Flow cytometry sorting of recombinant mycobacterial species yields bacterial clones with enhanced insert expression.
Recombinant mycobacteria hold promise as vectors for delivery of HIV-1 and other pathogen antigen inserts for inducing systemic and mucosal immune responses. In general, the immunogenicity of the recombinant mycobacterial insert is proportional to the level of insert expression. In this study, a novel flow cytometry-based assay has been developed to sort live recombinant mycobacterial mutants with high expression of foreign inserts and to enrich those sorted bacterial populations. Sorted recombinant mycobacterial clones expressed higher levels of the ovalbumin SIINFEKL epitope, and select sorted clones showed better immunogenicity than unsorted recombinant mycobacteria. Thus, flow cytometry-based sorting can isolate recombinant mycobacteria enriched for higher insert expression.
Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. yu000022@mc.duke.edu
Jae-Sung
JS
Yu
John
J
Whitesides
Sun-Hee
SH
Lee
Natalie
N
Taylor
William R
WR
Jacobs
Norman L
NL
Letvin
Barton F
BF
Haynes
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D004251:DNA Transposable Elements; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005822:Genetic Vectors; D007371:Interferon-gamma / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D009154:Mutation; D009161:Mycobacterium / Q000145:classification / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D009163:Mycobacterium bovis / Q000235:genetics / Q000378:metabolism; D010047:Ovalbumin / Q000737:chemistry; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000942:Antigens, Bacterial; D004251:DNA Transposable Elements; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D010047:Ovalbumin