Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.

Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step.

When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.

The Neisseria meningitidis outer membrane protein P1 produced in Bacillus subtilis and reconstituted into phospholipid vesicles elicits antibodies to native P1 epitopes.

Class 1 outer membrane protein (P1) of Neisseria meningitidis group B is considered a promising vaccine candidate because P1 subtype-specific antibodies have been shown to be protective in an animal model. We have previously described the production of P1 in the Gram-positive Bacillus subtilis as intracellular inclusion bodies, from which the protein (BacP1) is easily purified (Nurminen et al., Mol. Microbiol., 1992, 2499-2506). We show here that the purified BacP1 can be reconstituted into phospholipid vesicles with the formation of the native immunodominant surface epitopes. The detergent-solubilized, completely denatured BacP1 was fused with phospholipid-detergent micelles during detergent removal by dialysis or gel filtration to yield protein-lipid vesicles (liposomes). When mice were immunized with these liposomes, they produced high titers of antibodies reacting in a P1 subtype-specific manner with meningococcal cells indicating the presence of conformation-dependent P1-specific epitopes in the liposomes. The results suggest that a vaccine candidate for meningococcal disease could be developed from the BacP1-liposomes. They furthermore demonstrate the feasibility of refolding a denatured outer membrane protein, which has never been exposed to lipopolysaccharide, into a native-like conformation.

The antibody response to a prototype liposome vaccine containing Neisseria meningitidis outer membrane protein P1 produced in Bacillus subtilis.

Monoclonal antibodies to the class 1 outer membrane protein P1 of Neisseria meningitidis B:15:P1.7,16 have been shown to be bactericidal and protective in an infant rat meningitis model. We have produced the P1 protein in Bacillus subtilis as inclusion bodies. When the purified and denatured protein (BacP1) was reconstituted with phosphatidylcholine into liposomes, native antigenic epitopes were formed. Such liposomes were reproducibly immunogenic in mice and guinea pigs at a low dose (1-10 micrograms of BacP1 protein) and without any other adjuvant. The resulting antisera contained high titers (enzyme immunoassay) of antibodies directed to native P1 epitopes exposed on the surface of meningococcal cells. The sera were also active with live N. meningitidis in bactericidal assays and protective in the infant rat meningitis model; all these activities were specific to the serosubtype of the P1 protein.

Heterologous production of the P1 porin of Neisseria meningitidis in bacillus subtilis: the effect of an N-terminal extension on the presentation of native-like epitopes.

The major outer membrane protein P1 (class 1) of Neisseria meningitidis has been produced as inclusion bodies in Bacillus subtilis with the aim to develop a vaccine based on it. The protein produced in high yield in B. subtilis contained an N-terminal extension of 11 amino acid residues which was found to be necessary for expression in the production system. In the present study we asked whether or not the removal of this extension would effect the conformation of this protein in liposomes as judged by its immunogenic properties. A methionine was engineered in front of the mature P1 protein to provide a chemical cleavage site for CNBr to remove the extension. The CNBr-cleaved protein, complexed with phospholipids, elicited high titers of antibodies binding to the meningococcal cells similarly to the noncleaved protein. This suggests that the BacP1 protein can serve as an effective vaccine component irrespective of the presence, or absence, of this N-terminal extension.

Immunization with meningococcal class 1 outer membrane protein produced in Bacillus subtilis and reconstituted in the presence of Zwittergent or Triton X-100.

Vaccines against group B meningococcal infection tested in several field trials have all been extracts of the outer membrane of the bacteria. We have developed a single component vaccine based on the class 1 outer membrane protein P1 produced in a heterologous host Bacillus subtilis, and describe here its immunizing properties. The purified and denatured protein BacP1 was solubilized in SDS, followed by addition of an excess of a second detergent (Zwittergent 3-14 or Triton X-100). Immunization of mice showed that this process led to at least partial reconstitution of the native epitopes of the P1 protein. The immunogenicity of these BacP1 detergent preparations was further improved when administered together with adjuvants (aluminium hydroxide or monophosphoryl lipid A); high titers of antibodies were thus obtained with vaccine doses as low as 2 micrograms of protein. The antibodies elicited were essentially of IgG and reactive with protective epitopes present on the surface of meningococci. The bactericidal activity of the sera showed a good correlation to antibodies of the IgG1 and IgG2 isotypes, concomitantly increased in most sera.

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.