Jeremy Mark Wells

Lactococcus lactis: high‐level expression of tetanus toxin fragment C and protection against lethal challenge

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.

Heterologous Expression of an Immunogenic Pneumococcal Type 3 Capsular Polysaccharide in Lactococcus lactis

ABSTRACT In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogenStreptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter−1) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 × 106 CFU of live recombinant lactococci expressing a total of approximately 0.5 μg of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 μg of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.

A mitochondrial elongation factor-like protein is over-expressed in tumours and differentially expressed in normal tissues

The tissue‐specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have high levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF‐Tu and the mitochondrial EF‐Tu of S. cerevisiae with the structurally and functionally important amino acids of EF‐Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.

Progress in the development of mucosal vaccines based on Lactococcus lactis

Abstract This paper reviews recent work on the construction and characterisation of recombinant strains of Lactococcus lactis which are able to express immunogens derived from e.g. Clostridium tetani , H.I.V. — 1 and Schistosoma mansoni . The immunogenicity of these constructs has been tested in mice. In some instances it has been possible to elicit protective level systemic immune responses as well as significant mucosal response following certain types of mucosal route vaccination. These studies and the key issues which have emerged from them are discussed.

Mucosal Immunization with Recombinant Lactococcus lactis

The United States National Institutes of Health has recently pointed out that despite many years of intensive vaccine research and the development of sophisticated effective vaccines, thousands of people, mainly children, die each year as a result of vaccine-preventable diseases (Jordan Report 1995). A significant factor contributing to this mortality rate is the problem of implementing vaccination programs on a large scale due to the need for trained personnel to administer parenteral inoculations, repeat visits for booster inoculations, and concern over needle stick injuries. The scale of the problem is large, with a projected 125 million children requiring immunization annually by the year 2000. These problems have generated a great deal of interest in the development of new vaccines which could be mucosally administered, introducing the possibility of self-administration of booster inoculations. This would increase the probability of repeated doses being administered and decrease the need for trained personnel, and so decrease costs.

Cloning and Expression Vectors for Lactococci

In last decade there has been considerable interest in developing recombinant lactic acid bacteria with novel properties for applications in the health care industry and for their more traditional use in food fermentation and preservation. It is envisaged that genetically modified lactic acid bacteria will eventually be used to increase the production of certain flavour compounds or enzymes involved in various food processes or as cell factories for the production of antimicrobials, high value proteins and primary or secondary metabolites. A more recent development has been the growing interest in the use of non-pathogenic lactic acid bacteria as vaccine delivery vehicles for mucosal immunisation. A key component in the development of many of these applications is the requirement for efficient expression systems for the production of heterologous proteins in different species of lactic acid bacteria. This chapter summarises the progress of work on the development of general cloning vectors and inducible and constitutive expression systems for the lactococci.

Research articleFactors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

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 × 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.