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Development of attenuated shigella flexneri vectors as vaccines to elicit protection against mucosally transmitted infectious diseases


The aim of this project has been to develop an orally-delivered mucosal vaccine vector system based on rationally designed genetic attenuation of virulence in the invasive enteric pathogen S. flexneri. A proper vaccine vector is a strain attenuated to avoid reactogenicity in vaccinees, but retaining sufficient tissue invasiveness to elicit mucosal and possibly systemic immunity. Such vaccine strains should be able to achieve proper delivery of heterologous protective antigens to elicit protection against other mucosally transmitted pathogens.
Molecular and cellular bases of cell invasion
The Shigella proteins eliciting endocytosis by epithelial cells have been identified. They consist in a 17 kDa cytoplasmic chaperone IpgC, and four Ipa proteins, two of which, IpaB and IpaC, are the primary inducers of the host-cell cytoskeletal rearrangements causing the entry focus. Ipa proteins are secreted via a specialized Mxi-Spa translocon which is activated upon contact with target eukaryotic cells. Some of the Ipa-induced eukaryotic signals that trigger the massive cytoskeletal rearrangements leading to bacterial internalization have been identified and characterized, as well as the involvement of key actin-associated proteins. In summary, the entering bacterium intercepts signals essential for the establishment of cellular adhesion structures.
Intracellular life-style of Shigella. Actin dependent motility of Shigella has been further characterized. SopA, a surface protein of the OmpT family achieving cleavage of IcsA has been identified. Mutagenesis of the sopA gene shows that this protein affects polar localization of IcsA on the cell surface, thus modulating the motility process by enhancing asymetrical polymerization of actin on the bacterial surface. A bacterial surface protein, which interacts with components of the intermediate junction machinery, thus facilitating bacterial passage from one cell to another, has also been identified.
Bacterial factors governing intracellular multiplication of Shigella. Several mutants in different steps of major biosynthetic pathways (aroD, aroC, aroD, purE-aroB, purE-aroC, purE-aroD, thyA, purHD, dapB) have been constructed and tested for attenuated virulence. This provides a battery of candidates for construction of second-generation live vaccine candidates. In addition, strains harboring mutations in genes encoding other physiological functions are currently constructed. The chaA, B and C genes encoding a calcium/proton antiport system have been mutagenized. They will be analyzed for alteration in their capacity to establish proper interactions with eukaryotic cell components when bacteria invade epithelial cells.
Identification of promoters, construction of cassettes for expression of foreign epitopes and their secretion
Identification of promoters. A strategy has been developed to identify sequences regulating gene expression of intracellular bacteria. Our strategy is based on cat (i.e. chloramphenicol acetyl transferase) as reporting gene to monitor bacterial genes/ promoters activated intracellularly. The plaque assay, which explores the entry and cell to cell spread phenotypes, provides a positive selection assay within the host cell. On the basis of this screening strategy, over 2000 sequences were analyzed, among which two are able to express cat exclusively when bacteria are intracellular, and eight sequences confer chloramphenicol resistance in both situations. These sequences containing in vivo and in vitro activated promoters are currently studied. These in vivo promoters will be used to improve expression of the cassettes described below.
Construction of expression cassettes and expression and secretion of foreign epitopes. The S. flexneri 2a vaccine candidate SC602 has been used to express hybrid proteins in which epitopes of the V3 loop of the polio virus have been fused with the Shigella IpaC protein. Several sites have been characterized in which insertion of the epitope leads to stably expressed and properly secreted hybrid proteins. Current data indicate that immunization of mice leads to significant local and systemic immune response to the V3 loop epitope in addition to a strong response against the Shigella antigens.
Construction of vaccine candidates
SC602 is our reference vaccine candidate. A recent phase I trial carried out in the US Army has shown that this strain was well tolerated by volunteers after oral administration of up to 108 microorganisms, and that a good immune response was obtained with oral inocula of 105 microorganisms. An extended phase I-II trial is planned soon. However, further improvement of this candidate is required and other options need to be explored.
Based on the icsA mutation which appears pivotal to vaccine construction, other mutations have been introduced, encompassing several blocks in major metabolic pathways as described above. The aroC locus has been deleted in S. flexneri 2a icsA. The purEK locus has also been deleted in order to integrate the expression cassettes. This will generate other candidates presenting the combinations of icsA-purEK and icsA-aroC-purEK mutations.
Strains representative of these approaches have been tested in a guinea pig model of protection. Two mutants, M90T aroC and M90T purE-aroC, have been analyzed for their ability to induce a protective immune response in guinea pigs using either intragastric or intranasal immunization. Both the intragastric and the intranasal immunization protocols involved two 109-CFU doses (2 weeks apart). On day 28 after the first immunization, the animals were challenged with 5x108 CFU of the wild type strain M90T via the conjunctival route. Animals immunized intranasally with the wild type strain and M90T aroC were protective against a challenge by the wild type strain. In contrast, the majority of animals immunized with the purE-aroC double mutant were not protected. Conversely, animals immunized intragastrically with the double mutant were fully protected against the challenge, whereas only 50% of the animals immunized intragastrically with the aroC mutant showed protection. In general, guinea pigs immunized intranasally developed higher mucosal S-IgA and serum IgG titres when compared to the animals immunized intragastrically.
During the execution of the present contract, major scientific breakthroughs have been achieved:
-The major molecular and signalization pathways that account for Shigella invasion of epithelial cells and intestinal tissues have been identified and several of them characterized.
-Systems have been developed allowing expression and secretion of hybrid proteins presenting heterologous antigens in Shigella live vaccine candidates.
-New generation vaccine candidates have been generated which will serve as future Shigella vaccine candidates but also carriers to present heterologous antigens.

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