Antigenicity of Polymers of Glutamyl Peptide in Humans

Abstract

Antigenicity of glutamyl polypeptides has been the subject of many studies. Early interest arose from the fact that these peptides were one of the components of the capsule of Anthrax Bacillus and of Subtilis-Mesentericus group of organisms ( 1,2). Another more recent source of interest in antigenicity of these glutamyl peptides is the fact that polymers of them have been shown to have promise as plasma volume expanders (3). Interest has centered both on polymerized glutamyl peptides synthesized from straight chain peptide obtained from culture filtrates of B . mesentericus and on synthetic glutamyl peptides. The antigenicity of synthetic peptides has been studied by Stahman(4) and by Maurer ( 5 ) with somewhat discrepant results. This report presents data on antigenicity in humans of a branched chain glutamyl polymer which by actual test had molecular dimensions required for a satisfactory plasma volume expander in humans. As the question of antigenicity in humans of an otherwise suitable expander is of critical significance the findings are of considerable interest but must be confirmed by further studies.Materials and methods. The glutamyl peptide polymer was prepared by previously described methods (3,6). The backbone and side chains were gamma linked peptides obtained from B . mesentericus culture filtrates. The molecular weight was approximately 80,-000 by light scattering methods. The half life in blood stream of humans was approximately 15 hours. The material was essentially free of polysaccharide by the anthrone test, showing less than 2 pg of color-producing materia1/200 mg of peptide. It was dissolved as polysodium salt in isotonic saline to give a 2.5% solution. Immunization procedure in 24 human volunteers was essentially the same as that of Kabat and Berg (7) in testing mtigenicity of dextran preparations. After preimmunization bleeding, subjects were skin tested on one forearm with intracutaneous injection of 5'0 pg of polymer, and with a saline control on the other forearm. Skin tests were read in 20 minutes, 24 hours and 72 hours. After the 72-hour reading 2.5 mg of polymer in saline solution was injected subcutaneously. Another similar strength immunizing dose was given 12 days later. Two weeks later the skin test was repeated and read at the same time intervals. Then blood was taken for serological studies. Those subjects showing any evidence of reaction were skin tested again one month later. Quantitative precipitin tests were performed by the method of Heidelberger and McPherson (8) using the Folin-Ciocalteu tyrosine reagent as described by Kabat(9). Sera were kept in refrigerator for 2 weeks to remove complement. Three ml aliquots of pre-and postimmunization sera were mixed in 15 ml conical bottom centrifuge tubes with varying amounts of polymer (Table 11) and incubated at 37°C for 1 hour. They were then kept in refrigerator for 1 week with mixing twice a day. Any precipitate was collected by cold centrifugation and washed twice with cold saline prior to analysis. Complement fixation test ...

Immunochemical Studies on the Poly-γ-D-glutamyl Capsule of Bacillus anthracis. I. Characterization of the Polypeptide and of the Specificity of Its Reaction with Rabbit Antisera*

“…However, polymers composed of a single amino acid have in most instances been shown to be incapable of stimulating antibody formation (Maurer, 1957; Buchanan-Davidson et a!., 1959). In one study in which a high molecular weight polymer of glutamic acid was injected into humans, one of 24 subjects responded with a level of antibody which could be detected by serologic means (Ostroff et al, 1958). Homopolymers, on the other hand, if coupled to protein carriers, can elicit antibodies which have specificity directed against the polyamino acid portion of the complex (Sage et al, 1964;Arnon et al.…”

Poly(γ-d-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule ofBacillus anthracis: A potential addition to the anthrax vaccine

“…Other bacilli produce poly(␥-glutamic acid) (␥PGA) but only B. anthracis synthesizes it entirely in the D conformation (12). ␥DPGA is a surface structure (13), inhibits in vitro phagocytosis and, when injected, is a poor immunogen even as a bacterial component (14)(15)(16)(17)(18); the protective effect of anti-␥DPGA has not been reported. The capsule shields the vegetative form of B. anthracis from agglutination by monoclonal antibodies to its cell wall polysaccharide (19).…”

Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

“…Although the ␥DPGA capsule is relatively nonimmunogenic (55,75), recent immunization strategies have been successful in inducing anti-␥DPGA antibody responses (30,61,66,74), and passive immunization with monoclonal antibodies (MAbs) specific for the capsule of B. anthracis protected mice against a lethal pulmonary spore challenge with the fully virulent Ames strain of B. anthracis (9,33). However, the predominant strategy for designing vaccines against inhalation anthrax has focused on the toxins and utilizing PA as the primary antigen.…”