The class 1 outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity.

The class 1 outer membrane protein of Neisseria meningitidis B:15:P1.7,16 was expressed in Bacillus subtilis in high yield as intracellular aggregates. These were easy to isolate and the protein (called BacP1) could be solubilized under denaturing conditions. Sera of mice immunized with thus-solubilized BacP1 contained high titres of antibodies that reacted with the class 1 protein of the meningococcal envelope in immunoblots but did not react with native meningococcal envelope in enzyme immunoassays (EIA) or with intact meningococci in bactericidal assays. However, when the BacP1 protein was complexed with heterologous (Salmonella) lipopolysaccharide, the ensuing sera reacted with meningococcal envelope preparations in both EIA and immunoblots, showed subtype-specific bactericidal activity, and were protective in an infant rat meningitis model.

Class-3 porin protein of Neisseria meningitidis: cloning and structure of the gene.

The gene coding for the class-3 protein of Neisseria meningitidis was cloned and sequenced. The deduced amino acid (aa) sequence was highly homologous (50-78%) to those of other neisserial porin proteins. Alignment of the aa sequence of five neisserial porin proteins pinpointed several regions of identity or near identity. These are assumed to be membrane-spanning beta-strands. A comparison of the homologies between these neisserial porins showed that the class-3 protein is most closely related to the Neisseria gonorrhoeae P1A protein.

The 20 kDa C-terminally truncated form of pertussis toxin subunit S1 secreted from Bacillus subtilis.

The subunit S1 of pertussis toxin (PT) was purified as the recombinant product BacS1 from the culture supernatant of a Bacillus subtilis strain containing a secretion vector with a DNA fragment coding for the mature subunit S1 inserted downstream of the signal sequence of the alpha-amylase gene. The method of purification was successive ion exchange and adsorption chromatography. BacS1 occurred in two forms (28 and 20 kDa) of which the truncated 20-kDa peptide was the main one in the supernatant. The truncated BacS1 was purified and shown to have the same NH2-terminus as the full-size (28 kDa) BacS1. It was also enzymatically active indicating correct conformation. The truncated BacS1 was also shown to elicit neutralizing and protective antibodies when injected into mice or rabbits.

Immunogenicity and protective efficacy of pertussis toxin subunit S1 produced by Bacillus subtilis.

Pertussis toxin (PT) subunit S1 was produced in Bacillus subtilis as a secretory protein designated BacS1. BacS1 was partially purified and used to immunize mice. The sera were tested for PT-neutralizing antibodies and for protective capacity in a mouse model. Unlike previous findings with recombinant S1 from Escherichia coli, the recombinant BacS1 protein induced antibodies that were both neutralizing and protective. An adjuvant was necessary for efficient immunization with BacS1 but not with PT. Of the four adjuvants tested, aluminium phosphate gel was insufficient whereas Freund's incomplete adjuvant, Klebsiella lipopolysaccharide and Ribi's monophosphoryl lipid A-trehalose dimycolate emulsion all resulted in protective antibody production in NIH mice.

Expression of pertussis toxin subunit S4 as an intracytoplasmic protein in Bacillus subtilis.

The expression and secretion of pertussis toxin subunits S1 to S5 in Bacillus subtilis by the aid of a bacillary signal sequence has been reported. While secretion of subunit S1 was high, that of others was low. Ways have now been explored to improve the yield, using S4 as an example. The addition of a protease inhibitor was found to increase the amount of S4 in the culture supernatant, but the final amount was still much below that of S1. However, intracellular expression of S4 gave a high yield (500 mg l-1) and the aggregated protein could easily be isolated in a few simple steps.

Production and secretion of pertussis toxin subunits in Bacillus subtilis.

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.

Serum antibody response to B. pertussis Tn5 mutants, purified PT and FHA in two different mouse strains and passive protection in the murine intranasal infection model.

The protective capacities of antibodies to pertussis toxin (PT) were compared with antibodies to several other pertussis antigens in an experimental murine model of intranasal infection with Bordetella pertussis. Protection from lethal challenge was achieved by passive immunization with mouse antisera to whole cells of the virulent B. pertussis BP338(Vir+) and its Tn5-generated mutants, BP353(Fha-), BP348(Adc-Hly-) and to a lesser extent of BP347(Vir-). The immune sera were produced in two different mouse strains, a good PT antibody responder (NIH) and a poor responder (F1 of CBA x C57BL/6). The antisera produced in the F1 mice contained no detectable neutralizing antibodies to PT as measured by the CHO cell assay. In spite of this the anti-BP353(Fha-) and BP348(Adc-Hly-) sera of the F1 mice seemed as protective as those of the NIH mice. A strong dependence between PT neutralizing antibody and protection was seen only when comparing sera of NIH and F1 mice immunized with purified active PT. The protective capacity of sera of both mouse strains immunized with purified filamentous hemagglutinin (FHA) correlated with their anti-FHA titers measured by enzyme immunoassay. The data thus confirm the protective capacity of anti-PT and anti-FHA, but also show that antibodies of other specificities can confer protection.

Production of pneumolysin, a pneumococcal toxin, in Bacillus subtilis.

We have expressed the pneumolysin gene of Streptococcus pneumoniae in Bacillus subtilis, both from its own promoter and as a fusion protein. The level of expression of pneumolysin from its own promoter was low. The protein produced was hemolytically active. A higher level of expression (about 10 micrograms/ml of culture) was achieved when either one of two C-terminal fragments (corresponding to amino acids 265-471 and 55-471, respectively) or the entire coding part of the pneumolysin gene were fused to the promoter and signal sequence-coding region of the alpha-amylase gene of Bacillus amyloliquefaciens. The C-terminal fusion peptides reacted with anti-pneumolysin serum, but were not hemolytically active. In both cases most of the peptide remained cell-associated. When the entire pneumolysin gene was fused to the signal sequence, a hemolytically active form of pneumolysin could be detected, and most of the product was found in a processed form in the culture supernatant. The full-length pneumolysin secreted from B. subtilis was partially purified and used as antigen in an enzyme immunoassay with rabbit anti-pneumolysin serum.

Expression and secretion of pertussis toxin subunit S1 in Bacillus subtilis.

Pertussis toxin (PT) is an important virulence determinant of Bordetella pertussis and one of the major protective antigens against whooping cough. The genes coding for PT have recently been cloned, but attempts to express them in Escherichia coli have been unsuccessful. We therefore explored the possibility of expressing these genes in Bacilius subtilis for which efficient vectors are available. The lack of endotoxin in the Gram-positive Bacillus might be an additional advantage for the production of a vaccine component. A DNA fragment coding for S1, one of the subunits of pertussis toxin, was inserted into an alpha-amylase secretion vector and the recombinant plasmid was introduced into B. subtilis. This resulted in high expression of S1, most of which was secreted and therefore found in the culture supernatant. This supernatant had ADP-ribosylating activity similar to that of PT. Western blot with antiserum to B. pertussis holotoxin showed several proteins ranging in size from 28 kDa to 20 kDa reacting in specific manner. About 10% of the protein recognized by the antiserum was of the size expected for native-size S1. The total amount of S1 proteins (full size and truncated) in the culture supernatant was about 100 mg/l. S1 protein made in B. subtilis was partially purified using chromatography with P-cellulose and Blue Sepharose. This preparation was used to immunize rabbits; the immune serum thus obtained recognized subunit S1 of native pertussis toxin.