A bacteriophage-associated lyase acting on Klebsiella serotype K5 capsular polysaccharide.
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Bacteriophages↗
Biomedical subjects
H Snippe
Publications and source records attributed to H Snippe.
Combinations of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response.
Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.
Adjuvants, Immunologic↗
Effect of in vivo administration of different adjuvants on the in vitro candidacidal activity of mouse peritoneal cells.
The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 degrees C was called 1 CA50 unit. CA was expressed in CA50 units per 10(6) PC. Optimal reduction of the number of viable candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1-20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 (Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldioctadecylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymorphonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.
Adjuvants, Pharmaceutic↗
Surface-associated sialic acid is an immunological adjuvant.
The influence of neuraminidase on the immunogenicity of heterologous erythrocytes as determined by serum haemagglutination titres was investigated in mice. For this study sheep and rabbit erythrocytes were selected because of their high and low N-acetylneuraminic (sialic) acid content, respectively. Preincubation with neuraminidase resulted in a ten-fold reduction of the immunogenicity of sheep erythrocytes (ShE). By contrast, the immune response to rabbit erythrocytes appeared to be resistant to sialidase treatment. Addition of the extrinsic adjuvant dimethyldioctadecylammonium bromide largely restored the immunogenicity of neuraminidase-treated ShE, but did not change the response to control-treated ShE. The maximal antibody level induced by neuraminidase-treated ShE was lower than that provoked by control ShE. These results suggest that sialic acid is both an intrinsic immunological adjuvant and an antigenic determinant of ShE. The adjuvant effect of sialic acid does not depend on complement component C3 as judged by the response of cobra venom factor-pretreated animals. In genetically C5-deficient and in nude mice, however, sialic acid showed diminished and absent adjuvant activity, respectively.
Adjuvants, Immunologic↗
Rapid detection of Semliki Forest virus replication in cell culture with peroxidase-labeled monoclonal antibodies: determination of inhibitory concentrations of monoclonal antibodies and ribavirin.
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Animals↗
Aspects of monoclonal antibody-mediated protection of mice against infection with virulent Semliki Forest virus.
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Animals↗
Enzyme immunoassay of interferon with peroxidase-labelled virus-specific monoclonal antibodies.
To quantify the antiviral effect of interferon (IFN) we applied a mixture of two horseradish peroxidase-labelled monoclonal antibodies, specific for the E1 glycoprotein of Semliki Forest virus, in a direct enzyme immunoassay. This assay is suitable for detection of virus replication in L-cells, seeded as monolayers in 96-well plates. Inhibition of absorbance values caused by IFN was determined in a Flow Titertek Multiskan. Three IFN samples from different sources were titrated simultaneously in the enzyme immunoassay and in the vesicular stomatitis virus plaque reduction test in five consecutive experiments. Titres were calculated as the inverse value of the dilution of IFN causing 25% inhibition of absorbance values and 50% reduction of plaque counts respectively. The results show equality of precision and reproducibility between and within the two assays. However, the enzyme immunoassay is more convenient and objective than the plaque reduction assay.
Animals↗
Determination of inhibitory concentrations of antiviral agents in cell culture by use of an enzyme immunoassay with virus-specific, peroxidase-labeled monoclonal antibodies.
An enzyme immunoassay (EIA) to determine 50% inhibitory concentrations of drugs which suppress Semliki Forest virus replication is described. Inhibition of virus replication was measured in L-cells, seeded as monolayers in 96-well plates by use of horseradish peroxidase-labeled monoclonal antibodies directed against the E1 glycoprotein of Semliki Forest virus. The antiviral agents tested were cycloheximide, tunicamycin, NH4Cl, and disodium cromoglycate. The 50% inhibitory concentration of these antiviral agents was arbitrarily defined as the concentration of drug, in culture medium, associated with 50% reduction of the control absorbance value measured on Semliki Forest virus-infected cells without drug in the culture fluid. Twenty-two hours after infection the 50% inhibitory concentrations of the drugs were 0.2 microgram/ml for cycloheximide, 0.8 microgram/ml for tunicamycin, 0.3 mg/ml for NH4Cl, and 4.9 mg/ml for disodium cromoglycate. These values are similar to those determined by others with conventional methods of virus quantification. This test is sensitive and easy to perform and therefore is suited for large-scale experiments.
Ammonium Chloride↗
Immunogenic properties of octasaccharide-protein conjugates derived from Klebsiella serotype 11 capsular polysaccharide.
The tetrasaccharide repeating unit of the capsular polysaccharide of Klebsiella serotype 11, K11PS, comprises the following sequence: [----3)-beta-D-GlcpA-(1----3)-alpha-D-Galp-(1----3)-beta-D-Glcp-(1 ----] with a 4,6-O-(1-carboxyethylidene)-alpha-D-galactopyranosyl residue linked to O-4 of the glucuronic acid residue. Octasaccharide (OS) derived from K11PS by bacteriophage phi 11-associated glycanase, was coupled to bovine serum albumin and to keyhole limpet hemocyanin. The immunogenicity of various antigens after intraperitoneal immunization was studied by measuring the levels of circulating antibodies. Injection of BALB/c mice with K11PS resulted in induction of 2-mercaptoethanol-sensitive immunoglobulin M antibodies. The responses observed in BALB/c nu/nu mice and in male (CBA/N X C3H/HeN)F1 mice indicate that K11PS is a thymus-independent type 2 antigen. Immunization of BALB/c mice with either OS-bovine serum albumin or OS-keyhole limpet hemocyanin resulted in the induction of circulating 2-mercaptoethanol-resistant immunoglobulin G antibodies. Results in BALB/c nu/nu mice indicate that the OS-protein conjugates are thymus-dependent antigens. Since the OS-keyhole limpet hemocyanin conjugate induced antibodies in both (CBA/N X C3H/HeN)F1 females and males, we propose to refer to this kind of antigen as a thymus-dependent type 1 antigen, whereas OS-bovine serum albumin, which evoked immunoglobulins in (CBA/N X C3H/HeN)F1 females only, can be referred to as a thymus-dependent type 2 antigen.
Animals↗
Mechanisms of monoclonal antibody-mediated protection against virulent Semliki Forest virus.
Both neutralizing and nonneutralizing immunoglobulin G2a monoclonal antibodies (MAs) directed against the E2 glycoprotein of Semliki Forest virus (SFV) protected mice prophylactically and therapeutically against virulent SFV infection. The neutralizing MAs, however, conferred protection to mice at lower doses than did nonneutralizing MAs. The antibody-dependent, complement-mediated cytolysis of SFV-infected L cells was effectuated by both kinds of antibodies, but again neutralizing MAs were more effective. Removal of the Fc part of the neutralizing MA UM 5.1 by pepsin digestion resulted in a 100-fold reduction of the neutralization titer (10(4) versus 10(6)) and a complete loss of its capacity to mediate antibody-dependent, complement-mediated cytolysis. Passive protection of infected mice occurred only after administration of relatively high doses of F(ab')2 of MA UM 5.1 (30.0 micrograms versus 0.1 microgram). F(ab')2 fragments prepared from the nonneutralizing MA UM 4.2 had lost their protective capacity completely. Surprisingly, the nonneutralizing MA UM 4.2 retarded virus growth in mouse fibroblasts (L cells), although inhibition was at much higher doses than with the neutralizing MA UM 5.1. Furthermore, both MAs promoted the uptake of virulent SFV in the Fc receptor-bearing WEHI-3 cells. The results suggest that nonneutralizing MAs protect mice not only by antibody-dependent, complement-mediated cytolysis but also by growth inhibition and enhanced uptake of SFV in the nonpermissive macrophages of BALB/c mice. This hypothesis is supported by the absence of viremia in recipients of nonneutralizing MA UM 4.2 at 24 h after infection.
Animals↗
Immunomodulating properties of two synthetic adjuvants: dependence upon type of antigen, dose, and time of administration.
The effects of two synthetic adjuvants on the antibody response to sheep red blood cells (SRBC) as a thymus dependent (TD) antigen and to dinitrophenyl59-Ficoll as a thymus-independent (TI-1) antigen were investigated in mice. Both dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) augmented the humoral response to SRBC but not to dinitrophenyl59-Ficoll if injected simultaneously with antigen. Dose-response curves of both antigen and adjuvant revealed that DXS compared to DDA is a more effective adjuvant for the induction of a humoral response to SRBC. Intraperitoneal injection of DDA or DXS evoked a sequence of distinct immune responsive states in mice, measured by the capacity to develop an anti-SRBC response. A short immune-potentiating period (less than 6 hr) is followed by a suppressive, second immune-potentiating state. The immune suppressive state lasted for a period of about 8 days and was restricted to TD-antigens. Suppression could be totally overridden by injection of DDA or DXS simultaneously with antigen, suggesting that the suppressive state was reversible. The kinetics of the observed alteration of the immune response by DDA and DXS were very similar. It is concluded that differences in the modulation of the immune response by DDA and DXS are limited to the initial state. Long-term effects like the induction of a succession of distinct immune responsive states, are more or less similar for both adjuvants. Possible mechanisms by which these immunomodulators interfere with the immune system are discussed.
Adjuvants, Immunologic↗
Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.
Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes.
Animals↗
Identification of distinct antigenic determinants on Semliki Forest virus by using monoclonal antibodies with different antiviral activities.
Fifteen monoclonal antibodies (MAs) directed against either the E1 or E2 glycoprotein of Semliki Forest virus (SFV) were characterized by immunoglobulin subclass, pI traject, hemagglutination inhibition, neutralization of infectious virus, and protection against virulent infection in mice. All MAs except UM8.4 (immunoglobulin M [IgM]) belonged to various subclasses of IgG and predominantly to IgG2a, but all were unique as indicated by their banding patterns in isoelectric focusing. Competitive binding assays with these MAs revealed the presence of at least six distinct antigenic determinants (epitopes) on the E1 glycoprotein and five epitopes on the E2 glycoprotein. Two of the epitopes on E1, as defined by the properties of the MAs, were associated with hemagglutination inhibition (E1c and E1d), three were associated with neutralization (E1a, E1b, and E1f), and five were associated in various degrees with protection (E1a, E1b, E1c, E1e, and E1f) of mice against virulent SFV infection. With the MAs against E2, the epitopes on E2 were similarly defined. Epitopes E2b and E2e were associated with hemagglutination inhibition, E2c and E2d were associated with neutralization, and three epitopes were associated with in vivo protection (E2a, E2c, and E2d). Furthermore, for each MA the relative avidity to purified SFV was determined with an enzyme-linked immunosorbent assay. The binding of some MAs to purified SFV was enhanced by a second MA. The relative avidities of individual MAs did not correlate with their neutralizing capacities. From the results, we suggest that the amino acid sequence which makes up determinant E2d and is recognized by the highly protective MA UM5.1 is an excellent candidate for the production of a synthetic vaccine.
Animals↗
Delayed type hypersensitivity against semliki forest virus in mice: local transfer of delayed type hypersensitivity with thioglycollate-induced peritoneal exudate cells.
In mice, a strong delayed type hypersensitivity (DH) without detectable neutralizing antibodies in serum could be obtained after intracutaneous injection of inactivated Semliki Forest virus (SFV) mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). Thioglycollate-induced peritoneal exudate cells (PEC) from these mice were highly effective in passive transfer of DH against SFV locally in footpads of naive recipient mice. DH reactions were measured with a footpad swelling test. By contrast, either immune PEC from nonstimulated peritoneal cavities or thioglycollate-induced PEC from mice which had developed neutralizing antibodies were unable to transfer DH passively.
Animals↗
The effect of liposomal charge on the neutralizing antibody response against inactivated encephalomyocarditis and Semliki Forest viruses.
The primary neutralizing antibody response to encephalomyocarditis (EMC) virus and Semliki Forest virus (SFV) is enhanced by addition of either negatively or positively charged liposomes. The purified u.v. light inactivated viruses were merely mixed with liposomes and injected intraperitoneally (i.p.) into mice. In contrast, neutral liposomes were unable to enhance the primary response to these viruses. Furthermore primary immunization with inactivated SFV mixed with either neutral, positively or negatively charged liposomes was associated with an enhancement of the secondary humoral response after i.p. booster injection of mice with inactivated virus alone. But neutral liposomes seemed to be less effective in this respect than either positively or negatively charged liposomes.
Animals↗
Hapten-specific B cell blockade of the immune response to a thymus-independent-1 antigen produced by concomitant administration of a thymus-independent-2 antigen.
CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N X C3H/HeN F1 male greater than CBA/N female greater than CBA/N X C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate that T cell participation is not essential for the blockade of CBA/N PFC responses and they suggest that direct blockade of TI- and TD-responsive B cell populations is likely to occur.
Animals↗
Delayed-type hypersensitivity in mice after infection with avirulent Semliki Forest virus. Brief report.
After subcutaneous infection of mice with Semliki Forest virus, a delayed-type hypersensitivity (DH) could be demonstrated by footpad swelling. Pretreatment with cyclophosphamide resulted in enhanced DH if neutralizing antibodies were undetectable in serum.
Animals↗
Effect of the adjuvant dimethyl dioctadecyl ammonium bromide on the humoral and cellular immune responses to encephalomyocarditis virus.
The effects of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the immune responses to encephalomyocarditis (EMC) virus were studied in mice. The humoral response, as measured by appearance of neutralizing antibodies, was slightly enhanced in mice immunized by the intraperitoneal route. Intracutaneously, DDA almost did not affect the humoral response but resulted in distinct enhancement of delayed type hypersensitivity (DH), as measured by the footpad swelling test. DH to EMC virus was found to be antigen-specific and could be passively transferred to normal mice with peritoneal exudate cells from immunized mice. Dose-response curves for DH and humoral antibody responses to EMC virus were not concordant. Low doses induced DH on day 6 without measurable circulating antibodies; high doses gave good antibody responses but suboptimal DH reactions. Immunization conferred a state of resistance to infection with virulent EMC virus. Protection seemed more related to DH than to the prevalence of specific antibodies at the time of infection.
Adjuvants, Immunologic↗