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Construction of stable recombinant BCG and other live mycobacterial vaccines

Objective

- to construct insertion cassettes based on the pSAM2 integrative plasmid and the Ms6 temperate mycophage
- to study the replication of the pAL5000 plasmid vector
- to introduce foreign antigens from various pathogens and cytokines in BCG
- to study immune responses induced against foreign antigens produced by BCG recombinant strains
- to develop efficient methods for inactivating M.tuberculosis genes, either by allelic exchanges or Transposition
- Construction of a conjugative and thermosensitive vectors and their utilization for transposition mutagenesis
- Construction of a series of Mycobacterium/E.coli shuttle cloning vectors derived from the pAL5000 and pACYC184 plasmids
- New selection markers that are active in mycobacteria have been evidenced: a gentamycin antibiotic resistance gene and two markers other than antibiotic resistance: sac B and mercury resistance
- A fragment of 1750 bp containing the attP-int region of the Ms6 temperate mycophage was completely characterized (the complete nucleotide sequence was determined and a functional analysis was performed). It was used for the constructin of integrative cassettes
- A strong promoter of MS6 was evidenced
- Characterization of the 1437bp minimal replicating unit of pAL5000. It contains only two open reading frames that have been produced as recombinant proteins and will be used for in vitro studies of replication
- Construction of an insertion module based on pSAM2 and containing both the int gene and the attP site but not the xis gene
- Demonstration of allelic exchange in M. tuberculosis complex strains
- Construction of new vectors containing negative counterselective markers enabling efficient transposition and allelic replacement in fast growing and slow growing mycobacteria
- Construction of BCG strains with mutations (ure- and purC-) by allelic exchange
- Conditions for efficient transposon mutagenesis in M. smegmatis were performed
- Development of virulence tests in in vitro macrophage cultures and in mice
- Development of new technologies for studying the induction of systemic humoral and cellular immune responses and mucosal immunity induced by BCG recombinant strains expressing various foreign antigen genes
- Antigen presentation was investigated by isolating intracellular compartments of macrophages infected by BCG
- Study of the modulation of type 2 like responses by BCG expressing the major allergen Der P2 in association with different cytokines

MAJOR SCIENTIFIC BREAKTHROUGHS:
- Construction of Ms6 and pSAM2 integrative cassettes
- Definition of the minimal replicon of plasmid pAL5000
- Construction of new vectors: shuttle plasmid vectors with a multiple cloning site and conjugative and thermosensitive vectors
- Characterization of new markers for positive selection and negative counterselection. Two of them are non-antibiotic markers
- Demonstration of allelic replacement in M.tuberculosis complex strains
- Methods for the study of i.) the presentation of antigens from mycobacteria after interaction with macrophages ii.) the induction of systemic humoral and cellular immune responses and mucosal responses

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

INSTITUT PASTEUR
Address
Rue Du Docteur Roux 25
75724 Paris
France

Participants (4)

Imperial College of Science Technology and Medicine
United Kingdom
Address
Norfolk Place
W2 1PG London
UNIVERSIDAD DE CANTABRIA
Spain
Address
Pabellon De Gobierno, Avenida De Los Castros S/n
39005 Santander
UNIVERSIDAD DE ZARAGOZA
Spain
Address
Pedro Cerbuna, 12
50009 Zaragoza
UNIVERSIDADE DE LISBOA
Portugal
Address
Campo Grande
1699 Lisboa