The evaluation of the mutagenic activity of pertussis vaccinal preparations]

Two vaccine preparations obtained from Bordetella pertussis, whole-cell vaccine constituting one of the components of adsorbed DPT vaccine and acellular vaccine, were tested for mutagenicity. The doses of the preparations covered the range 1-100 ED50. Ames' test and the metaphase analysis of marrow cells of C57BL/6J mice were used. The acellular preparation was also tested on thymectomized mice, taking into consideration chromosomal aberrations in marrow metaphases. Whole-cell and acellular pertussis vaccines did not induce mutations in Salmonella typhimurium and chromosomal aberrations in mouse marrow cells.     

Comparative pathomorphologic study of the toxic properties of a corpuscular pertussis vaccine and of a cell-free pertussis preparation

The comparative study of morphological changes in the body of outbred mice under the action of corpuscular pertussis vaccine and acellular pertussis preparation has been made. The corpuscular vaccine has been shown to produce a pronounced, dynamically increasing toxic effect, thus causing the damage of lymphoid thymic and spleen cells, prolonged interstitial reaction in the lungs, destructive inflammatory process at the site of injection. The acellular pertussis preparation is less toxic, induces less pronounced changes in these organs at the early period of the experiment, stimulates the proliferation of lymphoid cells and lymphoblast transformation. As noted in this study, the damaging action of pertussis vaccine is mainly indicated by pathological phenomena appearing in the organs of the immune system, pulmonary parenchyma and muscular tissue (in the inoculation zone).     

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.     

Trial of pertussis toxin activity in vitro on CHO cells

The activity of B. pertussis toxin has been tested in the continuous culture of CHO (Chinese hamster ovary) cells. The in vitro method of testing B. pertussis toxin is rapid, highly sensitive and specific. The unit of activity of B. pertussis toxin is higher than in mouse tests by several orders. The specificity of the action of B. pertussis toxin on CHO cells has been confirmed by the test of the neutralization of the toxicity effect with antiserum.     

Effect of cucumarioside (a triterpene glycoside from the holothurian Cucumaria japonica) on the development of an immune response in mice to corpuscular pertussis vaccine

The influence of cucumarioside, triterpene glycoside obtained from Cucumaria japonica (Echinodermata, Holoturioidea), or sea cucumbers, on the resistance of mice to Bordetella pertussis infection (with the use experimental pertussis meningoencephalitis as a model) and on the development of immune response to corpuscular pertussis vaccine was studied. The preparation under test was shown to have greatly pronounced immunomodulating properties depending on both the concentration of cucumarioside and the route of its administration, as well as on the dose of pertussis vaccine. When administered orally in a dose of 4 micrograms per mouse and intraperitoneally in doses of 0.04 and 0.0004 micrograms, cucumarioside enhanced the protective effect of corpuscular pertussis vaccine. The use of cucumarioside in a dose of 0.001 micrograms per mouse abolished the suppressive action of large doses of pertussis vaccine in the background rosette-formation test at an early period after immunization and increased number of immune rosettes formed by lymphocytes in the spleen of mice immunized with different doses of the corpuscular vaccine.     

Investigation of an Aerosol Challenge Model as Alternative to the Intracerebral Mouse Protection Test for Potency Assay of Whole Cell Pertussis Vaccines

The current potency test for whole cell pertussis vaccines, the intracerebral mouse protection test, is still the only assay which has shown a correlation with protection in children. However, it has considerable disadvantages as it uses a severe challenge procedure and the results tend to show significant intra- and inter-laboratory variation. An alternative assay based on non-lethal aerosol challenge of mice has been investigated as a replacement for the current intracerebral mouse protection test. Evaluation of this indicated that the aerosol system allowed consistent inoculation of bacteria into mice and gave good reproducibility. The protective capacity of different vaccine preparations was distinguished by this assay. Furthermore, the viable counts of Bordetella pertussis in the lungs of challenged mice were immunisation dose-dependent, which allowed the relative potency of vaccines to be calculated. Comparison of potency of five batches of vaccine from different manufacturers assayed by both the intracerebral and the aerosol challenge methods ranked the vaccines in identical order. The results suggest that this method has potential for use as a potency test for whole cell pertussis vaccine which would result in a great reduction in the number of animals used. It would also replace the lethal challenge by a non-lethal procedure and thereby avoid the use of the severe intracerebral challenge procedure.     

The effects of different inactivating agents on the potency, toxicity and stability of pertussis vaccine

The effect of heat (56 degrees C for 10 min), formaldehyde (0.1% at 37 degrees for 24h), glutaraldehyde (0.05% at room temperature for 10 min), thimerosal (0.02% at 37 degrees C for 24h), acetone-I (three treatments at room temperature) and acetone-II (three treatments at room temperature and fourth treatment at 37 degrees C), when used as inactivating agents in the preparation of pertussis suspension, was studied with regard to potency, toxicity and stability. Five batches each of Bordetella pertussis strains 134 and 509 were used for the study. The thimerosal inactivated pertussis (TIP) preparation was 1.5-2 times more potent than the heat inactivated pertussis (HIP) preparation. The potency values of the formaldehyde inactivated pertussis (TIP) and glutaraldehyde inactivated pertussis (GIP) preparations were similar to those of the HIP preparation, while the potencies of the acetone-I treated pertussis (A(I)TP) and acetone-II treated pertussis (A(II)TP) preparations were about half those of the HIP preparation. The FIP preparation was the least toxic showing maximum weight gain in the mouse weight-gain test (MGWT), while the TIP preparation did not pass the MWGT. The weight gains shown the GIP, A(I)TP and A(II)TP preparations were greater than those shown by the HIP preparation. The potency of pertussis component in the adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was stable at 4-8 degrees C and 25 degrees C for three months for all types of pertussis vaccine. There was about 54-65% loss in the potency of the samples after three months at 35 degrees C. The inactivating agents used in the manufacture of pertussis preparations had no effect on the stability of the vaccine.     

Mitogenic action of the liquid phase of a microbial suspension of Bordetella pertussis

The suspension of B. pertussis cells in 0.15 M NaC1 solution, used for the preparation of corpuscular pertussis vaccine contains components loosely bound to microbial cells and producing pronounced mitogenic effect on mouse splenocytes at a concentration of 10 micrograms/ml. The mitogenic activity of B. pertussis is due to complex substances (lipopolysaccharide, protein, nucleic acids) with a wide range of molecular weights (70,000 to greater than 400,000). The mitogenic factor showing no leukocyte-stimulating and protective activity has been isolated by sedimentation with ammonium sulfate and gel filtration on Sephadex G-200. The mitogenic activity of B. pertussis lipopolysaccharide in the blast transformation test has been confirmed.     

The development of a new generation of pertussis vaccine

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.     

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.     

Alternative diphtheria, tetanus and whooping cough immunization schedule to evoke a Th2 tetanus and a Th1 pertussis immune response

In a previous study, using BALB/c mice, we found that while diphtheria (D), tetanus (T) and whooping cough (Pw, whole-cell Bordetella pertussis) immunization induces a Th1/Th2 tetanus response and memory T cells able to proliferate in response to in vitro stimulation with B. pertussis, DTPa immunization induces a Th2 tetanus immune response and no memory T cells that recognize B. pertussis as stimulus. Considering that a pro-inflammatory cytokine production is not necessary for protection against tetanus and therefore should be avoided, an alternative DTP immunization schedule with minimal Pw exposure was assessed in order to obtain a Th2 tetanus response and a Th1 pertussis response. BALB/c mice were primed with DT vaccine at day 0, with Pw vaccine at day 14 and boosted with DTPa vaccine at days 21 and 28. A control group was inoculated with saline. Antibodies against B. pertussis surface antigens, tetanus and diphtheria toxoids were produced by mice. Spleen cells stimulated in vitro with B. pertussis produced IL-6 and IFNgamma. Only IL-5 was produced by cells in response to tetanus toxoid stimulation. These results are in line with the low IgG1/IgG2a ratio for pertussis antibodies compared with those corresponding to tetanus and diphtheria. The immunization protocol presented herein succeeded in producing tetanus and pertussis immune responses of Th2 and Th1 type, respectively. In contrast to previous results obtained with DTPw immunization, no IL-12 production was observed. Our findings provide direct evidence that an immunization protocol with an interval of 14 days between DT and Pw primings, followed by DTPa boosters, can induce appropriate immune responses against DTP vaccine antigens.     

The effect of bacterial and synthetic peptidoglycans on the toxicity of a cell-free pertussis preparation

The influence of bacterial and synthetic peptidoglycans on the toxicity of acellular pertussis preparations (APP) has been studied in the mouse weight gain test and in the endotoxic shock development test on mice treated with Actinomycin D. The data obtained in these tests indicate that the immunomodulators (IM) under study are capable of changing (increasing or decreasing, depending on the dose) the toxic properties of APP. The antitoxic action of IM, established in this study, depends on the combination of the doses of IM and APP and the time elapsed from the time of immunization. The possibility of using these IM in a dose of 0.0001 microgram for reducing the LD50 of APP has been established. The use of IM belonging to the group of bacterial synthetic peptidoglycans for the development of acellular pertussis vaccines with reduced reactogenicity has been shown to hold much promise.     

The action of pertussis vaccines and GMDP on the change in the enzymatic activity of macrophages from peritoneal exudate

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.     

Methods of determination of toxicity of pertussis vaccine. 1. Change in the weight of the thymus gland and the spleen in mice after administration of pertussis vaccine]

Experiments were conducted on mice, strain NIH, line HSFS/N, immunized with pertussis vaccine. Determination of thymocyte pool proved to be a more sensitive method of detection of the stress effect of the vaccine than determination of the thymus weight. The test can be used for the elaboration of the method of assessment of the toxicity of pertussis preparations.     

Antigen-nonspecific suppression of the immune response in mice as affected by pertussis vaccine

A study was made of the suppressorgenic action of killed whole-cell pertussis vaccine prepared from B. pertussis strains 475 and 305. Thymic and splenic lymphocytes of CBA mice 3-7 days following intraperitoneal or intravenous inoculation of pertussis vaccine were shown to inhibit in an antigen-nonspecific manner the plaque-forming cell (PFC) production in the adoptive transfer experiments. Suppression of graft-versus-host reaction was also observed, estimated by the survival of irradiated (CBA X C57BL/6) Fl mice, or by measuring the endogenous colony formation. Suppression-mediating cells were found to be susceptible to complement-dependent lysis by the anti-I-Jk alloantiserum against the specific marker of suppressor T cells, antigen I-J. Furthermore, thymocytes of pertussis vaccine-treated mice were shown to inhibit the endogenous colony formation in syngeneic mice irradiated in sublethal dose. Thus, B. pertussis vaccination of CBA mice resulted in appearance of suppressor T cells that exerted various inhibitory activities in several experimental test-systems.     

Interaction of T-activin with peritoneal macrophages

The action of T-activin on peritoneal macrophages of CBA mice after its introduction into the animals has been studied. In intact mice the phagocytic activity of macrophages and their resistance to the cytopathogenic action of Salmonella typhimurium live cells remains unchanged. The injection of corpuscular pertussis vaccine into mice leads to a decrease in the resistance of macrophages to the action of salmonellae. The simultaneous injection of T-activin into mice in doses of 0.1 and 1.0 microgram per animal abolishes the damaging action of the vaccine. The analysis of the in vitro action of T-activin on macrophages of intact mice revealed that the preliminary incubation of cells with the preparation sharply increases their resistance to the action of salmonellae, while its introduction simultaneously with bacteria or after them rapidly leads to the death of macrophages. The action of T-activin is supposed to be linked with triggering the biosynthetic processes mediating the resistance of macrophages to the cytopathogenic action of salmonellae.     

The modified leukocytosis promoting factor (LPF)-test: A valuable supplement to the mouse weight gain (MWG)-test in toxicity control of whole cell pertussis vaccine

For the safety testing of pertussis vaccine, many in vivo assays have been developed, but none of these assays, except the Mouse Weight Gain (MWG)-test, are obligatory. Leukocytosis Promoting Factor (LPF) test, performed in mice, is one of the tests to examine the toxicity. However, due to lack of standardization, this test has not been implemented in the regular safety testing of the vaccine. Our investigations demonstrate that the LPF-test becomes more reproducible and sensitive if preparations are administered subcutaneously on day 0 and and counting of the leukocytes are done on day 6. Therefore, it is suggested to include the revised LPF-test in the quality control panel for the assessment of the toxicity of whole-cell pertussis vaccine.     

Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.     

Effect of pertussis vaccine on the functional activity of the peritoneal and splenic macrophages in 2 mouse strains genetically differing by the intensity of their immune response

The immunostimulating effect of corpuscular pertussis vaccine on the antigen-presenting and bactericidal functions of peritoneal and splenic macrophages in CBA and C57BL/6 mice, differing in the intensity of immune response to sheep red blood cells and Salmonella typhimurium, has been studied. The study has revealed that the injection of pertussis vaccine alters the functional activity of the cells under study, the effect depending on the immunizing dose, the strain of mice and the time elapsed from the moment of immunization. Pertussis vaccine enhances the low capacity of macrophages for antigen presentation in C57BL/6 mice with low responsiveness and alters the resistance of peritoneal and splenic macrophages to the cytopathic action of salmonellae.     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.