Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNA(ala )T-loops in slow- and fast-growing mycobacteria

BACKGROUND: Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA(ala )genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA(ala )loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA(ala )genes. RESULTS: The three tRNA(ala )genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNA(alaU )and tRNA(alaV )containing the attB site, a single base difference was observed between the two species. We observed that the tRNA(alaU )gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNA(alaV )gene was used as the target. No integration occurred in the BCG tRNA(alaU )T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNA(ala )T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNA(alaU )and tRNA(alaV )T-loops of the same BCG chromosome. CONCLUSION: Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNA(ala )genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies

Mucosal administration of three recombinant Mycobacterium bovis BCG-SlVmac251 strains to cynomolgus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination

International audienceThe widely administered Mycobacterium bovis BCG is an attractive live vector for the development of AIDS vaccines. We explored immune responses induced in cynomolgus macaques tor BCG-SIV3, a mixture of three recombinant BCG strains expressing the SIVmac251 nef, gag and env genes. After a single intradermal (ID) inoculation, circulating blood cells from rBCG-SIV3-vaccinated monkeys exhibited CTL responses targeted against the three antigens and interferon-gamma (IFN-gamma) secretion was observed. A rectal or oral boosting dose of rBCG-SIV3 elicited anti-SIV IgAs in the rectum of vaccinated monkeys and increased IFNgamma secretion by circulating blood cells. Despite a good response against the vector, rBCG-SIV3 administration did not induce IgG antibody responses or lymphoproliferation against the SIV antigens in blood. This could be due to the lack of in vivo persistence of the recombinant BCG strains that were used. Rectal challenge with fully pathogenic SIVmac251-infected all animals. However, after viral challenge, anti-SIV cellular and antibody responses were higher in rBCG-SIV3 monkeys than in controls indicating that the vaccine induced anti-SIV CD4(+) T-cell memory. (C) 2003 Elsevier Ltd. All rights reserved