Making Vaccines On Demand

The integrated US Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) has made great strides in strategic preparedness and response capabilities. There have been numerous advances in planning, biothreat countermeasure development, licensure, manufacturing, stockpiling and deployment. Increased biodefense surveillance capability has dramatically improved, while new tools and increased awareness have fostered rapid identification of new potential public health pathogens. Unfortunately, structural delays in vaccine design, development, manufacture, clinical testing and licensure processes remain significant obstacles to an effective national biodefense rapid response capability. This is particularly true for the very real threat of “novel pathogens” such as the avian-origin influenzas H7N9 and H5N1, and new coronaviruses such as hCoV-EMC. Conventional approaches to vaccine development, production, clinical testing and licensure are incompatible with the prompt deployment needed for an effective public health response. An alternative approach, proposed here, is to apply computational vaccine design tools and rapid production technologies that now make it possible to engineer vaccines for novel emerging pathogen and WMD biowarfare agent countermeasures in record time. These new tools have the potential to significantly reduce the time needed to design string-of-epitope vaccines for previously unknown pathogens. The design process—from genome to gene sequence, ready to insert in a DNA plasmid—can now be accomplished in less than 24 h. While these vaccines are by no means “standard,” the need for innovation in the vaccine design and production process is great. Should such vaccines be developed, their 60-d start-to-finish timeline would represent a 2-fold faster response than the current standard

In Vitro Antimycobacterial Activities of Capuramycin Analogues▿

Translocase I inhibitor compounds derived from capuramycin demonstrated rapid bactericidal activity against several different mycobacterial species. SQ641 was the most active of the compounds, with a MIC of 0.12 to 8 μg/ml, a postantibiotic effect of 55 h, and interesting synergistic effects with other antitubercular drugs

In Vitro Interactions between New Antitubercular Drug Candidates SQ109 and TMC207▿

The in vitro interactions of two new antitubercular drugs, SQ109 and TMC207, with each other and with rifampin (RIF) were evaluated. The combination of SQ109 with TMC207 (i) improved an already excellent TMC207 MIC for M. tuberculosis H37Rv by 4- to 8-fold, (ii) improved the rate of killing of bacteria over the rate of killing by each single drug, and (iii) enhanced the drug postantibiotic effect by 4 h. In no instance did we observe antagonistic activities with the combination of SQ109 and TMC207. Rifampin activates cytochrome P450 genes to reduce the area under the curve (AUC) for TMC207 in humans. The presence of RIF in three-drug combinations did not affect the synergistic activities of SQ109 and TMC207, and SQ109 also dramatically decreased the MIC of RIF. SQ109 was active by itself, and both its activity was improved by and it improved the in vitro activities of both RIF and TMC207

Antitubercular Nanocarrier Combination Therapy: Formulation Strategies and in Vitro Efficacy for Rifampicin and SQ641

Tuberculosis (TB) remains a major global health concern, and new therapies are needed to overcome the problems associated with dosing frequency, patient compliance, and drug resistance. To reduce side effects associated with systemic drug distribution and improve drug concentration at the target site, stable therapeutic nanocarriers (NCs) were prepared and evaluated for efficacy in vitro in Mycobacterium tuberculosis-infected macrophages. Rifampicin (RIF), a current, broad-spectrum antibiotic used in TB therapy, was conjugated by degradable ester bonds to form hydrophobic prodrugs. NCs encapsulating various ratios of nonconjugated RIF and the prodrugs showed the potential ability to rapidly deliver and knockdown intracellular M. tuberculosis by nonconjugated RIF and to obtain sustained release of RIF by hydrolysis of the RIF prodrug. NCs of the novel antibiotic SQ641 and a combination NC with cyclosporine A were formed by flash nanoprecipitation. Delivery of SQ641 in NC form resulted in significantly improved activity compared to that of the free drug against intracellular M. tuberculosis. A NC formulation with a three-compound combination of SQ641, cyclosporine A, and vitamin E inhibited intracellular replication of M. tuberculosis significantly better than SQ641 alone or isoniazid, a current first-line anti-TB drug

<i>In Vitro</i> Characterization of the Anti-Bacterial Activity of SQ109 against <i>Helicobacter pylori</i>

<div><p>The most evident challenge to treatment of <i>Helicobacter pylori</i>, a bacterium responsible for gastritis, peptic ulcers and gastric cancer, is the increasing rate of resistance to all currently used therapeutic antibiotics. Thus, the development of novel therapies is urgently required. <i>N</i>-geranyl-N'-(2-adamantyl) ethane-1, 2-diamine (SQ109) is an ethylene diamine-based antitubercular drug that is currently in clinical trials for the treatment of tuberculosis (TB). Previous pharmacokinetic studies of SQ109 revealed that persistently high concentrations of SQ109 remain in the stomach 4 hours post oral administration in rats. This finding, combined with the need for new anti-<i>Helicobacter</i> therapies, prompted us to define the <i>in vitro</i> efficacy of SQ109 against <i>H. pylori</i>. Liquid broth micro-dilution was used for susceptibility studies to determine the antimicrobial activity of SQ109 against a total of 6 laboratory strains and 20 clinical isolates of <i>H. pylori</i>; the clinical isolates included a multi-drug resistant strain. All strains tested were susceptible to SQ109 with MIC and MBC ranges of 6-10 µM and 50-60 µM, respectively. SQ109 killing kinetics were concentration- and time-dependent. SQ109 killed <i>H. pylori</i> in 8-10 h at 140 µM (2MBCs) or 4-6 h at 200 µM (~3MBCs). Importantly, though the kinetics of killing were altered, SQ109 retained potent bactericidal activity against <i>H. pylori</i> at low pH. Additionally, SQ109 demonstrated robust thermal stability and was effective at killing slow growing or static bacteria. In fact, pretreatment of cultures with a bacteriostatic concentration of chloramphenicol (Cm) synergized the effects of typically bacteriostatic concentrations of SQ109 to the level of five-logs of bacterial killing. A molar-to-molar comparison of the efficacy of SQ109 as compared to metronidazole (MTZ), amoxicillin (AMX), rifampicin (RIF) and clarithromycin (CLR), revealed that SQ109 was superior to MTZ, AMX and RIF but not to CLR. Finally, the frequency of resistance to SQ109 was low and electron microscopy studies revealed that SQ109 interacted with bacterial inner membrane and cytoplasmic content(s). Collectively, our <i>in vitro</i> data demonstrate that SQ109 is an effective monotherapy against susceptible and multi-drug resistant strains of <i>H. pylori</i> and may be useful alone or in combination with other antibiotics for development as a new class of anti-<i>Helicobacter</i> drugs.</p> </div

Activity of SQ641, a Capuramycin Analog, in a Murine Model of Tuberculosis ▿

New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue α-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis

Molar-to-molar comparison of the antibacterial activity of SQ109, amoxicillin, metronidazole, and clarithromycin.

<p>Time-kill assays for two drug concentrations, 70 µM (<b>A</b>) and 140 µM (<b>B</b>), were used to compare the bactericidal activity of SQ109 to those of conventional antibiotics currently used for the treatment of <i>H. pylori</i> infection. The data are representative results from three independent experiments. The horizontal dashed line on each graph indicates the limit of detection (500 bacteria).</p

TEM analysis of SQ109-induced morphological and ultra-structural changes in <i>H. pylori</i>-cell wall, membranes, and cytoplasm.

<p>Approximately 6 x 10⁷<i>H. pylori</i> cells were sampled following 2 h (top panels) or 8 h- (bottom panels) of culture in the presence of PBS (<b>A</b> and <b>B</b>) as a negative control or 140 µM SQ109 (C, D, <b>E</b>, <b>F</b>, <b>G</b>, and <b>H</b>). Bacterial cells cultured in the presence of 100 µM AMX or 20 µM C ₁₂K-2β₁₂ were included as positive controls (data shown in Figure S3). PBS-treated negative control cells showed evidence of normal spiral-comma rod shaped morphology in addition to smooth homogenous cytoplasm (<b><i>sc</i></b>) and intact cell walls (<b>A</b>, <b>B</b>). In contrast, after 2 h of culture, SQ109 induced the formation of spindle actin-like cytoskeleton structures (<b><i>a</i></b>) in some cells (<b>C</b>) that appeared to condense cytoplasmic contents and lead to detachment of the IM from OM (arrows; <b>C</b>, <b>E</b>, <b>F</b>, <b>G</b>, and <b>H</b>) of nearly all cells resulting in complete disappearance of the periplasmic region; 95-99% of these cells also showed a deformed coccoid morphology (<b>E</b>, <b>G</b>, and <b>H</b>). After 2h and 8 h treatment with SQ109, the cells further showed blebs (<b><i>b</i></b>), electron-dense structures (<b><i>e</i></b>), evidence of complete loss of the IM and part of the cell wall with only the OM remaining intact (<b>D</b>), formation of two aberrant cytoplasmic bodies from a single cell (arrowheads) (<b>G</b> and <b>H</b>), and formation of outer membrane vesicles (omv) and inner membrane vesicles (imv) (<b>C</b>, <b>F</b>, <b>G</b>, and <b>H</b>). All scale bars are of 200 nm except <b>B</b> and <b>E</b> which measure 1500 nm. Representative images from two independent experiments are presented.</p

Effect of temperature and low pH on the stability and bactericidal activity of SQ109.

<p><b>A</b>) The effect of temperature on bactericidal activity of SQ109 was examined by pre-incubation of the drug at various temperatures (22^oC, 37^oC, 60^oC and 95^oC) for 1 h prior to use in the time-kill assay. <b>B</b>) In order to determine the effect of pH on SQ109, the time-kill assays were performed in pH-adjusted culture medium. The antibacterial activity of 140µM SQ109 against <i>H. pylori</i> cultured in pH 4.5 medium was compared to bactericidal activity at pH 6.8. The plotted data are representative results from three independent experiments. The horizontal dashed line on each graph indicates the limit of detection (500 bacteria).</p