Antigens in penicillin allergy. II. The influence of the number of penicilloyl residues on the antigenicity of macromolecules as determined by radioimmunoassay (RIA), passive cutaneous anaphylaxis (PCA) and antibody induction.

The present communication reveals a relationship between the epitope density of penicilloylated protein antigens and their antigenic activities in a radioimmunoassay (RIA), in passive cutaneous anaphylaxis (PCA) and in inducing antibody formation in mice. In the RIA and PCA a critical number of 2-4 penicilloyl residues per protein molecule was noted. At this level small changes in the number of substituents considerably influenced the antigenic activities. The molecular weight and the nature of the carrier proteins, myoglobin, bovine serum albumin (BSA) and dimeric BSA also affected the threshold concentration for efficient antigenic activity. The results with the RIA and PCA were significantly correlated to each other. Using penicilloylated BSA as immunizing antigen in mice it was found that an epitope number higher than 11 penicilloyl residues per protein molecule induced significant antibody formation after a single injection. Antigens with a lower degree of penicilloyl substitution were less immunogenic. An antigen carrying 0.6 penicilloyl residues per BSA molecule did not induce penicilloyl-specific antibodies even after three injections. The capacity of heavily penicilloylated proteins to induce and elicit penicillin allergy as revealed by the present results stresses the importance of limiting their presence in penicillin preparations.     

Oral induction of the secretory antibody response by soluble and particulate antigens

The present experiments have been performed in order to study the immunogenicity of antigen taken up by peritoneal macrophages using the hapten-carrier model and to investigate the role of macrophages in the antigenic competition between hapten and carrier moieties of the antigen molecule we have previously described. Guinea pigs were immunized with peritoneal cells collected from guinea pigs previously injected intraperitoneally with soluble or glutaraldehyde-polymerized hapten-carrier conjugates in Freund's incomplete adjuvant. Delayed hypersensitivity reactions to the carrier and anaphylactic reactions to both the hapten and the carrier were studied 14 and 16 days after immunization. After immunization with macrophage-associated hapten-carrier conjugate, delayed hypersensitivity reactions to the carrier were first detected in the absence of anaphylactic reactions which appeared later. The capabilities of macrophage-ingested antigen to induce delayed hypersensitivity reactions, but not anaphylaxis, decreased when increasing the incubation time of macrophages with antigen. The antigenic competition between hapten and carrier was confirmed to be a transient phenomenon occurring in the macrophage.     

Induction of immunological tolerance to the penicilloyl antigenic determinant. II. Evaluation of stable and unstable penicilloyl dextrans.

A specific tolerant state to the major antigenic determinant of penicillin allergy, the penicilloyl group, was induced in C3H mice primarily immunized with penicilloylated bovine gamma globulin in complete Freund's adjuvant. Tolerance was obtained by intraperitoneal administration of either of two penicilloyl-bearing dextrans of molecular weight 2 X 10(6). One conjugate contained penicilloyl groups stably bound via a 1,6-diaminohexane spacer, the other bore the penicilloyl groups directly bound to the hydroxyl groups of the carrier. These directly bound penicilloyl groups undergo hydrolytic cleavage within 3 days under physiological conditions in neutral aqueous solution. Model experiments showed that the rapid cleavage into carrier and haptenic derivatives also applies to penicilloylated dextran in receptor-bound and particulate form, as may be expected from the highly hydrophilic character of the conjugate. The stable conjugate at 1 mg and the cleavable conjugate at 4 mg doses induced comparable tolerance lasting for at least 8-12 weeks.     

Antigens in penicillin allergy. I. A radioimmunoassay for detection of penicilloylated protein contaminants in penicillin preparations.

This communication presents a sensitive and discriminative method for detection of protein impurities in penicillin preparations. Antibodies against various penicilloyl derivatives of high avidities and specificities raised in rabbits were coupled to microcrystalline cellulose. The amount of penicilloyl antigen present in a sample was calculated from the relative uptake of a radioiodinated penicilloylated albumin competing with the sample for binding to the antipenicilloyl immunosorbent. As little as 0.048 pmol/ml of penicilloylated human serum albumin could be detected. The accuracy of the determination was within +/- 23% (p less than 0.05). The pronounced specificities against the penicillin side chains demonstrated by the various immunosorbents were not displayed by the antibodies in passive cutaneous anaphylaxis experiments in guinea pigs. Furthermore, the immunosorbents showed the same pattern of specificity against monomeric penicillins as for penicilloylated proteins, but the former were considerably less efficiently recorded. The relatively small quantities of protein impurities in penicillin preparations, however, necessitated a separation from penicillin prior to analyses with the RIA. This was accomplished by fractionation on Sephadex G-50 fine, ginving a recovery of 80-90% of 0.1-2.5 ppm of penicilloylated protein.     

Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Immunological Activities of Purified Preparations of Enterobacterial Common Antigen

The immunological activities of three purified preparations of enterobacterial common antigen (ECA) obtained by different procedures were studied. ECA-Ma (method of A. Marx) was from Salmonella typhimurium TV149 (Ra mutant), ECA-My (method of H. Mayer) was from S. montevideo, and ECA-Ro (method of E. Romanowska) was from Shigella sonnei phase I. These preparations, on a weight basis, neutralized similar amounts of ECA antibodies, indicating that the serological activities were comparable. Neither ECA-My nor ECA-Ro elicited specific delayed-type hypersensitivity skin reactions at 24 or 48 h in immunized guinea pigs. ECA-Ma, as well as the nonpurified preparations of the antigens used for immunization, elicited reactions at 24 h but not at 48 h. Thus, ECA-specific delayed-type hypersensitivity was not detected in immunized guinea pigs. Striking differences were noted in the immunogenicity of these antigens, ECA-Ma being highly immunogenic in the rabbit in contrast to ECA-My and ECA-Ro. ECA-Ma was a potent mitogen for guinea pig spleen cells, stimulating high levels of DNA synthesis; ECA-My was only slightly active. The three antigens were mitogenic to spleen cells from both CBA/J and C3H/HeJ mice, although not to the same degree, indicating that this effect is not due to contaminating lipopolysaccharide, since the latter strain of mice is resistant to endotoxin. Since an ECA-Ma extract made from an ECA-negative mutant proved to be mitogenic to murine spleen cells, the mitogenicity is not due to the ECA haptenic determinant. The mitogenic effect is polyclonal in nature, ECA-Ma producing a maximum response on day 3. Thus, the ECA preparations are both B-cell mitogens and polyclonal activators in murine spleen cells. From these studies it is evident that the biological and immunological activities of these purified antigens depend not only on the haptenic determinant but also on associated or bound components of the preparations.     

Immunogenicity of hapten-carrier conjugates associated with liposomes

Immunogenicity of liposome-entrapped hapten-carrier conjugates was studied in guinea pigs. Animals immunized intravenously with a subimmunogenic dose of a hapten-carrier complex entrapped in liposomes exhibited cutaneous anaphylactic reactions to the hapten, even when the liposomes were treated with trypsin, but not delayed hypersensitivity (DH) reactions to the carrier. Animals immunized with a mixture of empty liposomes and antigen showed anaphylactic reactions to the hapten that appeared to be elicited by antigen adsorbed on the outer membrane of liposomes. The induction of anaphylaxis to the hapten by liposome-associated antigen was not due to an adjuvant property of liposomes. DH reactions to the carrier after injection of a liposome-entrapped subimmunogenic dose of a hapten-carrier conjugate could be observed only after activation of macrophages by Bacillus Calmette-Guérin.     

In Vitro Induction of Specific T Cells Able to Help in the Generation of Delayed-Type Hypersensitivity by Thymus-Dependent and Type-II 'Thymus-Independent' Antigens

An in vitro system is described which supports the primary induction of T cells able to help in the induction of delayed-type hypersensitivity. This system is used to demonstrate that hapten-Ficoll conjugates induce potent hapten-specific T cells with this function. These type-II 'thymus-independent' antigens are thus able to induce specific regulatory T cells. These helper T cells are similar to those induced by classical thymus-dependent antigens in that they are specific for antigen rather than idiotype and act by a linked mechanism.     

Dermal granulomatous hypersensitivity in Q fever: comparative studies of the granulomatous potential of whole cells of Coxiella burnetii phase I and subfractions

Dermal granulomatous reactivity to Q fever antigens in guinea pigs has been described as a model for vaccine reactions seen in previously sensitized humans. This model has now been applied to study the ability of subfractions of Coxiella burnetii to produce granulomas. Q fever organisms in phase I, trichloroacetic acid-soluble and -insoluble fractions, and the extract and residue of chloroform-methanol extraction were tested for their relative ability to elicit and immunize for dermal granulomatous reactions and specific lymphocyte proliferative responses in guinea pigs. The results suggest that a determinant(s) causing granulomas can be removed by chloroform-methanol extraction of phase I whole cells. The chloroform-methanol residue elicited strong delayed-type hypersensitivity without subsequent granuloma formation. The chloroform-methanol residue appears to possess a determinant(s) for lymphocyte stimulation equivalent to that of whole phase I organisms.     

Antigenic properties of polymers formed by beta-lactam antibiotics.

Purified polymers have been isolated from 6-aminopenicillanic acid and from semi-synthetic penicillins and cephalosporins. All the polymers were shown to react with rabbit antibody of penicillins and cephalosporins. All the polymers were shown to react with rabbit antibody of penicilloyl specificity, as demonstrated by passive cutaneous anaphylaxis in guinea pigs, and the reactions were shown to be penicilloyl-specific by hapten inhibition experiments. The cephalosporin-derived polymers in addition reacted with rabbit antibodies raised to the corresponding cephalosporin conjugates of bovine gamma-globulin. Using direct skin tests, passive cutaneous anaphylaxis and an in vitro assay, no evidence was obtained that the polymers induced the formation of specific antibodies in baboons, guinea pigs and rabbits, but in baboons the induction of cell-mediated immunity, demonstrable by delayed skin test reactions, was shown.     

Carrier and hapten functions in immune deviation. III. Possible antigenic competition between hapten and carrier determinants at the macrophage level

The effects of the intravenous injection of DNP-Ficoll on delayed hypersensitivity reactions to bovine gamma globulins (BGG) and anaphylactic reactions to the hapten induced by immunization with DNP-BGG in Freund's adjuvant were studied in guinea pigs. Animals treated with DNP-Ficoll exhibited stronger delayed hypersensitivity to BGG and lower anaphylactic reactions to the hapten than control animals treated with saline, Ficoll alone or with an unrelated protein antigen. These results could be explained by antigen competition between hapten and carrier determinants occurring at the macrophage level.     

Studies on hypersensitivity. I. Delayed and Arthustype skin reactivity to protein conjugates in guinea pigs

A study has been made of the immunization of guinea pigs with proteins conjugated with picryl, acetyl and ethoxymethylene-phenyloxazolone groups. Immunization by means of complexes of these substances with anti-protein and anti-hapten antisera have also been studied. Antibody production, anaphylactic and Arthus-type sensitivity and delayed skin sensitivity to the conjugates, to the carrier proteins and to unrelated proteins carrying the same haptenic group have been investigated.

Immunization with conjugates is found to be followed by the appearance of delayed hypersensitivity to the protein `carrier' in the absence of detectable antibodies against it, although antibodies are produced at that time against the haptenic group itself. Delayed hypersensitivity to the haptenic group has not been detected at any time: blocking it with specific antibody does not lead to the appearance of delayed sensitivity, but merely suppresses antibody formation against that group.

Pure delayed sensitivity has been produced against gelatin, both alone and as a conjugate with picryl. Conjugates with homologous serum proteins are shown to provoke only Arthus-type sensitivity and antibody against the haptenic group.

These findings are discussed in view of the light they may throw upon the relation of delayed hypersensitivity to antibody production and upon the process of immunization.

     

Effective protection against Listeria monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4+ and Lyt 2+ T cells.

Selected L3T4- and Lyt 2- T-cell subpopulations from Listeria monocytogenes-infected mice were transferred into syngenic recipients, and their capacity to adoptively mediate protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens was determined. Both functions were markedly reduced by pretreatment of cells with either anti-L3T4 or anti-Lyt 2.2 antibodies plus complement, but they could be restored by admixture of the two selected T-cell subsets. Thus, after systemic cell transfer effective protection against L. monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4+ and Lyt 2+ T cells.     

The use of hapten-polysaccharide conjugates for the induction of B-cell tolerance involving IgE responses. I. Preparation, characterization and specific tolerogenic activity of penicilloyl-substituted levans affecting IgE responses in normal and sensitized mice

Benzyl penicillin was coupled directly to the hydroxylic groups of dextrans and levans, and indirectly to the amino groups of diaminopropyl-carboxymethyl-levans. Derivatives of both types were tested for their capacity to react with anti-penicilloyl antibodies, either in vitro (immunoprecipitation and its inhibition) or in vivo (inhibition of PCA reaction). Only derivatives coupled to diaminopropyl-carboxymethyl-levans were active by both criteria. High molecular weight penicilloyl-diaminopropyl-carboxymethyl-levans were tested for their immunogenicity (IgM response) and capacity to induce tolerance in the IgE component of anti-penicilloyl responses in DBA/2 and CBAT6T6 mice. The IgM response to the levan carrier was very much depressed by penicilloyl substitution of the levan, whilst that to the penicilloyl determinants was also very poor. These derivatives induced a state of IgE antibody tolerance when given before or after the induction of an allergic response with penicilloyl-ovalbumin. A higher degree of substitution was required for the induction of tolerance after immunization.

The tolerance induced was specific, since it did not affect IgE responses against ovalbumin and persisted for at least 3 months. Further challenges with penicilloyl-ovalbumin did not break the tolerant status.

     

Reverse Passive Cutaneous Anaphylaxis in the Guinea Pig with Horse, Sheep or Hen Antibodies

Reverse passive cutaneous anaphylaxis experiments were performed in guinea pigs with rabbit gamma globulin and human gamma globulin as antigens and horse, sheep or hen sera containing antibodies against these antigens. These antibodies were unable to directly sensitize the guinea-pig skin but when the reverse technique was used, namely, the antigen (gamma globulin) was injected before the antibody, characteristic anaphylactic reactions were obtained. It is concluded that, if a suitable antigen is used, which can be fixed to the host by the reverse technique, sera from species which cannot directly sensitize the guinea pig can nevertheless give typical anaphylactic reactions.     

Preferential induction of cell-mediated immunity by chemically modified sheep erythrocytes

Sheep red blood cells (SRBC) almost exclusively induce humoral antibodies when injected in saline into adult rats. Chemical modification of SRBC by either periodate oxidation or acetoacetylation resulted in preparations which provoked lower antibody responses than did normal SRBC, but induced much higher levels of delayed-type hypersensitivity. Furthermore, SRBC which had been both periodate oxidized and acetoacetylated induced even higher delayed responses, but were unable to stimulate detectable antibody formation. Thus, by two simple chemical treatments, SRBC have been converted from an antigen which predominantly induces humoral antibodies tc an antigen which exclusively provokes cell-mediated immunity. The chemically modified SRBC had some notable immunological properties. (I) Antigenically, they were still strongly agglutinated by anti-SRBC antiserum. (II) They very effectively induced delayed-type hypersensitivity when injected either in saline or Freund's complete adjuvant. (III) They lost their immunogenicity when lysed just prior to injection. (IV) They induced delayed-type hypersensitivity which cross-reacted with goose, rabbit and horse red blood cells, a result consistent with the notion that antigens cross-react more broadly at the cell-mediated immune level than at the antibody level. (V) Comparatively high levels of both humoral and cell-mediated immunity were achieved when a mixture of periodate oxidized SRBC and acetoacetylated SRBC was injected. The theoretical and practical implications of these findings are discussed.     

Immune and non-immune responses to monovalent low molecular weight penicilloyl—polylysines and penicilloyl—bacitracin in rabbits and guinea-pigs

Rabbits and guinea-pigs have been immunized with low molecular weight penicilloyl—polylysines (BPO₁—PLL_{1 2}, BPO₆—PLL_{1 2}) with varying degrees of substitution and with monovalent penicilloyl—bacitracin (BPO₁—BAC). The immune response to the penicilloyl (BPO) determinant has been followed by skin tests, passive haemagglutination, passive cutaneous anaphylaxis (PCA), immunodiffusion and immunoelectrophoresis.

Maximally substituted BPO₆—PLL_{1 2} was found to be non-immunogenic. Apparently monovalent BPO₁—PLL_{1 2}, which inhibits precipitation of anti-BPO antibodies in vitro, was found to induce delayed-type hypersensitivity and antibodies for BPO in most of our random bred and in all strain 2 guinea-pigs. The delayed hypersensitivity in some of the sensitized guinea-pigs was hapten (BPO)-specific. BPO₁—PLL_{1 2} was very poorly immunogenic in rabbits.

Monovalent BPO₁—BAC induced BPO-specific delayed hypersensitivity and anti-BPO antibodies in some random bred guinea-pigs but not in strain 2 animals. BPO₁—BAC induced good levels of haemagglutinating anti-BPO antibodies in most random bred rabbits.

Truly monovalent compounds, such as BPO₁—BAC or BPO—ε-aminocaproate, do not elicit antibody-dependent reactions such as anaphylactic or Arthus reactions, but inhibit them.

On the other hand, apparently monovalent BPO₁—PLL_{1 2} was found to elicit BPO-specific PCA and Arthus reactions in immunized rabbits and guinea-pigs. When administered in higher doses, similar reactions were produced in non-immunized guinea-pigs. The possibility is discussed that some of the reactions observed with basic polypeptide antigens are due to the formation of mixed `specific antibody—polypeptide—non-specific protein' complexes formed in vivo by electrostatic interaction.

     

5-Hydroxytryptamine releasing activity in culture supernatants of guinea pig lymphocytes

5-Hydroxytryptamine (5-HT) is not only involved in anaphylactic reactions but also in delayed-type hypersensitivity (DTH) reactions. Supernatants of cultivated guinea pig spleen lymphocytes were investigated for possessing 5-HT releasing activity. Both fractionated supernatants of mixed lymphocytes unspecifically stimulated by Concanavalin A (Con A) and fractionated supernatants of non-stimulated mixed lymphocytes decrease the 5-HT content of rabbit thrombocytes. Fractions of a molecular weight smaller than 12,500 daltons are more effective than fractions of a higher molecular weight range.The 5-HT releasing activity of the supernatants is discussed in comparison to a cell migration stimulating activity.     

Modulation of the immune response by passive antibodies. IV. Effects of IgG1 and IgG2 anti-hapten antibodies

The effects of IgG1 and IgG2 anti-hapten antibodies waere studied on celluar and humoral reactions induced by immunization with a hapten-carrier complex. IgG2 was shown to depress delayed hypersensitivity reactions to the carrier and to enhance anaphylactic reactions to both the hapten and the carrier whereas IgG1 appeared to have no activity except a little enhancing effect on antibody synthesis to the carrier. The regulatory role of IgG2 antibodies, which were cytophilic for macrophages, in the immune response to a hapten-carrier conjugate is discussed.     

Group B streptococcal type II and III conjugate vaccines: physicochemical properties that influence immunogenicity

Recent efforts toward developing vaccines against group B streptococci (GBS) have focused on increasing the immunogenicity of GBS polysaccharides by conjugation to carrier proteins. However, partial depolymerization of GBS polysaccharides for the production of vaccines is a difficult task because of their acid-labile, antigenically critical sialic acids. Here we report a method for the partial depolymerization of type II and III polysaccharides by mild deaminative cleavage to antigenic fragments with reducing-terminal 2,5-anhydro-d-mannose residues. Through the free aldehydes of their newly formed end groups, the fragments were conjugated to tetanus toxoid by reductive amination. The resulting conjugates stimulated the production in animals of high-titer type II- and III-specific antibodies which induced opsonophagocytic killing of type II and III strains of group B streptococci. For the type II conjugates, immunogenicity increased as oligosaccharide size decreased, whereas for type III conjugates, the size of the oligosaccharides did not significantly influence immunogenicity. When oligosaccharides of defined size were conjugated through sialic acid residues, the resulting cross-linkages were shown to affect immunogenicity. When oligosaccharides were conjugated through terminal aldehyde groups generated by deamination, modification of the exocyclic chain of sialic acid did not influence immunogenicity.