Background: TLR trafficking must be regulated to control ligand accessibility for effective pathogen recognition. The TLR1 I602S polymorphism is deficient in surface trafficking. Results: A TLR1 trafficking motif is interrupted by the 602S polymorphism. PRAT4A and PRAT4B differentially regulate TLR trafficking. Conclusion: A mechanistic basis for deficient TLR1 602S surface trafficking is provided. Significance: Differential receptor trafficking is a potential mechanism for controlling inflammation, based on TLR availability.
Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette–Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4+ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy.
The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ⌬leuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freezethaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ⌬leuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10 24 -fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10 68 -fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ⌬leuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. The immense global burden of human immunodeficiency virus (HIV) infection necessitates the development of an efficacious vaccine. There is increasing interest in the use of live recombinant bacterial vectors as HIV vaccines due to the inherent advantages of utilizing a replicating antigen delivery system that is itself an effective adjuvant (1, 2). Previous studies have examined the use of live Gram-positive and Gram-negative bacterial vectors, including recombinant Salmonella, Listeria, Streptococcus, and Escherichia coli, for heterologous expression of HIV antigens, with varying success (3-8).Mycobacterium bovis BCG is the most widely administered vaccine in the world (9). Its extensively documented safety in immunocompetent individuals, relatively low production cost, and well-established infrastructure for vaccine administration make it an ideal candidate for use as an anti-HIV vaccine vehicle (10-12). In addition to the logistical advantages of using BCG, mycobacterial antigen delivery systems possess inherent adjuvant properties which activate innate immunity (13,14). Mycobacteria such as BCG ...
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