With the increasing incidence of tuberculosis and drug resistant disease in developing countries due to HIV/AIDS, there is a need for vaccines that are more effective than the present bacillus CalmetteGué rin (BCG) vaccine. We demonstrate that BCG vaccine can be dried without traditional freezing and maintained with remarkable refrigerated and room-temperature stability for months through spray drying. Studies with a model Mycobacterium (Mycobacterium smegmatis) revealed that by removing salts and cryoprotectant (e.g., glycerol) from bacterial suspensions, the significant osmotic pressures that are normally produced on bacterial membranes through droplet drying can be reduced sufficiently to minimize loss of viability on drying by up to 2 orders of magnitude. By placing the bacteria in a matrix of leucine, high-yield, freeflowing, ''vial-fillable'' powders of bacteria (including M. smegmatis and M. bovis BCG) can be produced. These powders show relatively minor losses of activity after maintenance at 4°C and 25°C up to and beyond 4 months. Comparisons with lyophilized material prepared both with the same formulation and with a commercial formulation reveal that the spray-dried BCG has better overall viability on drying.aerosolization ͉ bacillus Calmette-Gué rin ͉ spray drying ͉ dry powder ͉ mycobacterium B acillus Calmette-Guérin (BCG), the most widely administered childhood vaccine in the world with 100 million infant administrations annually (1), is presently delivered by needle injection, requires refrigerated storage, and has shown in different parts of the world variable degrees of protection against tuberculosis (TB) ranging from 0% to 80% (2). Given the increasing global burden of TB, its coupling to the HIV/AIDS epidemic, and the emerging problem of drug resistance, there is a clear need for alternatives to the traditional BCG vaccine and current treatment strategies (2, 3). An optimal new vaccine would obviate needle injection, not require refrigerated storage, and provide a safe and more consistent degree of protection.Current commercial BCG vaccine preparations are filled as bacterial suspensions in vials, dried through lyophilization, and stabilized through refrigeration for up to 1 year on the shelf (4). Lyophilization, or freeze-drying, however, is known to produce significant losses of BCG viability after freezing (5). Moreover, lyophilized BCG fails to exhibit satisfactory shelf stability at room temperature, a major liability for transport and distribution, particularly in low-income environments, where the TB epidemic is most seriously concentrated (4, 6). Most importantly for our work, lyophilized BCG does not exhibit a particle form conducive for potential noninjectable delivery, notably through an aerosol route.In the search for a more effective vaccine against TB, spray drying provides an attractive alternative to lyophilization. It is less costly to operate (6), can be scaled up in sterile conditions, avoids cell freezing (7), and has the potential to be inhaled (8). Previous published att...
Recombinant adenoviruses are used extensively as delivery vectors in clinical gene therapy and in molecular biology, but little is known about how the viral carrier itself contributes to cellular responses. Here we show that infection with an E1/E3-deleted adenoviral vector (Adv) sensitizes human epithelial cells to tumor necrosis factor (TNF)-induced apoptosis. To explore the mechanism of Adv-mediated sensitization, we measured activity time courses for three protein kinases (MK2, IKK and JNK1) centrally involved in the TNF-receptor signaling network, as well as two kinases (Akt and ERK) activated by growth factors. Both the pro-apoptotic signal MK2 and the anti-apoptotic signal Akt were upregulated when Adv-infected cells were stimulated with TNF, and MK2 and Akt each contributed significantly to TNF-induced cell fate. Surprisingly, further activation of Akt in Adv-infected cells via insulin treatment did not significantly reduce apoptosis or MK2 activity. We show that the ineffectiveness of insulin-mediated anti-apoptotic signaling through Akt is due to saturation of Akt-effector substrate phosphorylation in Adv-infected cells. Normalizing Akt signaling relative to its Adv-induced baseline activity identified a global dose-response curve that relates Akt signaling to cellular survival. Thus, the background Akt activity induced by Adv limits the transmission of anti-apoptotic signals in response to further cytokine or growth-factor stimulation. The phenotypic and intracellular synergy between Adv and TNF may have implications for interpreting cellular responses in gene-therapy and laboratory applications.
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