Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Studies on γM and γG Antibody Response of Mice to Bovine Serum Albumin

Response of CBA mice with γM and γG antibodies to bovine serum albumin (BSA) was studied in relation to a variety of conditions of antigen administration. The variables in the conditions were doses and physical forms of antigen, and injection routes. It was realized that γG antibody response to soluble BSA and both γM and γG antibody responses to particulate forms of BSA were augmented as the dose was increased. The γM response to soluble BSA was not elevated by an increase in the amount of antigen up to 1 mg. The soluble form was not so immunogenic as the particulate forms, in which alum-precipitated BSA was capable of inducing both γM and γG antibodies to high titers, and heat-denatured BSA elicited preferably antibody. Alum-precipitated BSA and the emulsified BSA were strong inducers for γG antibody response when injected subcutaneously. In any antigen form, γM response was markedly influenced by changing the injection route, the order of decreasing efficiency for antibody being intravenous, intraperitoneal and subcutaneous. The γG antibody response was hardly affected by the injection route. The effect of a single intravenous injection of 0.01 mg of endotoxin, given 1 to 2 hr after antigen injection, on γM and γG antibody production differed according to the antigen administration procedures. Generally speaking, this agent had an enhancing effect when the antigen was given in the particulate forms, and it depressed the response when the antigen was given in the soluble form.     

Further studies on amplification of cell-associated immunological memory by secondary antigenic stimulus.

Using the hapten-carrier system in which the dinitrophenyl group (DNP) served as a B cell reactive hapten and bovine serum albumin (BSA) or human gammaglobulin (HGG) as a T cell reactive carrier, changes in the hapten-specific memory (B cell-associated memory) and the carrier-specific memory (T cell-associated memory) after a secondary antigenic stimulus were analyzed in mice. Since an immunological adjuvant was indispensable in the induction of the primary increase in memory, antigen used for the primary antigenic stimulus was injected together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) which has already been shown to exhibit a potent adjuvant effect. With the cell-transfer technique, it was found that the cell-associated hapten-specific memory for anti-DNP antibody response to DNP-BSA was truly amplified by the secondary injection of DNP-HGG into mice primed with DNP-HGG, and that the cell-associated carrier-specific memory as judged by the helper effect on anti-DNP response to DNP-BSA was also truly amplified by the secondary injection of BSA into mice primed with BSA. However, when memory was assessed in actively immunized mice, the secondary injection of BSA into mice primed with DNP-BSA and HGG decreased anti-DNP responsiveness to the tertiary injection of DNP-BSA, whereas the secondary injection of DNP-HGG secondarily increased anti-DNP responsiveness. In mice primed with DNP-BSA the titers of serum antibodies to BSA increased after the secondary injection of DNP-BSA or BSA. From these results it has been concluded that, like B cell-associated memory, T cell-associated memory is also amplified by a secondary antigenic stimulus, although its expression is inhibited in actively immunized mice through negative control by their antibodies.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The sequence of histological changes in the regional lymph node and other lymphoid organs of mice injected with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) or bacterial lipopolysaccharide (LPS) was followed. Injection of CPS-K, but not LPS, induced the following characteristic histological changes in the regional lymph node. In the early stage there was a marked decrease in the number of small lymphocytes, accompanied by the appearance of scattered fragmented nuclei and infiltration of polymorphonuclear neutrophilic leukocytes, and in the late stage there was marked proliferation of macrophage-like cells and pyroninophilic cells. Histological changes in the thymus and spleen and changes in cell populations in the bone marrow and peripheral blood after CPS-K injection were essentially the same as after LPS injection. Since CPS-K has a much stronger adjuvant action on antibody response than does LPS, it is suggested that the characteristic histological changes in the regional lymph node after injection of CPS-K are closely related to its extraordinarily strong adjuvant action.     

Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 μg per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 μg per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

Adjuvant actions of polyclonal lymphocyte activators. I. Comparison and characterization of their actions in antibody response to deaggregated bovine serum albumin.

Various polyclonal lymphocyte activators (PLA) were compared in their effects to trigger the initiation of the immune response and the amplification of the once-initiated immune response. When PLA were injected into mice subcutaneously together with deaggregated bovine serum albumin (DBSA), all of the nine kinds of PLA tested, such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide, dextran sulfate, concanavalin A, pokeweed mitogen, phytohemagglutinin, polyadenylic-uridylic acid, and polyinosinic-cytidylic acid, exhibited more or less the adjuvant action to trigger the antibody response and to increase immunological memory to DBSA. Among the PLA tested the action of CPS-K was the strongest. On the other hand, none of these PLA, including CPS-K, acted to augment the secondary antibody response to the optimal dose of DBSA in mice primed with DBSA together with CPS-K. It was concluded that PLA act generally to trigger the initiation of the antibody response, although the intensity of their actions distributes in a wide range of diversity, but that they do not stimulate the amplification of the response.     

Effect of Capsular Polysaccharide of Klebsiella pneumoniae on Host Resistance to Bacterial Infections

In normal mice, the total count of peritoneal leukocytes was markedly decreased after intraperitoneal (i.p.) injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) depending on the dosage injected. This decrease was mainly due to the depletion of macrophages, and a decrease in the number of lymphocytes occurred to a lesser extent. CPS-K in relatively smaller doses mobilized polymorphonuclear neutrophilic leukocytes (PMN) into the peritoneal fluid but it decreased them transiently in larger doses. In mice infected i.p. with a virulent strain of Salmonella enteritidis, there was an abundant emigration of PMN into the peritoneal fluid. When 200 μg of CPS-K was injected i.p. immediately before bacterial challenge, emigration of PMN was markedly delayed for 48 hr after infection. Associated with this suppressed emigration of PMN, the numbers of macrophages and lymphocytes in the peritoneal fluid were significantly less in mice treated with CPS-K than those in untreated control mice for 48 hr after infection. The numbers of both cell-associated and extracellular bacteria in the peritoneal fluid were markedly greater in mice treated with CPS-K than those in untreated control mice. In both in vivo and in vitro experiments, ingestion of bacteria by macrophages and PMN was not blocked by CPS-K or neutral CPS-K, the active substance responsible for the infection-promoting effect of CPS-K. It appeared that CPS-K somehow impaired the intraphagocytic bactericidal activity.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was made to characterize the active substance for the extraordinarily strong adjuvant effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain. CPS-K was fractionated into acidic and neutral CPS-K by the addition of cetyl-pyridinium chloride. Neutral CPS-K exhibited an extremely strong adjuvant effect. The active substance in neutral CPS-K was precipitable when mixed with a rabbit homologous antiserum. The neutral CPS-K antigen was serologically distinct from the O antigen and from the acidic CPS-K which was the type-specific capsular antigen. Among preparations of neutral CPS-K from eight different strains of K. pneumoniae tested, the preparation from only one strain (MH-2) exhibited a strong adjuvant effect comparable to that of the neutral CPS-K from the Kasuya strain. The neutral CPS-Ks from Kasuya and MH-2 strains were antigenically identical. This antigen was not found in all preparations of neutral CPS-Ks obtained from seven different strains. Preparations of acidic CPS-Ks from all strains of K. pneumoniae tested with various serologic types including Kasuya and MH-2 strains were found to exhibit only weak adjuvant effects. The active substance (neutral CPS-K antigen from Kasuya strain) was shown to form a single peak upon analyses by gel filtration (Sephadex G-100) and ultracentrifugation. Sedimentation coefficient of the substance was approximately 20 S at a concentration of 5 mg per ml in 0.1 M NaCl. The active substance finally purified by gel filtration contained 65% sugars (as glucose equivalents), 6.8% hexuronic acids, 2.6% hexosamine, 2.3% proteins, and very small amounts of lipids.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

Changes in the number of cells and the weight of various lymphoid organs of mice, such as the regional lymph node (right inguinal node), spleen, thymus, bone marrow, and peripheral blood, were followed after the subcutaneous injection of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K). For comparison, the changes after injection of various polyclonal lymphocyte activators (PLA) including various preparations of bacterial lipopolysaccharide (LPS) were concurrently studied. The number of cells of all of the lymphoid organs tested and that of nucleated cells in the peripheral blood decreased significantly within a few days after injection of CPS-K, and increased later. Above all, the increase in the number of cells and in the weight of the regional lymph node was most prominent (about 10 times larger than that of the normal control). Such a marked increase in the number of cells of the regional lymph node was not induced by the injection of any preparation of LPS or any other PLA tested. The initial decrease in the number of cells after CPS-K injection was most marked and long lasting in the thymus. Although LPS prepared by Westphal's method from Escherichia coli O55 or Salmonella enteritidis exhibited a stronger decreasing effect on the number of cells of the thymus, the effect of LPS prepared by Westphal's method from E. coli O111 or that by Boivin's method from E. coli O55 was similar to that of CPS-K. It is concluded therefore that CPS-K has the ability to decrease the number of cells of various lymphoid organs, especially that of the thymus, initially after injection, which is a property in common with LPS, and CPS-K has a unique ability to increase markedly the cells of various lymphoid organs, especially those of the regional lymph node, at later stages after injection. Considering that CPS-K exhibits a much stronger adjuvant effect on the antibody response than does LPS or other polyclonal lymphocyte activators, it is suggested that this extraordinarily potent activity of CPS-K in increasing the number of cells of the regional lymph node is closely related to its strong adjuvant action.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

Immunogenicity of Nanogram to Milligram Quantities of Inactivated Foot-and-Mouth Disease Virus. I. Relative Virus-neutralizing Potency of Guinea Pig Sera

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.     

Further studies on generation of macrophages in in vitro cultures of mouse spleen cells and its inhibition by the capsular polysaccharide of Klebsiella pneumoniae.

Although the number of macrophages detected in cultures of mouse spleen cells at the start of the culture was very small, it markedly increased during further incubation. Macrophages were generated not only from the glass-adherent cell fraction of spleen cells, but also from the nonadherent cell fraction obtained after removal of adherent cells either by incubating in glass petri dishes or by passing through a glass bead column. The generation of macrophages from the nonadherent cell fraction occurred even when it was separated as late as 48 hr after the start of the culture. The phagocytic activity of macrophages newly generated from the nonadherent cell fraction was relatively weak, but it was activated during further incubation. Based on these results, the maturation process of macrophages can be divided into at least the following four stages; glass-nonadherent nonphagocytic precursor cells, glass-adherent nonphagocytic precursor cells, immature macrophages with low phagocytic activity, and mature macrophages with full phagocytic activity. The addition of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) to cultures of spleen cells markedly suppressed the generation of macrophages. The suppressive effect of CPS-K depended on its dosage, and the minimum concentration of CPS-K showing a definite effect was 0.05 μg/ml. CPS-K inhibited further generation of macrophages in either the nonadherent or adherent cell fraction at any time after the start of the culture. The suppressive effect of CPS-K on the generation of macrophages could not be reversed by simple washing of spleen cells which had been kept in contact with CPS-K for 3 hr. There was no evidence which showed that CPS-K exhibited direct cytotoxic effects on spleen cells in the culture.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on host resistance to bacterial infections. I. Induction of increased susceptibility to infections in mice.

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 mug per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 mug per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund's complete adjuvant in rabbits

Ethylene-vinyl acetate copolymer (EVAc) was evaluated as an antigen delivery device in laboratory rabbits. Bovine serum albumin (BSA) was incorporated with EVAc in a pellet, which was implanted subcutaneously. Serum antibody titers to BSA in four implanted rabbits were equal to titers in four rabbits injected twice with BSA in complete Freund's adjuvant. Three of four rabbits implanted with EVAc displayed no inflammation or systemic illness in response to the pellet. The fourth rabbit repeatedly developed a small abscess at the implantation site, but the lesions were less severe than complete Freund's adjuvant injection sites. The EVAc pellet is recommended as a non-inflammatory alternative method to Freund's adjuvants for producing serum antibody in rabbits.     

Microbial adjuvant and autoimmunity. II. Production of lesions in mice immunized with syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae.

The target organs of mice immunized with the respective syngeneic tissue extracts together with the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a powerful adjuvant were examined for production of lesions. In 15 out of 24 mice injected three times or more with syngeneic eyeball extracts and CPS-K adjuvant at intervals approximately 30 days, severe eyeball lesions developed in which the normal structure was almost completely lost. A large part of the eyeball tissue of these mice was replaced by infiltration with cells such as lymphocytes, plasma cells and other mononuclear cells and by connective tissue. No definite eye lesions developed in mice injected with CPS-K alone, eyeball extracts alone or eyeball extracts emulsified in complete Freund's adjuvant (CFA). In all of mice injected four times with thyroid gland extracts and CPS-K at intervals of approximately 30 days, definite thyroid gland lesions were produced. In three out of five mice of this group, the thyroid lesions were so severe that the normal thyroid follicular structure was almost completely lost, and a large part of the thyroid gland was replaced by infiltration with lymphocytes, plasma cells and other mononuclear cells and in part by connective tissue. In only one out of five mice injected with thyroid gland extracts emulsified in CFA, definite but milder thyroid gland lesions developed. No definite thyroid lesions developed in the remaining four mice of this group and also in any of the mice injected with thyroid gland extracts alone or CPS-K alone. Repeated injections of lymphoid tissue extracts and CPS-K also induced pathological changes in the spleen and lymph nodes, although less marked than those in the cases of the eyes and thyroid gland. The most remarkable change was a decrease in numbers of small lymphocytes at the areas surrounding the central arterioles in the white pulp of the spleen and the post-capillary venules in the cortex of the lymph nodes. From these results it has been concluded that our system can provide new and useful models for autoimmune diseases in man.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

The neutral fraction (neutral CPS-K) of Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, has already been reported as the active substance responsible for the strong adjuvant effect of CPS-K. The present results demonstrate that neutral CPS-K exhibits further common biological activities with lipopolysaccharide (LPS) isolated from Salmonella enteritidis. The intensity of the lethality in mice of neutral CPS-K by the intraperitoneal route is very similar to that of LPS. Its lethality for mice by the intravenous (i.v.) route is significantly stronger than that of LPS, because the degree of increase in the sensitivity to their lethality by i.v. challenge is smaller for LPS than for neutral CPS-K. The intensity of the pyrogenicity of neutral CPS-K in rabbits is approximately one-tenth of that of LPS as judged by the minimal pyrogenic doses and fever indices. The skin-preparatory potency of neutral CPS-K for the dermal Shwartzman phenomenon in rabbits is also approximately one-tenth of that of LPS compared on the basis of the minimal skin-preparatory doses. When injected i.v., neutral CPS-K exhibits a provocative effect on hemorrhagic reactions in skin sites prepared with neutral CPS-K or LPS.     

Conjugation of protein antigen to microparticulate β-glucan from Saccharomyces cerevisiae: a new adjuvant for intradermal and oral immunizations

Immunostimulatory glucose polymers known as β-glucans have been studied for many years. Our laboratory has prepared and characterized a novel microparticulate β-glucan (MG) from the budding yeast Saccharomyces cerevisiae. Because MG particles are rapidly phagocytized by murine peritoneal macrophages and induce the expression of B7 costimulatory molecules, we hypothesized that MG could serve as a vaccine adjuvant to enhance specific immune responses. Here, we describe a procedure for conjugating the test vaccine antigen bovine serum albumin (BSA) to MG via water-soluble carbodiimide linkage. Conjugates with up to 0.4 mg of BSA/mg MG were prepared. MG/BSA conjugates were still actively phagocytized by mouse peritoneal macrophages. When used to immunize mice by the intradermal route, these conjugates enhanced the primary IgG antibody response to BSA in a manner comparable to the prototypic complete Freund’s adjuvant. Although primary oral immunization with MG/BSA caused no increase in serum anti-BSA antibody titers, booster immunization elicited a significant anti-BSA antibody response. These results suggest that protein antigens can be conjugated to MG via a carbodiimide linkage and that these conjugates provide an adjuvant effect for stimulating the antibody response to the protein antigens.     

Adjuvant actions of polyclonal lymphocyte activators: III. Two distinct types of T-initiating adjuvant action demonstrated under different experimental conditions

We demonstrated that each of various polyclonal lymphocyte activators (PLA) exhibits two types of adjuvant action to initiate the carrier-specific helper T-cell response to otherwise nonimmunogenic antigen. Type 1 action was characterized as that to initiate the T-cell response to subcutaneous injection of soluble bovine γ-globulin (BGG), and type 2 as that to initiate the response to intravenous injection of aggregated BGG. Each of various PLA showed these two types of adjuvant action in a dissociated fashion. The capsular polysaccharide of Klebsiella pneumoniae (CPS-K) showed both types of action to the highest degrees. Lipopolysaccharide of Escherichia coli exhibited type 2 action as markedly as CPS-K, but failed to show type 1 action. Concanavalin A showed definite type 1 action, but not type 2 action. Polyadenylic-uridylic acid showed definite type 2 action, but not type 1 action. Type 1 and type 2 actions of dextran sulfate were minimal. A hypothetical view is presented to consider that type 1 adjuvant action is directed to two mutually independent sites whereas type 2 action is directed to one site.     

Cationization of protein antigens. VI. Effects of cationization on the immunoregulatory properties of a bovine serum albumin peptide, a.a. 506-589.

Cationization of bovine serum albumin (BSA) causes a profound increase in its immunogenicity. To establish if immunoregulatory properties of an immunosuppressive peptide are affected by cationization, a BSA peptide, a.a. 506-583, was cationized and tested for its immunogenic properties. A greatly reduced amount of cationized peptide compared to native peptide was required to stimulate BSA-primed T cells to proliferate in vitro. Mice primed with the cationized peptide administered with an adjuvant responded with a significantly greater anti-BSA response than mice immunized with the native form of the peptide. In the absence of an adjuvant i.v. or i.p. administration of the native peptide was immunosuppressive, while the cationized form was immunoenhancing. Both forms of the peptide stimulated in vivo induction of L3T4+ (CD4), and Lyt-2+ (CD8) T cells. Removal of Lyt-2+ T cells from lymph node cultures following immunization with the native peptide caused a significant increase in the proliferation of the remaining T cells. This increase was not observed when the mice were immunized with the cationized peptide. No major BSA B cell determinants were present within the peptide sequence. Mice immunized with the peptide exhibited a negligible anti-BSA antibody response compared to those immunized with the whole BSA molecule. Furthermore, the peptide did not inhibit anti-BSA antibody binding to BSA. We demonstrated that cationization modifies immunoregulatory properties of an immunosuppressive BSA-derived peptide.