Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 (pat-1)) on chromosome 1 of B. patellaris.

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 (pat-1). Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11800, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11800, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11800 loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 (pat-1) locus. Two copies of the middle-repetitive OPX21100 marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.

Nonionic block polymer surfactants enhance the avidity of antibodies in polyclonal antisera against Streptococcus pneumoniae type 3 in normal and Xid mice.

Avidities of antibody (sub)classes in polyclonal antisera against Streptococcus pneumoniae type 3 (S3) can be (semi) quantitatively determined with a specific inhibition ELISA. A hexasaccharide was isolated from the hydrolyzed S3 capsular polysaccharide and coupled to a protein-carrier. Mixtures containing these conjugates and nonionic block polymer (NBP) surfactants were used for immunization. After various immunizations of these conjugates without NBP the anti-S3 specific antibodies of IgM and IgG2a isotype decreased in both antibody level and avidity. The adjuvants NBP 1501 and L121 not only enhanced the hexasaccharide-protein induced IgM and IgG antibody levels but also clearly increased the avidity of the two antibody (sub)classes IgM and IgG2a. This effect was observed in normal (data not shown) and X-linked immunodefective mice. A maturation of the IgG antibody response was realized by the second immunization with hexasaccharide-protein conjugate whereas the third immunization showed no further increase in antibody level and avidity.

Modulation of the immune response to pneumococcal type 14 capsular polysaccharide-protein conjugates by the adjuvant Quil A depends on the properties of the conjugates.

Streptococcus pneumoniae type 14 capsular polysaccharide-bovine serum albumin (S14PS-BSA) conjugates were prepared by water-soluble-carbodiimide-mediated condensation with or without the use of N-hydroxy-sulfosuccinimide. The immunogenicities of the capsular polysaccharide (S14PS) and of the conjugates were studied in (CBA/N x BALB/c)F1 mice and in female BALB/c mice. The response in these mice indicates that S14PS could be classified as a thymus-independent type 2 antigen. Coupling of S14PS to BSA improved the immunogenicity of this polysaccharide, and an immunoglobulin G memory response was evoked. Conjugation with N-hydroxysulfosuccinimide resulted in a product with a higher polysaccharide/protein ratio. This conjugate induced a greater immune response than did the classical conjugate. Quil A enhanced the immune response to S14PS and to most S14PS-BSA conjugates. The enhancement of the immune response to the conjugates seemed to depend on the coupling procedure. Our results indicate that for the construction of immunostimulating complexes based on polysaccharide or oligosaccharide-protein conjugates, attention should be paid to the degree of cross-linking of the antigens involved.

Estimation of the avidity of antibodies in polyclonal antisera against Streptococcus pneumoniae type 3 by inhibition ELISA.

The reliability of the determination of antibody avidity in polyclonal sera by indirect sandwich ELISA was studied. Binding of IgM and IgG (sub)classes in unpurified serum to Streptococcus pneumoniae type 3 capsular polysaccharide, which was coated onto ELISA plates, was inhibited with different inhibitors. The inhibitor concn at which 50% inhibition of antibody binding to the ELISA coat was achieved, was used as a measure for antibody avidity. As this 50% inhibition value is dependent upon the dilution of the serum and thus upon the initial amount of free antibody, it is necessary to define (a narrow range of) final ELISA absorbance values to which the dilutions of non-inhibited sera have to be adjusted. The shapes of the serum dilution curves have a good correlation with the numerical 50% inhibition values of the antibody avidity. The inhibition ELISA is suitable to compare the avidity values of the different antibody isotypes, but two remarks should be made: (1) antibody heterogeneity should be considered to influence the results and prevent the accurate measurement of absolute numerical avidity values. Because in the ELISA system merely antibody "activity" is measured, comparison of the efficacy of vaccines by means of the 50% inhibition (avidity) value of various antibody (sub)classes can still be performed in a reliable way; (2) results of the determination of the 50% inhibition values of the different antibody (sub)classes showed them to be dependent on the molecular ratio between antibody (sub)class levels. More aspects of the determination should be taken into account, like shapes of simple dilution curves, influences of various inhibitor concns in the diluent and whole (extended) inhibition curves.

Nonionic block polymer surfactants enhance immunogenicity of pneumococcal hexasaccharide-protein vaccines.

Incorporated in oil-in-water emulsions, nonionic block polymer surfactants change the kinetics of generated antibody responses against pneumococcal hexasaccharide-protein conjugates: prolonged immunoglobulin M and immunoglobulin G responses are realized. Nonionic block polymer surfactants favor the immunogenicity of hexasaccharide-protein conjugates in young mice in such a way that a single injection yields long-lasting protection.

Deletion analysis of the proteinase gene of Streptococcus cremoris Wg2.

The Streptococcus cremoris Wg2 proteinase gene, cloned in S. lactis, specified a proteinase which exhibited the same specificity toward casein as did the proteinase isolated from the original host. Although the cloned gene lacked the last 130 codons, the proteinase still specifically degraded beta-casein. Deletion of the C-terminal 343 amino acids from the proteinase did not influence this specificity. Cell-free transcription-translation studies of plasmids carrying deletion derivatives of the proteinase gene showed that the 100-kilodalton C-terminally truncated proteinase still exhibited proteolytic activity. Crossed immunoelectrophoresis revealed that proteins A and B identified in the proteolytic system of S. cremoris Wg2 are both encoded by the proteinase gene. A working model based on integration of available genetic, immunological, and biochemical data is presented to explain this result.

Measurement of the humoral immune response against Streptococcus pneumoniae type 3 capsular polysaccharide and oligosaccharide containing antigens by ELISA and ELISPOT techniques.

A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.

Serum amyloid P component induction by immunomodulators.

The capacity of immunoadjuvants to enhance serum amyloid P component (SAP) levels and to modulate the humoral immune response to sheep red blood cells in a number of different mouse strains was investigated. Although the synthetic adjuvants dimethyldioctadecylammonium bromide, dextran sulphate and bacterial-derived lipopolysaccharide did not enhance SAP levels in some of the mouse strains tested, these strains responded normally to the immunomodulating effects of the adjuvants. We conclude that increased SAP levels and modulation of immune responses are induced via at least partially different pathways. For these reasons, it is impossible to screen drugs for potential adjuvant activity by only measuring SAP levels in mice.

Immunogenic properties in mice of hexasaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 3.

Hexasaccharide (HS) containing 3 U of cellobiuronic acid was isolated from Streptococcus pneumoniae type 3 capsular polysaccharide S3 and coupled to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or tetanus toxoid (TT). The immunogenicity of these HS-protein conjugates in BALB/c mice was studied by measuring the production of circulating antibodies and the induction of protective immunity to viable S. pneumoniae type 3. Immunization of BALB/c mice with 0.5 micrograms of S3 resulted in the induction of immunoglobulin M (IgM) antibodies and complete protection against 25 U of a mean lethal dose of S. pneumoniae type 3 for 19 weeks after immunization. BALB/c mice immunized with 100 micrograms of HS9-BSA (containing 12 micrograms of HS) were also protected due to circulating IgM antibodies. Repeated injections with either 100 micrograms of HS9-BSA (three immunizations) or 100 micrograms of HS6-KLH (two immunizations) resulted in high levels of circulating IgG antibodies. These HS-protein conjugates induced complete protection which lasted at least 14 (HS9-BSA), 23 (HS6-KLH), or 8 (HS16-TT) weeks after the last immunization. Protection against viable S. pneumoniae type 3 could be passively transferred to nonimmunized mice by antisera containing IgM or IgG antibodies or both. Sera containing both IgM and IgG antibodies gave better protection than sera containing only IgM antibodies. The specificity of the induced protection was confirmed by challenge with the non-cross-reacting S. pneumoniae type 11.

Impaired macrophage functions as a possible basis of immunomodification by microbial agents, tilorone and dimethyldioctadecylammonium bromide.

Four microbial and two chemically defined immunomodulating agents namely viable BCG, killed Mycobacterium butyricum, killed Lactobacillus plantarum, zymosan, tilorone, and dimethyldioctadecylammonium bromide (DDA) were studied for their effects on macrophage functions in vitro and in vivo. All agents induced a dose-dependent mortality of macrophages as determined by trypan blue exclusion. DDA and especially tilorone were rather toxic for macrophages in vitro. All agents except tilorone and DDA inhibited phagocytosis of yeast cells and uptake of acridine orange in vitro at doses which killed up to about 30% of the macrophages. DDA and tilorone had no effect at similar doses. All agents but zymosan inhibited the spreading of macrophages. No interference with the fusion of lysosomes and yeast cell-containing phagosomes could be observed. The activity of the mononuclear phagocytic system (MPS) in vivo as measured by carbon clearance was stimulated by all substances within twenty-four hours. All agents but DDA and tilorone enhanced non-specific bacterial resistance. As demonstrated previously for DDA, tilorone could serve as adjuvant for induction of specific resistance to Listeria monocytogenes. The results are discussed in relation to other data on influencing of macrophage functions and on immunomodification. It is concluded that hampered antigen destruction by local macrophage suppression attended with MPS stimulation might be a basic mechanism for adjuvanticity exerted by these agents.

Adjuvanticity of dimethyl dioctadecyl ammonium bromide in guinea pigs. I. Skin test reactions.

The effect of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the induction of cellular immunity in guinea pigs was studied. DDA, a surface-active lipid, was mixed with the antigen bovine serum albumin (BSA) or with a conjugate of BSA and dinitrophenol (DNP22--BSA) and injected into the footpads of guinea pigs. At varying intervals skin tests were performed to test the immediate and delayed hypersensitivity (DH) reactions. Optimal DH reactions to BSA were observed from 3 to 6 weeks after immunization with BSA in DDA. The hapten-specific response to DNP22--BSA had an optimum at 3 weeks and was highly specific for the homologous antigen. Histological examinations of skin test sites confirmed that the reaction was rather of the tuberculin type than of the cutaneous basophil hypersensitivity type. When guinea pigs were immunized with DNP22--BSA in Freund's complete adjuvant (FCA) a long-lasting DH to both carrier and hapten groups developed but the DH was always complicated by an Arthus reaction due to antibodies to the DNP hapten. In conclusion, DDA is superior to FCA as adjuvant for the induction of a state of pure DH in guinea pigs.

Delayed-type hypersensitivity in rabbits. Comparison of the adjuvants dimethyl dioctadecyl ammonium bromide and Freund's complete adjuvant.

Injection of rabbits with antigen mixed with the cationic surface-active lipid dimethyl dioctadecyl ammonium bromide (DDA) induced delayed-type hypersensitivity (DH), which could be measured as skin reactions and was confirmed by histology of the skin test sites. 1 week after injection of a conjugate of bovine serum albumine (BSA) with dinitrophenol (DNP30-BSAj) mixed with DDA, DH was detectable in most but not in all rabbits. Similar results were obtained using FCA as adjuvant. The animals treated with the latter adjuvant, however, produced a long-lasting DH (1-3 weeks) complicated by circulating anti-DNP antibodies (Arthus-type reactions). Skin testing with heterologous hapten-carrier complexes revealed that individual rabbits immunized with DNP30-BSA in DDA expressed DH with different reaction patterns, either to hapten, carrier or both. In conclusion, DDA is a useful adjuvant for the induction of a state of pure DH in rabbits. However, not all rabbits do respond, or respond similarly.

Effects of lactobacilli on parameters of non-specific resistance of mice.

The viability of Lactobacillus plantarum in vivo and the effects of viable and heat-killed lactobacilli on parameters of non-specific resistance were studied. After intravenous administration of 10(8) viable lactobacilli, which is a dose with optimal adjuvant activity, viable lactobacilli could be isolated from spleens for more than 1 week and from livers and lungs for more than 3 weeks. Both viable and heat-killed lactabacilli stimulated the clearance of colloidal carbon, viable bacteria stimulated initially to a higher extent. Doses of 10(8) viable and heat-killed lactobacilli, but not less, stimulated non-specific resistance to Listeria monocytogenes and caused splenomegaly. Doses as small as 10(5) viable and heat-killed lactobacilli induced substantial natural killer (NK) cell activity in the peritoneal exudate 4 days after i.p. administration. Higher doses generally caused a dose-dependent increase of NK cell activity. Viable lactobacilli injected in the paw and to a lesser extent heat-killed bacteria caused a proliferative response in the draining popliteal lymph node, which peaked at day 5. Results are discussed in relation to adjuvanticity and comparisons are made with bacterial agents already used in immunotherapy.

Adjuvant effect of nonionic block polymer surfactants in humoral and cellular immunity.

The adjuvant activities of four chemically similar, but physicochemically different nonionic surface-active agents called pluronic polyols F 68, L 31, L 101 and L 121 were studied. These four agents were tested in mice using an experimental model developed for studying the adjuvant activity of the cationic surface-active agent dimethyl dioctadecyl ammonium bromide (DDA). L 121 and DDA enhanced the primary antibody response to sheep red blood cells (SRBC) while F 68, L 31 and L 101 suppressed this response. The secondary humoral response to SRBC was enhanced by the polyol L 121 while the secondary response to dinitrophenylated bovine serum albumin (DNP22-BSA) was enhanced by both L 121 and L 101. DDA and the polyol L 101 were very effective adjuvants for induction of delayed-type hypersensitivity (DTH) to SRBC and DNP22-BSA after intracutaneous immunization of mice with a mixture of antigen and adjuvant. Since the four pluronic polyols were composed of identical chemical constituents, we proposed that difference in their activities as adjuvants were due to variation in their physicochemical properties. A correlation was found between a physicochemical parameter, the hydrophilelipophile balance (HLB), and the adjuvant activities of the pluronic polyols and several other types of nonionic surface-active agents. The agents which were strong adjuvants all had HLB values within a narrow range which classified them as spreading agents.

Influence on macrophage functions as a possible basis of immunomodification by polyanions.

The effects of the polyanions, carrageenan, dextran sulphate, polyanetholesulphonate and suramin, on macrophage functions and on the complement system were studied. The function of phagosomes and lysosomes in vitro was inhibited by all polyanions but carrageenan whereas phagocytosis in vitro was enhanced by small doses of all polyanions. Interaction with complement occurred already at very low doses polyanion. On intraperitoneal administration, the polyanions inhibited the mononuclear phagocytic system as measured with the carbon clearance test. In some instances this was followed within 72 h by a stimulation. While polyanion injections caused an increase in spleen weight, the effects on the liver weight diverged. Our results and data of other authors suggest that the immunomodifying properties of the polyanions studied can be explained partly by drug-macrophage interaction, partly by indirectly mediated modulation of macrophage function.

Regulation of the immune response by macrophages.

Regulation of the immune response by macrophages was studied with cellular resistance to Listeria monocytogenes as parameter. The use of agents which suppress macrophage activity during the induction-phase of immunity enabled the induction of protective immunity with killed listeria. Fractionation of the cell content of listeria yielded an RNA'se sensitive fraction which in a dose of 300 ng and in combination with the cationic surfactant dimethyl dioctadecyl ammonium bromide induced protective immunity against listeria.

Bacteriophage MS-2 in the immune response.

Several aspects of the immune response to bacteriophage MS-2 were studied. In thymectomized, irradiated and bone marrow-reconstituted (TXBM) mice, the response was normal when a high dose of antigen was used. With a 50-fold lower dose of MS-2, the response was impaired, indicating a T-cell involvement in antibody formation. More evidence for the (partial) T-cell dependence of MS-2 was obtained from experiments with anti-thymocyte serum or cyclophosphamide-treated mice. (3H)-thymidine incorporation experiments demonstrated that both B and T cells were active upon in vitro stimulation with MS-2. The dose of MS-2 which was able to induce a normal response in TXBM mice, proved to be optimal for both sensitization and elicitation of a DH. It is concluded that MS-2 is a thymus-dependent antigen which is only thymus independent in high doses.