No abstract

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-␥) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-␥ production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-␥) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. To date, vaccine strategies against HPV-16 are based on the ability of the E7 oncoprotein to elicit an immune response against this virus. In this study, the use of an inducible or a constitutive system to produce the HPV-16 E7 protein in Lactococcus lactis, a non-pathogenic and non-invasive Gram-positive bacterium, was compared. The highest E7 production was obtained with the inducible system. When mice were immunized intranasally with recombinant lactococci expressing either inducible or constitutive E7, an antigen-specific cellular response (i.e. secretion of IL2 and IFN-ª cytokines) was evoked and was substantially higher in mice receiving L. lactis expressing E7 with the inducible system. As bacterial antigen location may influence the immune response, recombinant L. lactis strains that produced E7 in three cellular locations, intracellular, secreted or cell-wall-anchored were evaluated. The highest immune response was elicited by administration of L. lactis producing an inducible cell-wall-anchored form of E7 protein. These promising results represent a step towards the development of a new, safe mucosal vector to treat HPV-related cervical cancer. INTRODUCTIONEpidemiological data have clearly shown that human papillomavirus type 16 (HPV-16) infection is the main aetiological factor for cervical cancer (CxCa) (Furumoto & Irahara, 2002). Worldwide, $400 000 women die annually from CxCa (Parkin et al., 1999). A prophylactic and/or therapeutic vaccine against this virus is thus a priority to prevent or to treat, respectively, CxCa. A prophylactic vaccine based on highly purified virus-like particles has recently been successfully used in trials in women, with a significant reduction observed in the incidence of both HPV-16 infection and related CxCa (Koutsky et al., 2002). However, such vaccines could probably not be used therapeutically in alreadyinfected patients because the virion capsid proteins are not detected in CxCa. The HPV-16 E7 protein, constitutively produced in cervical carcinomas, is required for the transformation process (Baker et al., 1987;Bedell et al., 1987;Dyson et al., 1989;Tanaka et al., 1989) and is considered a good antigen candidate for the development of a therapeutic vaccine against CxCa.Several studies have investigated the use of bacteria as E7 antigen delivery vehicles to elicit an immune response against HPV-16. In these studies, the vectors used were attenuated strains of pathogenic bacteria such as Salmonella and Mycobacterium spp. (Londoño et al., 1996; Jabbar et al., 2000). Although these recombinant strains elicited immune responses, invasiveness of the vectors and risks of reversion to pathogenicity limit their use in vulnerable groups such as immunocompromised patients or children. There is thus a need for the development of a new generation of safe delivery vehicles.Lactic acid bacteria (LAB) are promising candidates as safe vehicles for in vivo delivery of antigens. Compared with att...

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.Infection with human papillomavirus type 16 (HPV-16) is the main factor associated with development of cervical cancer (42). The HPV-16 E6 and E7 proteins are constitutively produced in cervical carcinomas, and E7 was shown to interact with several cell compounds, causing deregulation of the cell cycle and cell transformation (43). E7 is a 98-amino-acid nuclear phosphoprotein that is devoid of any known enzymatic activity (36). In eukaryotic cells, E7's half-life is short (30 to 40 min); its degradation is mediated by the ubiquitin-proteasome pathway (31). E7 protein is widely studied because of its implication in carcinoma onset. It is also considered to be a good antigen candidate for the development of new vaccines against cervical cancer.E7 production systems have been developed in both eukaryotes (1, 39) and prokaryotes (27,34). Since the 1990s, several workers have investigated the use of bacteria as E7 antigen delivery vehicles to elicit an immune response against 22). The gram-positive and generally regarded as safe (GRAS) commensal bacterium Streptococcus gordonii was used for this purpose to display E7 protein at the cell surface in fusion with export signals (30). These recombinant S. gordonii strains could elicit an immune response in mice and monkeys (23,26). Although encouraging, these results rely on a commensal, GRAS but non-food-grade bacterium. One risk of commensal, and hence persistent, microorganisms is the induction of immunotolerance. Thus, a transient presentation of the antigen to the immune system by a noncommensal bacterium may be needed to avoid this risk.None of the systems mentioned above seems to provide the combination of safety, sufficient yields, and simplified methods that would allow both purification and eventual oral immunization using E7. We therefore considered an alternative system for native E7 production based on a food-grade lactic acid bacterium. The best-known lactic acid bacterium, Lactococcus lactis, has been extensively engineered for the production of heterologous proteins (5,6,10,18,20,21,28,29,35,37). Protein production in L. lactis offers advantages: L. lactis is a food-grade gram-positive bacterium that produces very low amounts of native exoproteins. It is therefore a good candidate for heterologous protein secretion in different applications ranging from industrial production of high-added-value proteins to in vivo use as a live vaccine. As L. lactis is a noncommensal and transient bacterium in the digestive tract (12), t...