during antigen presentation.     

Improved production and function of llama heavy chain antibody fragments by molecular evolution

The aim of this study was to improve production level of llama heavy chain antibody fragments (V_HH) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V_HH fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V_HH with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V_HH fragments were selected. We found that these shuffled V_HH fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V_HHs point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90°C were compared with parental V_HHs. One shuffled V_HH was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V_HH displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90°C. Structural analysis suggested that improved antigen binding is associated with the A24→V24 substitution, which reduces the size of the hydrophobic pit at the llama V_HH surface. We demonstrate that it is possible to improve desired characteristics of the same V_HH fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.     

Immunization with a plant-produced colorectal cancer antigen

Cancer vaccination has become an important focus of oncology in recent years. Active immunization with tumor-associated antigens such as colorectal cancer antigen GA733-2 is thought to potentially overcome the reoccurrence of metastasis. As recombinant protein production in bioreactors is costly and subject to growing safety concerns, we tested plants as an alternative for the expression of a potential colorectal cancer vaccine. Comparing colorectal cancer antigen GA733-2 produced in tobacco plants with the same antigen produced in insect cell culture, we found a similar humoral immune response to injection of either of the two antigen preparations into mice. Some minor differences were observed in the cellular response that might be due to impurities. Our studies compare for the first time, immunization with the same antigen expressed in either plants or insect cell culture. This will provide important data for use of plants as production systems of therapeutics.     

A Modification of the Indirect Immunofluorescence Test for Detection of Ehrlichia phagocytophila Antibodies

A modified technique for production of antigen and performance of the test is described. A suspension of infected neutrophils was directly applied to multiwell slides. Multichannel pipettes may be used for dilution and application of sera. The modification inreases the capacity both by production of the antigen and by performance of the test. This paper also gives a quantitative determination of the antibodies.     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.