The effects of purified pertussis components and lipopolysaccharide on the results of the mouse weight gain test

The effects of highly purified components of Bordetella pertussis, that is pertussis toxin (PT) and filamentous haemagglutinin (FHA), and of lipopolysaccharide (LPS) were studied in the active mouse weight gain test (MWGT). The PT when given alone or with other components in various combinations caused weight losses and deaths 2-3 days after inoculation but FHA was not toxic in the MWGT. When FHA was given with PT, the toxic effect of PT was reduced. The LPS caused weight losses at 24 h which decreased when LPS was given with PT. The toxic effects of PT as indicated by late deaths and late weight losses or failure to gain weight continued until 14 days after inoculation. The various components had similar effects on mouse weight gain in both LACA and NIH strains of mice. The doses of PT used in the MWGT caused marked leucocytosis but FHA and LPS did not. No agglutinins appeared in the sera of mice inoculated with various purified components. The components were thus pure and did not contain agglutinogens.     

The effects of purified components of Bordetella pertussis in the weight gain test for the toxicity testing of pertussis vaccines

The effects of highly purified preparations of three Bordetella pertussis components--pertussis toxin (PT), lipopolysaccharide (LPS) and filamentous haemagglutinin (FHA)--were examined in the mouse weight gain test, a toxicity test for pertussis vaccine. When these components were administered alone, PT enhanced initial weight gains of the mice, LPS produced an initial weight loss and FHA had no detectable effect on the weights of the mice. However, testing the components in combinations revealed that the effect of PT and LPS together was not simply the sum of their individual effects. This combination generally produced lower weights than LPS alone, particularly in the later stages of the test.     

Trypanosoma cruzi: involvement of IgG isotypes in the parasitemia control of mice immunized with parasite exoantigens of isoelectric point 4.5.

In a previous work we demonstrated that Trypanosoma cruzi exoantigens of pI 4.5 (Ea 4.5), whose most important epitopes are glucidic, are able to induce a partially protective immune response in mice. To ascertain the involvement of antibody isotypes in this protection, we immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The analysis of immune response by skin test revealed the occurrence of specific immediate type hypersensitivity on Day 15 after the last immunization. By ELISA and using Ea 4.5 as antigen, specific IgG1 antibody was detected. When formaldehyde-fixed epimastigotes were used as antigen, binding of IgG1 and IgG2 was observed. Trypomastigotes incubated for 1 hr at 33 degrees C with the immune sera and then injected in normal syngeneic mice produced a significantly lower parasitemia than trypomastigotes incubated with the control sera. This capacity of anti-Ea 4.5 sera was resistant to 56 degrees C for 2 hr and was diminished after the absorption of immune sera with the carbohydrate moiety of Ea 4.5. The assay with the immune IgG1 and IgG2, separated through protein A-Sepharose affinity chromatography, showed that IgG1 retains most of this capacity. Purified immune IgG1 revealed two antigenic bands of molecular weight between 50 and 55 kDa in SDS-PAGE of Ea 4.5.     

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough

The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.     

Bordetella pertussis filamentous hemagglutinin delivered by mucosal routes enhances immunoglobulin levels in serum and mucosal fluids

Free Bordetella pertussis filamentous hemagglutinin (FHA) can act as an adjuvant for mucosally administrated antigens. Here, we show that independently of the adjuvant properties of FHA toward an unrelated antigen, total IgG or IgA concentrations in serum and mucosal fluids are enhanced by the administration of FHA. Oral administration of FHA increases both total IgG concentrations in serum and total immunoglobulin concentrations in intestinal lavages. Nasal administration of FHA increases total IgA concentrations in broncho-alveolar lavages. FHA induces Langerhans cell recruitment and MIP-3alpha mRNA expression within hours after administration. These observations shed a new light on the potential molecular mechanisms of FHA-induced adjuvanticity.     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Interaction of Bordetella pertussis virulence components with neutrophils: effect on chemiluminescence induced by a chemotactic peptide and by intact bacteria

The effect of secreted virulence components of Bordetella pertussis on chemiluminescence (CL) of rabbit peritoneal neutrophils was determined with the chemotactic peptide N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or intact B. pertussis as the stimulus. Pertussis toxin (PT) inhibited the response to fMLP in a dose-dependent manner, although only after the neutrophils had been exposed to the toxin for greater than 15 min. Both filamentous haemagglutinin (FHA) and lipopolysaccharide (LPS) markedly enhanced the CL response to fMLP after greater than or equal to 15 min incubation with the neutrophils. Similar effects to those of B. pertussis LPS were also seen with smooth and rough LPS from Salmonella minnesota. With the lowest dose of each component which elicited a maximal effect on CL, the inhibitory effect of PT overrode the enhancing effect of FHA and B. pertussis LPS. Pre-incubation of neutrophils with PT, FHA or B. pertussis LPS caused a slight reduction in the subsequent CL response to virulent B. pertussis Tohama. Virulent (phase I, or X-mode) organisms of B. pertussis 18334 and B. pertussis Tohama induced greater neutrophil CL than their avirulent (C-mode) derivatives. There appeared to be an inverse correlation between bacterial hydrophilicity and the ability to induce neutrophil CL: X-mode bacteria were significantly less hydrophilic than C-mode organisms. Three mutants, the adenylate cyclase (AC)- and haemolysin (HLY)-deficient B. pertussis BP348, the FHA-deficient B. pertussis BP353, and the PT-deficient B. pertussis BP357, generated similar levels of CL and had similar hydrophilicity values. The hydrophilicity value of the avirulent mutant B. pertussis BP347 (deficient in AC, HLY, FHA and PT) and the CL induced by this strain were similar to those of B. pertussis C-mode organisms. Thus, the interaction of B. pertussis with neutrophils appears to be complex, reflecting both the alteration of leucocyte function by secreted virulence components of the organism and, in the absence of opsonins, the surface properties of the bacterium.     

Adjuvant and immunogenic properties of bacterial lipopolysaccharide in IgE and IgG1 antibody formation in mice

The ability of Gram-negative lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in IgE and IgG₁ immune responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG₁ under a variety of experimental conditions. Both isolated LPS and whole heat-killed bacteria were capable of enhancing IgE and IgG₁ antibody formation to a protein antigen, egg albumin (EA). The LPS-induced anti-EA, IgE, and IgG₁ antibody titers exhibited a cycling phenomenon with time. In the presence of LPS, IgE, and IgG₁ antibodies specific for EA did not occur in athymic nude (BALB/c-nu/nu) mice, demonstrating the inability of LPS to substitute for the stringent requirement for T cells in homocytotropic antibody formation.     

Purification and Immunogenicity of a Recombinant Bordetella pertussis S1S3FHA Fusion Protein Expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.     

Effect of myeloma IgE injections on passive and active cutaneous anaphylaxis in rats

The ability of injected rat IgE myeloma protein IR162 to inhibit passive and active cutaneous anaphylaxis in Lewis rats was investigated. IgE injected i.p. 24 hr before the sensitization with IgE anti-ovalbumin (OVA) completely inhibited both IgE- and IgG2a-induced passive cutaneous anaphylactic (PCA) reactions at a dose (2.5 mg/100 g body weight) that resulted in peak serum concentrations of 150 micrograms IgE IR162/ml. Peak IgE IR162 serum concentrations of 20 to 60 micrograms/ml inhibited the PCA reaction in approximately 50% of the rats. Intracutaneous injection of a mixture of myeloma IgE and anti-OVA IgE in a ratio of 100:1 or more also inhibited the PCA reaction. In contrast, the PCA reaction was not inhibited by seven daily doses of IgE beginning 24 hr after passive sensitization. Likewise, the cutaneous anaphylactic reaction elicited in rats 14 days after immunization with OVA and Bordetella pertussis was not prevented by daily injections of myeloma IgE despite a 1000- to 3000-fold excess of the myeloma IgE to anti-OVA IgE serum concentration. The data demonstrate that parenteral administration of myeloma IgE inhibits the PCA reaction only when given before passive sensitization and does not prevent cutaneous anaphylaxis in actively immunized rats. Because myeloma IgE failed to inhibit anaphylactic reactions in actively immunized rats, it is questionable whether administering human IgE-derived synthetic peptides or recombinant DNA-produced IgE fragments will be able to prevent allergic diseases by blocking the IgE Fc receptors on mast cells.     

Mice are protected against Bordetella pertussis infection by intra-nasal immunization with filamentous haemagglutinin

Abstract Intra-nasal immunization of mice with purified Bordetella pertussis filamentous haemagglutinin (FHA) or a crude cell sonicate was shown to protect against subsequent B. pertussis aerosol challenge. Immunization with FHA was found to be the most effective and resulted in complete clearance of the bacterial infection from the lungs within 14 days. Serum IgG and lung IgA anti-FHA antibodies were detectable within 4 weeks of the first immunization and anamnestic responses were seen following secondary immunization and subsequent challenge with B. pertussis . Nasal administration of pertussis is a route which induces good systemic serum, as well as local secretory, antibody responses.     

Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera

Abstract To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli α-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with α-hemolysin.     

Humoral response to the injection of acellular Pertussis vaccine

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.     

The polyclonal and antigen-specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant

BALB/c mice were injected ip with 1 microgram ovalbumin (OVA) in alum or complete Freund's adjuvant (cFA) and the changes of the IgE and IgG subclass serum levels and isotypes of the anti-OVA specific antibodies determined by radioimmunoassays. By Day 10, OVA in alum had induced a 5- to 10-fold increase of the IgE serum level and an initial decrease of the IgG subclass levels which subsequently increased to two to threefold over the preinjection level. OVA in cFA induced a gradual twofold increase of the IgE serum level, a rapid fourfold increase of the IgG2a level occurring by Day 7, and a gradual two to threefold increase of the other IgG subclasses. Over 90% of the anti-OVA antibodies were of the IgGl isotype with both adjuvants; OVA in alum induced slightly more IgGl anti-OVA antibodies than cFA. In contrast, the OVA in alum injected mice formed significantly more (5- to 10-fold) IgE anti-OVA antibodies than the cFA-injected mice. OVA in alum also induced a large nonspecific increase of the IgE serum level because only approximately 40% of the increase observed on Day 14 was absorbable with OVA, whereas approximately 90% the IgE increase in cFA injected mice was absorbable with OVA. The data demonstrate that mice form mainly IgGl and IgE antibodies to OVA irrespective of the adjuvant. The low specific and lack of nonspecific IgE formation by mice injected with OVA in cFA may be the result of cFA-induced interferon-gamma (IFN-gamma) production because IFN-gamma has been shown to stimulate IgG2a and inhibit IgE secretion in vitro.     

Strong adjuvant action of Klebsiella O3 lipopolysaccharide and its inhibition of systemic anaphylaxis

Abstract Immunization with lipopolysaccharide from Klebsiella O3 as an immunological adjuvant did not cause the death of mice in systemic anaphylaxis to bovine serum albumin. On the other hand, most mice immunized with lipopolysaccharide from Escherichia coli O111, Klebsiella O4 and Salmonella minnesota did die. Klebsiella O3 lipopolysaccharide enhanced IgM and IgG antibody response to BSA more markedly than Escherichia coli O111 lipopolysaccharide, while it affected the production of IgE antibody only slightly. Therefore, it is suggested that the inhibition of systemic anaphylaxis by Klebsiella O3 lipopolysaccharide adjuvant might be related to its strong adjuvant action on IgM and IgG class antibody production, and that high levels of circulating IgM and IgG antibodies might act as blocking antibodies in the development of IgE-mediated systemic anaphylaxis.     

Humoral imunity to pertussis in children with diabetes of type 1

To determine the state of humoral immunity to pertussis in children with insulin-dependent diabetes, IgG antibodies to pertussis toxin (PT) were determined in blood serum samples by means of EIA. In a group of children aged up to 6 years the highest percentage (100%) received the complete course of vaccination against pertussis with Russian adsorbed DPT vaccine, containing whole-cell pertussis monovaccine, while in a group over 6 years the complete vaccination course (3 vaccinations and 1 revaccination) had 53.4% of children. Pertussis morbidity was considerably higher in nonvaccinated subjects than in children with 4-fold vaccination (p < 0.001). The coefficient of association (Q) was 0.84. Children of all age groups were found to have low and average titers of antibodies to PT. The regressive analysis showed a decrease in antibodies in persons completely immunized against pertussis by the age of 6 years old. The presence of antibodies in nonimmunized persons showed that cases of pertussis or carrier state took place among the population. High titers of antibodies, indicative of recent cases of pertussis, were registered in all age groups, but high titers of antibodies were registered mostly in the group of children over 13 years old (p < 0.05), which confirmed an increase in pertussis morbidity in adolescents. Thus, vaccination against pertussis effectively protected children with diabetes of type 1, aged up to 6 years. For more prolonged protection the vaccination and revaccination of children aged over 4 years old is necessary.     

Production and properties of reaginic antibodies in rabbits infected with Clonorchis sinensis or Schistosoma japonicum

The production of reaginic antibodies detected by homologous passive cutaneous anaphylaxis (PCA) was demonstrated in all rabbits experimentally infected with either Clonorchis sinensis or Schistosoma japonicum. The antibodies appeared in the sera as early as 3 weeks after exposure and persisted with relatively high titers for at least 7 weeks in some animals. The antisera of rabbits infected with C. sinensis were found to be cross reactive against heterologous trematode antigens, although PCA titers were less than 3% of the titer by the homologous antigen; no cross reaction was observed between S. japonicum antiserum and the heterologous antigens. PCA activity of the antisera was completely destroyed in some samples by heat treatment at 56 C for 2 hr, but partially in the others even after heating for 6 hr. However, the physicochemical properties of these antibodies were analogous to human IgE; the PCA activity was eluted with 0.035 M phosphate buffer from a DEAE-cellulose column and recovered in the ascending portion of the IgG peak by Sephadex G-200 gel filtration. PCA activity was found in a β region in preparative agar electrophoresis.     

Cell-mediated and humoral immunity in mice: cross reaction between lysozyme and S-carboxymethylated lysozyme studied by a modified footpad test.

The mouse sensitized by subcutaneous (sc) injection of lysozyme in emulsion of Freund's complete adjuvant (FCA) was shown by a modified footpad test to develop three kinds of hypersensitivities. Injecting lysozyme in 2.5-mul emulsion of Freund's incomplete adjuvant (FIA) into the footpad elicited strong footpad swelling in 30 min (anaphylactic reaction), in 3 hr (Arthus-type reaction) and in 24 hr (delayed-type hypersensitivity; DTH). The mice showing anaphylactic reaction in the footpad test manifested severe active systemic anaphylaxis, and the sera of these animals showed high IgG1 antibody titers with only sparingly detectable or no IgE antibody titers. In the sensitizing system with the use of FCA, the antigenicity of S-carboxymethylated lysozyme (CM-lysozyme) devoid of the three-dimensional conformation of lysozyme was compared with that of the native molecule. CM-lysozyme and lysozyme completely cross-reacted to each other in DTH, but not at all in the anaphylactic or Arthus-type reaction or in IgG1 antibody production. CM-lysozyme was shown also to have the ability to bestow immunological memory for the induction of humoral immunity against lysozyme; intravenous (iv) injection of lysozyme in saline or sc injection of CM-lysozyme-FCA alone failed to induce immediate hypersensitivities and IgG1 antibody production against lysozyme, but pre-sensitization by sc injection of CM-lysozyme-FCA enabled the animal to induce these responses to significant levels when iv injection of lysozyme in saline was given as a booster.     

一株减毒百日咳鲍特菌的构建及生物学特性分析

百日咳是传染性强、感染率高的急性呼吸道传染病,主要感染婴幼儿,是婴儿死亡的主要原因之一。百日咳鲍特菌（Bordetella pertussis）是引起百日咳的最主要病原菌。近年来世界各地多次出现百日咳暴发,迫切需研制更加有效的新型百日咳疫苗。本研究构建了一株减毒百日咳活疫苗BPTM1,利用同源重组方法敲除编码百日咳鲍特菌主要毒力因子百日咳毒素（pertussis toxin,PTX）和皮肤坏死毒素（dermonecrotic toxin,DNT）的基因,并用大肠埃希菌的同源基因置换了负责气管细胞毒素（tracheal cytotoxin,TCT）转运的基因ampG。通过聚合酶链反应验证了毒素及相关基因的敲除和置换,蛋白免疫印迹法检测表明PTX的S1亚基未表达。体外生长曲线和体内定植曲线均表明,相比于野生型百日咳鲍特菌BPMM,减毒BPTM1的生长和定植能力未受影响,其所致肺部病理效应减轻,而所诱导的百日咳鲍特菌特异性IgG、IgG1、IgG2a抗体保持高水平。本研究表明,减毒百日咳鲍特菌BPTM1有可能成为百日咳疫苗的候选疫苗。