The antibacterial effect of Bordetella pertussis antisera

Antisera to Bordetella pertussis are bactericidal for some strains of B. pertussis in the presence of complement and lysozyme. Phase I bacilli, virulent for mice by the intracerebral and intranasal routes, are sensitive to the bactericidal effect; most mouse avirulent strains are not.

Most of the bactericidal antisera belong to one of two types according to the concentration of antibody and complement that are optimally lethal. Type 1 antisera are bactericidal in the range 1/30 to 1/900 with guinea-pig serum as the complement source at 1/18 to 1/100. Type 2 antisera are characterized by a zone without bactericidal activity over similar antiserum and complement dilutions. Their bactericidal activity is made evident by diluting the antiserum further, by increasing the amount of complement, or by adding tissue homogenates, body fluids or crystalline lysozyme. The two types are further characterized by the differing requirements of the bactericidal system for the individual components of haemolytic complement.

The combined lethal action of antibody, complement and lysozyme is rapid. In the early stages it is partly inhibited by substances in the medium used for estimating viable counts indicating that in its initial stages the reaction is bacteriostatic rather than bactericidal.

     

Biological activities of crystalline pertussigen from Bordetella pertussis.

We studied various biological activities of crystalline pertussigen and found that in mice as little as 0.5 ng of pertussigen induced hypersensitivity to histamine, 8 to 40 ng induced leukocytosis, 2 ng increased production of insulin, 0.1 ng increased production of immunoglobulin E and immunoglobulin G1 antibodies to hen egg albumin, 9.5 ng increased susceptibility to anaphylactic shock, and 0.5 ng increased the vascular permeability of striated muscle. We also found that in Lewis rats 20 ng of pertussigen promoted the induction of hyperacute experimental allergic encephalomyelitis. Pertussigen given intraperitoneally was toxic to mice at a dose of 546 ng. Treatment of pertussigen with glutaraldehyde eliminated this toxicity. Mice immunized with 1,700 ng of detoxified pertussigen were protected against intracerebral challenge with 3 x 10(4) viable Bordetella pertussis cells. When as little as 0.5 ng of pertussigen was given intravenously to mice, the increased susceptibility of the animals to histamine could still be detected 84 days later. The biological properties of crystalline pertussigen indicate its similarity to leukocytosis-promoting factor, Islet-activating protein, late-appearing toxic factor, and mouse-protective antigen of B. pertussis.     

Mouse-protecting and histamine-sensitizing activities of pertussigen and fimbrial hemagglutinin from Bordetella pertussis.

We compared the protective activities of fimbrial hemagglutinin (FHA) and pertussigen and their respective antibodies in mice infected intracerebrally with Bordetella pertussis. We found that mice were protected by a 1.7-microgram/mouse dose of pertussigen which was free of detectable FHA and was detoxified by treatment with glutaraldehyde. A pertussigen preparation made from cells grown in shake cultures that did not contain demonstrable FHA protected mice at a dose of 1.4 microgram/mouse. FHA at a dose of 10 microgram/mouse protected mice from intracerebral infection, but it also sensitized mice to histamine at a dose of 2 micrograms/mouse, which indicated that it was contaminated with pertussigen. When FHA was obtained free of demonstrable pertussigen, it failed to sensitize mice to histamine at a dose of 30 micrograms/mouse and to protect mice from infection at a dose of 12 micrograms/mouse (largest doses tested). Passive protection tests with antisera known to contain antibodies to pertussigen protected mice from intracerebral infection, whereas sera lacking anti-pertussigen antibodies but containing high concentrations of anti-FHA antibodies did not protect mice. The most important antigen for the immunization of mice against intracerebral infection with B. pertussis appears to be pertussigen.     

Serum antibody responses to the outer membrane proteins of Bordetella pertussis

The serum antibody responses to the outer membrane proteins, purified filamentous hemagglutinin, and leukocytosis-promoting factor of Bordetella pertussis were examined in mice and children immunized with pertussis whole-cell vaccine. It was found that, although there were many similarities in the responses of mice and children, there were important differences. Sera from vaccinated mice reacted strongly with purified filamentous hemagglutinin and gave weak or undetectable responses to the components of purified leukocytosis-promoting factor. The converse was found with sera from vaccinated children. Antibodies to leukocytosis-promoting factor may thus be of importance in protecting children against pertussis, although they appear to play no role in the active mouse protection test for vaccine efficacy. These results cast doubt on the value of the mouse as an animal model for the potency testing of extracted acellular pertussis vaccines.     

Characterization of the antibodies responsible for the `bactericidal activity patterns' of antisera to Bordetella pertussis

Antisera to fractions of Bordetella pertussis have been investigated to determine the nature of the antibodies responsible for bactericidal activity and the cause of prozoning observed with some (type 2) sera. Bactericidal activity in both type 1 and type 2 sera tends to be correlated with the titre of agglutinins, anti-haemagglutinin and intracerebral protective antibody; it is not constantly associated with any other function. It is almost certainly not a property of antitoxin, antibody to histamine sensitizing factor or intranasal protective antibody.

The inhibition zone of type 2 antisera appears to be due to an antibody present in the crude β-globulin fraction of antiserum. The antigen stimulating this antibody is probably a protein but is not the normal agglutinin, haemagglutinin or the histamine sensitizing factor.

     

Serum IgA antibody to Bordetella pertussis as an indicator of infection

The levels of pertussis-specific IgA antibodies in sera from vaccinees and from children with Bordetella pertussis infection were compared by an enzyme-linked immunosorbent assay (ELISA). Serum IgA antibodies were produced only after natural contact with the pathogen and, therefore, their presence can be used as an indicator of infection. However, in view of the relatively long interval between infection and the appearance of antibodies, and the prolonged antibody response, their presence cannot be used as proof of recent infection. The finding of these antibodies in a high percentage of the normal adult population may indicate a constant circulation of B. pertussis without symptoms of disease.     

Proinflammatory and proapoptotic activities associated with Bordetella pertussis filamentous hemagglutinin

Filamentous hemagglutinin (FHA) is a dominant cell surface-associated Bordetella pertussis adhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other activities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-type B. pertussis resulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-alpha), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-alpha monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-alpha receptor 1 inhibited FHA-associated apoptosis by 47.7% (P = 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in the B. pertussis-host interaction.     

Characterization of antibody inhibiting adherence of Bordetella pertussis to human respiratory epithelial cells

We have recently established the topographic specificity of the adherence of Bordetella pertussis to human ciliated respiratory epithelial cells. For this study, we employed the same quantitative, immunofluorescent adherence assay to test the possibility that sera of patients recovering from naturally acquired whooping cough or immunized with pertussis vaccine may contain activity capable of interfering with this specific adherence. Evaluation of paired sera from six children with culture-proven pertussis demonstrated that antiadherence activity appeared in serum during convalescence from disease. Nine children immunized with diptheria-pertussin-tetanus vaccine also showed activity against adherence, although it was significantly less than in those with clinical disease. Naturally acquired serum antiadherence activity was identified in both immunoglobulin G (IgG) and IgA antibody classes, whereas, as expected, only IgG antibody was present in children receiving the parenteral vaccine. The findings suggest that natural infection or vaccination are associated with the acquisition of serum activity inhibiting the adherence of B. pertussis to ciliated cells. Immunization may fail to elicit IgA antiadherence activity.     

Preferential enhancement of IgE antibody formation by Bordetella pertussis.

Preferential enhancement of IgE antibody response was observed in BALF/c mice by the administration of Bordetella pertussis with antigen (DNP-Salmonella). Correlation between B cell mitogenic activity and adjuvant action among B. pertussis, Salmonella, lipopolysaccharide of Escherichia coli and Ficoll was examined but was not found. Thymus-derived cells seemed necessary to develop adjuvant action of B. pertussis since antibody response in athymic nude mice was not influenced by B. pertussis. Helper function of adoptively transfered spleen cells was enhanced by immunization of the donor mice with carrier antigen in the presence of B. pertussis. The magnitude of enhancement was greatest in IgE class. The results indicated that preferential enhancement of IgE antibody formation by B. pertussis is mediated by the augmentation of carrier-specific helper function.     

Adjuvant for IgE antibody and islet-activating protein in Bordetella pertussis.

A new purified protein, with a molecular weight of 77,000, isolated from the supernatant of Bordetella pertussis culture fluid and called islet-activating protein (IAP), had adjuvant activity for IgE antibody in addition to leukocytosis-promoting, histamine-sensitizing and islet-activating activities. None of the three subunits (F1, F2 and F3) was biologically active by itself. All of the four activities were recovered in the complexes of subunits F1.3 and F2.3. These results suggest that the activities are brought about by a common factor inherent in B. pertussis.     

Crystallization of pertussigen from Bordetella pertussis.

A method is described for crystallizing pertussigen from Bordetella pertussis. The crystalline material induced histamine hypersensitivity in mice at a dose of 0.5 ng of protein and leukocytosis at a dose of 100 ng and was toxic at a dose of 429 microgram. The histamine-sensitizing activity and the toxicity were as high as ever reported.     

Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase.

The Bordetella pertussis cyaA gene encodes a virulence factor which is a bifunctional protein exhibiting calmodulin-sensitive adenylate cyclase and hemolytic activities (P. Glaser, H. Sakamoto, J. Bellahov, A. Ullmann, and A. Danchin, EMBO J. 7:3997-4004, 1988). We characterized the hemolytic and toxin activities of the 200-kilodalton (kDa) bifunctional (CyaA) protein and showed that, whether cell associated or secreted, the 200-kDa CyaA protein carries hemolytic and toxin functions. The catalytically active 45-kDa form of adenylate cyclase released by proteolytic digestion of the 200-kDa CyaA protein displayed neither hemolytic nor toxin activities. We constructed in-phase deletions in the 3' region of the cyaA gene, which presumably carries the hemolytic determinant, and showed that the resulting proteins exhibited wild-type adenylate cyclase activity and were secreted without processing into culture supernatants. The hemolytic activities of these mutant CyaA proteins were severely reduced, and their toxin activities were abolished. These results suggest that the structural integrity of the 200-kDa CyaA protein is necessary for toxin activity and that distinct structural determinants within the CyaA protein are involved in secretion, pore formation, and entry into target cells.     

Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase.

Bordetella pertussis organisms secrete adenylate cyclase, at least one form of which can invade host cells and appears to be a virulence factor. Treatment of urea extracts containing invasive cyclase of B. pertussis with trypsin, chymotrypsin, or subtilisin abolishes the ability to increase intracellular cyclic AMP levels in CHO cells (invasiveness) at concentrations that have minimal or no effects on adenylate cyclase activity. Higher protease concentrations can inhibit catalytic activity, and 1 microM calmodulin protects this catalytic activity, but not invasiveness, against proteolytic inhibition. Rabbit immunoglobulin G (IgG) fractions from antisera prepared against urea extracts inhibited invasiveness at 10-fold-lower concentrations than inhibited catalytic activity. One IgG from a rabbit immunized against a partially purified, noninvasive form of the B. pertussis adenylate cyclase inhibited catalytic activity but was ineffective against invasiveness. We conclude that these two properties of the adenylate cyclase are independent functions that reside on different domains of the same protein or on different proteins.     

Serum immunoglobulin G antibody responses to Bordetella pertussis lipooligosaccharide and B. parapertussis lipopolysaccharide in children with pertussis and parapertussis

Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussis were measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.     

Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.     

Enhancement of antibody production after treatment with actinomycin-D: interrelationships between 7S and 19S antibody

Administration of actinomycin-D to adult male CF 1 mice immunized with sheep erythrocytes led to enhanced production of haemolytic antibody during the primary response. Haemolytic antibody was shown to be of the 19S variety, while haemagglutinating antibody belonged to the 7S class. The enhancement effect was dependent on the dosage of actinomycin-D and on the relative times of administration of drug and antigen.Increase in 19S antibody production was shown to be associated with and probably dependent on delay and depression of production of 7S antibody, although attempts to reverse the effects of actinomycin-D by passive transfer of 7S antibody were not successful.