The purification and protective capacity of Bordetella pertussis outer membrane proteins.

The whole cell vaccine (WCV) of Bordetella pertussis is protective in the intracerebral (i.c.) mouse protection assay. We found a correlation between the i.c. mouse protection assay potency and the presence of the virulence-associated outer membrane proteins (OMPs) in outer membrane complexes (OMC). The virulence-associated 92, 32 and 30 kDa OMPs were purified and the N-terminal amino acid sequences were determined. The N-terminal amino acid sequences of the 30 and 32 kDa OMPs show homology with the C-terminal fragment of the P.93 precursor of the 69 kDa OMP (pertactin). The purified 32 kDa OMP was protective in the i.c. test when presented as mixed protein-detergent micelles. The 92 kDa OMP became a protective antigen when nonprotective levels of pertussis toxin were added. We found a correlation between the i.c. mouse protection value and the 92 kDal38 kDa (porin) ratio in OMC preparations.

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid.

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.

In vitro induction of antigen specific antibody synthesis and proliferation of T lymphocytes with acellular pertussis vaccines, pertussis toxin and filamentous haemagglutinin in humans.

The in vitro response of human B- and T-lymphocytes to the acellular vaccines JNIH-6 (containing pertussis toxoid and filamentous hemagglutinin), and JNIH-7 (containing pertussis toxoid), and to the purified components JNIH-4 (filamentous hemagglutinin) and JNIH-5 (pertussis toxin) was investigated. Pertussis toxoid and filamentous hemagglutinin induced specific Ig synthesis in vitro in lymphocytes obtained from convalescent pertussis patients as target cells. The antigen-dependent Ig production was demonstrated in lymphocyte culture supernatants by ELISA techniques and by a chinese hamster ovary cell toxin neutralization assay. Particularly with JNIH-4, -6 and -7, high antibody titers were obtained. At optimal antigen concentrations a marked lymphocyte blast transformation was found in lymphocyte cultures from whooping cough patients, but not in cultures of lymphocytes obtained from healthy volunteers. At high concentrations native pertussis toxin as well as the B oligomer (S2-5) of the toxin induced a strong proliferation of patient as well as control lymphocytes, indicating non-specific mitogenic activity. At lower concentrations lymphocyte blast transformation was seen in patient cultures only, which indicates an antigen-specific T-cell response. The A protomer (S1), dimer 1 (S2 + 4) and dimer 2 (S3 + 4) induced proliferation of patient lymphocytes, which demonstrates the presence of T-cell epitopes on these peptides. The in vitro B-cell response and the lymphocyte blast transformation assay are both useful tools for estimating the potency of acellular pertussis vaccines in man. Spontaneously acquired and vaccine induced immunity to Bordetella pertussis can be investigated at the level of B- and T-lymphocytes.

Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.

HLA and the rubella-specific immune response following vaccination.

A group of 88 seronegative 11-year-old Dutch girls was selected to be tested for human leukocyte antigens (HLA) and for both the in vivo and in vitro immune response to rubella virus, 6 and 12 weeks after rubella vaccination. Although a slight influence of HLA-associated factors on the antibody levels, as measured by the enzyme-linked immunosorbent assay could not be excluded, no evidence for HLA-associated control of the rubella-specific immune response following vaccination was obtained. From these data it is concluded that HLA-associated factors cannot be expected to hamper the effectiveness of large-scale rubella vaccination procedures.

Induction of antigen-specific antibody response in human peripheral blood lymphocytes in vitro by a dog kidney cell vaccine against rabies virus (DKCV).

In the present report an in vitro method for obtaining a secondary human antibody response to a dog kidney cell vaccine against rabies virus (DKCV) is described. Cultures of peripheral blood mononuclear cells from normal rabies-immune and nonimmune donors were stimulated in vitro by DKCV. The production of virus-specific antibody in supernatant fluids was monitored by ELISA. Antibody was produced by lymphocytes from rabies-immune individuals, whereas those of nonimmune subjects consistently failed to produce anti-rabies antibodies after in vitro stimulation with DKCV. The generation of the anti-rabies virus antibody response of lymphocytes stimulated with DKCV was shown to be an antigen-dependent, as well as an antigen-specific process. Optimal antigen-specific responses were observed at relatively low concentrations of antigen (10(-1) to 10(-2) micrograms/culture). At increasing concentrations of antigen in culture (greater than 1 microgram/culture), the anti-rabies virus response was suppressed. Antibody produced upon stimulation was capable of neutralizing rabies virus. The response to rabies virus requires T cell help because lymphocytes depleted of SE rosetting cells did not respond to an antigenic stimulus. Studies in which the same individuals were followed over time showed a sequential development of circulating B cell subsets. The system may provide a model for the study of human B cell differentiation in vivo and in vitro and may be valuable for testing the potency of rabies vaccines in vitro.

Stimulation of cynomolgus peripheral blood lymphocytes with tetanus toxoid and smallpox vaccine.

The cynomolgus monkey was studied as an animal model to investigate the cell-mediated immunity induced by vaccines. Optimal conditions are described to isolate peripheral blood lymphocytes. Lymphocyte transformation tests were performed with tetanus toxoid and smallpox vaccine. Antigen-specific lymphocyte transformations with smallpox vaccine could only be demonstrated when lymphocytes were obtained from vaccinated monkeys. Tetanus toxoid appeared to be a weak antigen. However, after adsorption of the toxoid to aluminum phosphate, a significant antigen-specific lymphocyte transformation was observed.

Absence of a correlation between humoral and cellular responses to a vaccinia virus and products of the major histocompatibility complex in rhesus monkeys.

Sixty-two Rhesus monkeys were tested at different times after vaccinia virus infection for virus-specific induction of lymphocyte proliferation in vitro or antibody production in vivo. No association was found between identifiable RhLA-controlled antigens and the strength of the cellular proliferative and/of humoral response.

In vitro immune responsiveness to vaccinia virus and HLA.

Because several lines of evidence suggest that HLA products might have an important function in the immune response to infectious agents, we studied the possible relation between immune response to vaccinia virus and HLA phenotype in 79 soldiers who received a primary vaccination. A low in vitro response to vaccinia virus was associated with HLA-Cw3 both in 49 subjects tested three to four weeks after vaccination (P less than 0.001) and in the remaining 30 subjects tested five to 11 weeks after vaccination (P = 0.035). Responses to unrelated antigens and phytohemagglutinin of lymphocytes tested before, three to four weeks and five to 11 weeks after vaccination indicated that this association was specific for vaccinia virus and suggested that differences in immune response to vaccinia were reflected in temporarily altered immune responsiveness to unrelated antigens. Our results indicate that HLA-Cw3 or an HLA product associated with Cw3 is involved in the cellular immune response to vaccinia virus.

The limulus amebocyte lysate test micromethod and application in the control of sera and vaccines.

To estimate the amount of endotoxin in sera and vaccines relatively high quantities of limulus lysate are necessary. Because this makes the control rather expensive, a micromethod was developed in which the amount of limulus lysate was reduced fivefold. This method was used to estimate endotoxin in typhoid vaccines. The relation between the reactions in man and the amount of endotoxin in the vaccines was examined. In these experiments it appeared that the limulus amebocyte lysate (LAL) test demonstrates predominantly the endotoxin in solution. The test was also used as a control test for calf sera. The culture quality of these calf sera as estimated by the lymphocyte stimulation test appeared to be influenced by previous bacterial contamination as could be demonstrated with the LAL test.

B- and T-cell markers on human lymphoblasts after stimulation with mitogen or antigens.

Receptors for sheep erythrocytes and for the third component of complement were demonstrated on human lymphoblasts after stimulation with various antigens and mitogens. With these markers B- and T-cell stimulation could be differentiated. Phytohaemagglutinin (PHA) and purified protein derivative (PPD) proved to activate predominantly T cells, whereas foetal calf serum (FCS) was shown to be a B-cell stimulator. Candida albicans and allogeneic cells, on the other hand, stimulated both B and T cells.