To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form.

A defined allelic-replacement mutant of the sly gene, encoding a thiol-activated cytolysin, from a European isolate of Streptococcus suis serotype 2 was generated and characterized. Unlike the parental strain, it is nonhemolytic, noncytotoxic for cultured macrophage-like cells, avirulent in a mouse infection model, yet only slightly attenuated in a porcine model of systemic infection. Streptococcus suis has been described as the etiological agent for a number of infectious disease syndromes in pigs, including arthritis, septicemia, meningitis, and pneumonia. S. suis produces a secreted hemolysin (suilysin) which has been suggested as playing a role in virulence (2,(4)(5)(6)(7)10). In order to investigate the role played by suilysin in the pathogenesis of a European serotype 2 isolate of S. suis, we generated a defined allelic-replacement mutant of the sly gene and compared the wild-type organism with the sly mutant in a number of assays.Bacterial strains and media. S. suis type 2 strain P1/7 was grown on Columbia agar (Oxoid) containing 10% defibrinated horse blood or in liquid cultures of Todd-Hewitt broth (Oxoid) supplemented with 7% fetal calf serum (FCS) (Gibco). Allelicreplacement mutants of S. suis were maintained on 1 g of erythromycin (Sigma) per ml.Mutagenesis of the suilysin gene from S. suis type 2. An erythromycin resistance gene cassette was introduced into an EcoRV site within a 1,278-bp fragment of the sly gene, contained in the vector pT7-Blue (Stratagene), amplified by PCR using primers suis1 (5Ј-AGCTTGACTTACGAGCCACAAG AG-3Ј) and suis2 (5Ј-CCACCATTCCCAAGCTAATCCTGT-3Ј) with chromosomal DNA from P1/7 as a template. The resulting plasmid, pSUI-erm, contains the erm gene in the same orientation as the sly gene. Plasmid pSUI-erm was introduced into P1/7 by electroporation (22.5 V/cm, 25 F, and 1,000 ⍀). A transformant which had undergone a doublecrossover event, confirmed by Southern hybridization and PCR, with concomitant insertion mutation of the sly gene, was isolated and named S7c.Phenotypic analysis of S7c. Overnight growth of suilysin mutant S7c on Columbia blood agar plates revealed no ␤-hemolysis. Secreted proteins from anaerobically grown overnight cultures of P1/7 and S7c were concentrated 100-fold by ammonium sulfate (50%, wt/vol) precipitation; then 10 l each of these preparations was spotted onto a Columbia horse blood agar plate and incubated at 37°C for 30 min. The proteins from P1/7 show clear zones of hemolysis, whereas there is a complete absence of hemolytic activity in the proteins obtained from S7c (Fig. 1a). The hemolytic activity from the wild-type parental bacteria was enhanced by the addition of ␤-mercaptoethanol, as previously reported (7) (Fig. 1a).The lack of expression of suilysin was confirmed by Western blotting. Supernatants from aerobically grown cultures of both P1/7 and S7C (sterilized using a 0.22-m-pore-size filter and concentrated 25-fold using Amicon filters) were probed with a monoclonal antibody (INT-STS-28-02; A. C. Jacobs, Intervet) raised against purified s...

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The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 x LD50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10-20 fold) more immunogenic than the alternative forms of the protein.