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Early Protection against Pertussis Induced by Live Attenuated Bordetella pertussis BPZE1 Depends on TLR4
Anne-Sophie Debrie, Nathalie Mielcarek, Sophie Lecher, Xavier Roux, Jean-Claude Sirard and Camille Locht
J Immunol December 15, 2019, 203 (12) 3293-3300; DOI: https://doi.org/10.4049/jimmunol.1901102
Anne-Sophie Debrie
Centre d'Infection et Immunité de Lille, CNRS UMR8204–INSERM U1019, Institut Pasteur de Lille, F-59019 Lille, France; and
Nathalie Mielcarek
ORCID record for Nathalie Mielcarek
Sophie Lecher
Xavier Roux
Jean-Claude Sirard
ORCID record for Jean-Claude Sirard
Camille Locht
Live pertussis vaccine BPZE1 protects against B. pertussis infection within days.
Early protection induced by BPZE1 does not depend on adaptive immune responses.
Early protection induced by BPZE1 depends on TLR4-mediated innate immune responses.
Pertussis is a severe respiratory disease mainly caused by Bordetella pertussis. Despite wide global vaccination coverage with efficacious pertussis vaccines, it remains one of the least well-controlled vaccine-preventable diseases, illustrating the shortcomings of the current vaccines. We have developed the live attenuated nasal pertussis vaccine BPZE1, currently undergoing clinical evaluation in human phase 2 trials. We have previously shown that in mice, BPZE1 provides strong and long-lasting protection against B. pertussis challenge by inducing potent Ab and T cell responses as well as secretory IgA and IL-17–producing resident memory T lymphocytes in the nasal cavity. In this study, we show that BPZE1 induces protection in mice against B. pertussis within days after vaccination, at a time when Ab and T cell responses were not detectable. Early protection was independent of T and B cell responses, as demonstrated by the use of SCID mice. Instead, it was due to TLR4-dependent signaling through the MyD88-dependent pathway of the innate immune response, as demonstrated in experiments with TLR4-deficient and MyD88-knockout mice. TLR2-dependent signaling did not play a major role in early protection. In addition, this study also shows that even at high doses, BPZE1 is safe in the severely immunocompromised MyD88-deficient mice, whereas virulent B. pertussis caused a severe pathological condition and death in these mice, even at a low dose. Finally, coadministration of virulent B. pertussis with BPZE1 did not cause exacerbated outgrowth of the virulent strain, thereby adding to the safety profile of this live vaccine candidate.
Despite the widespread use of efficacious vaccines, pertussis, or whooping cough, is still a major global health problem. The most recent model, based on data from 2014, provided estimates of 24.1 million cases and 160,700 pertussis-linked deaths per year in children <5 y old (1), which is almost certainly an underestimation (2, 3). Although the disease is most severe and life-threatening in infants <6 mo of age (4), it affects all age groups (5). Furthermore, incidences have been increasing over the last decade, even in countries with high vaccination coverage (6–10).
Two types of vaccines are currently available: the first-generation, whole-cell vaccines (wP) and the more-recent, second-generation, acellular vaccines (aP) (11). Both types provide good protection after three doses, given usually at 1- to 2-mo intervals, starting at the age of 6–8 wk. This results in suboptimal protection of the most vulnerable age group despite good full vaccination coverage. Beginning vaccination earlier than 6 wk, including vaccination at birth, has been proposed (12, 13). However, vaccine efficacy may be limited by the incomplete maturation of the neonate immune system (14), especially with respect to Th1 responses (15) and a concurrent Th2 bias (16). Yet, both Ab and Th1/Th17 type responses are known to contribute to optimal protection against pertussis (17). Despite the incomplete maturation of the neonatal immune system, strong IFN-γ responses have been documented following vaccination at birth with the antituberculosis vaccine bacillus Calmette-Guérin (18) or after natural infection in early life by Bordetella pertussis (19), indicating that it is possible to induce Th1 responses in neonates.
As an alternative to neonatal immunization with current vaccines, maternal immunization with aP during the second or third trimester of pregnancy is now recommended in several countries and has been shown to be very effective in reducing severe pertussis in neonates (20). However, blunting by maternal Abs of the immune response to primary vaccination, especially with wP, has been reported in several studies (21–23), although the clinical relevance of this blunting is still unknown. Furthermore, even in high-income countries, maternal immunization appears to be difficult to implement for a variety of reasons, including perceived safety concerns and limited usefulness (24). Finally, current vaccines have limited impact on infection by B. pertussis, the main causative agent of whooping cough, and its transmission, as evidenced by epidemiological (summarized in Ref. 25) and experimental animal studies (26).
In contrast to the current vaccines, natural infection with B. pertussis induces strong Th1 responses to B. pertussis Ags, even in very young children (19), and prevents secondary infection in a nonhuman primate model (26). Furthermore, immunity induced by natural infection is of longer duration than that induced by vaccination (27, 28).
Therefore, we have developed the live attenuated nasal pertussis vaccine BPZE1, which is able to mimic natural infection without causing disease (29). BPZE1 produces genetically inactivated pertussis toxin (PT) and carries a deletion of the dermonecrotic toxin gene and the replacement of the B. pertussis ampG gene by that of Escherichia coli, thereby reducing the production of tracheal cytotoxin to background levels. Extensive preclinical studies have shown that BPZE1 is safe in several immune-competent (29–31) and immune-deficient models (32) and is able to induce robust humoral and cellular immune responses both systemically and at mucosal sites, leading to strong protection against challenge with virulent B. pertussis in mice (29, 30, 33, 34) and nonhuman primates (35). BPZE1 is now in clinical development and has successfully undergone phase 1 safety trials in humans (36).
In previous studies, we and others have shown that, in contrast to vaccination with aP, a single intranasal (i.n.) administration of BPZE1 provides long-lasting protection in mice (34, 37, 38). However, the ultimate goal of BPZE1 would be to offer early protection to prevent pertussis disease as early as possible after vaccination. In this study, we thus determined the rapidity of protection induced by BPZE1 and found that early protection is independent of the adaptive immune system but depends partially on TLR4 signaling.
Bordetella strains and growth conditions
Virulent B. pertussis BPSM, a streptomycin-resistant Tohama I derivative (39); BPBCTA1, a gentamicin-resistant BPSM derivative (34, 40); and the attenuated vaccine strain BPZE1 (29) were grown on Bordet-Gengou (BG) agar (Difco, Detroit, MI) supplemented with 1% glycerol, 10% defibrinated sheep blood, 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO), and 10 μg/ml gentamicin (Sigma-Aldrich) for 2 d at 37°C. The bacteria were then harvested and suspended in sterile PBS at the desired density. CFUs were quantified by plating the bacteria in serial dilutions onto BG blood agar and counted after 4 d of incubation at 37°C.
Vaccination and challenge infection
Three-week-old and 8-wk-old BALB/C, C57BL/6, and SCID mice, purchased from Charles River Laboratories (l'Abresle, France); C3H/HeN and C3H/HeJ (TLR4-deficient) (41) mice purchased from Harlan (Gannat, France); Myd88−/− mice (42, 43) obtained from S. Akira and bred inhouse; and Tlr2−/− and Tlr4−/− mice purchased from the Centre de Développement des Techniques Avancees pour l'expérimentation animale (Orléans, France) were maintained in individually ventilated cages and handled in a vertical laminar flow cabinet (Class II A2; ESCO, Hatboro, PA). All experiments complied with current national and institutional regulations and ethical guidelines (C59-350009; Institut Pasteur de Lille, Lille, France). Mice were i.p. sedated with a mix of Ketamine/valium/atropine and infected or immunized i.n. by pipetting 20 μl of PBS containing the indicated amounts of bacteria (BPBCTA1 and/or BPZE1), and the lung colonization was evaluated as described (44). For the coadministration experiments, 20 μl of BPZE1 was instilled in one nostril and 20 μl of BPBCTA1 was instilled in the other. For challenge infection, mice were i.n. infected with ∼1.6 × 106 BPBCTA1 in 20 μl of PBS. Lung colonization was determined 3 h and 7 d later.
Ab determination
Sera were collected, and Ab titers were estimated by ELISA as described (44). Briefly, 96-well plates (Maxisorp; Nunc) were coated overnight at 4°C with 100 μl of whole BPSM cell lysate diluted at 10 μg/ml. BPSM lysates were prepared from a 200-ml bacterial culture grown in modified Stainer Scholte medium, as described (45), until stationary phase. The bacteria were then harvested by centrifugation, resuspended in 10 ml of PBS with a mixture of antiproteases complete mini (Roche), and lysed by serial passages through a French pressure cell. The protein concentration was quantified using BCA test (Thermo Fisher Scientific), following manufacturer instructions. Serum samples were then added in 2-fold serial dilutions and incubated overnight at 4°C. Goat anti-mouse IgG (H+L) horseradish or goat anti-mouse IgA peroxidase-conjugated Abs (Southern Biotechnologies Associates, Birmingham, AL) were then added for 1 h at 37°C. The ELISAs were developed using tetramethylbenzidine dihydrochloride (TMB; Interchim), according to the manufacturer's specifications. The results are expressed in titers defined as the reciprocal of the dilution giving an OD at 450 nm three times that of the conjugate control.
Evaluation of T cell responses
Spleen cells from individual naive or BPZE1-vaccinated mice were harvested 6 or 28 d after BPZE1 administration and stimulated with whole BPSM cell lysate at 10 μg/ml or were left unstimulated, as described (29). Supernatants were harvested from triplicate cultures after 72 h incubation at 37°C in the presence of 5% CO2, and IFN-γ and IL-5 concentrations were determined by immunoassays (BD OptEIA Set; Pharmingen, San Diego, CA).
The results were analyzed using the unpaired Student t test and the Kruskal-Wallis test followed by the Dunn posttest (GraphPad Prism Program), when appropriate. Differences were considered significant at p < 0.05.
Early protection against challenge induced by BPZE1
To determine how fast BPZE1 is able to protect infant mice against challenge with virulent B. pertussis, 3-wk-old BALB/C mice were vaccinated with 20 μl of PBS containing 1 × 106 CFU of BPZE1 by the i.n. route. Control mice received 20 μl of PBS without bacteria. One, two, three, or four weeks later, 10 mice of each group were challenged with virulent B. pertussis BPBCTA1. Five mice per group were sacrificed 3 h after challenge to validate that the groups were similarly infected (results not shown). Seven days later, the five remaining mice per group were sacrificed to assess the BPBCTA1 colonization in the lungs. As shown in Fig. 1, already 1 wk after vaccination, BPZE1-immunized mice had the bacterial load reduced by approximately two orders of magnitude compared with the nonvaccinated controls. This clearance was further increased by approximately two and three orders of magnitude 2 and 4 wk after vaccination, respectively.
BPZE1-induced early protection against mouse lung infection by B. pertussis. BPZE1-vaccinated (n = 5 per time point, black bars) or mock-vaccinated (n = 5 per time point, white bars) BALB/C mice were infected 1, 2, 3, or 4 wk after vaccination, as indicated, with virulent B. pertussis BPBCTA1, and lung homogenates were plated in serial 10-fold dilutions on gentamicin-containing BG blood agar plates 1 wk postinfection. The bacteria were counted after 4 d of incubation at 37°C. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01.
Early protection provided by BPZE1 is independent of B. pertussis–specific Ab or T cell responses
Vaccine-induced protection against B. pertussis has been shown to be mediated by both anti–B. pertussis Abs and a Th1-type cellular immune response (17, 33). To investigate whether the protection obtained with BPZE1 1 wk after vaccination was linked to a significant rise in Ab levels, anti-BPSM lysate IgG and IgA were measured in the blood of vaccinated mice 1 d before each challenge time point. As shown in Fig. 2A, 2B, 1 wk after immunization with BPZE1, anti-BPSM IgG and IgA were undetectable. IgG and IgA levels against B. pertussis extracts started to rise significantly only after, respectively, 2 and 3 wk in the BPZE1-treated mice.
Ab and cytokine responses after BPZE1 vaccination. Serum IgG (A) and IgA (B) titers were measured against a total B. pertussis BPSM lysate 1, 2, 3, and 4 wk after BPZE1 administration, as indicated (black bars), and compared with the respective titers in mock-vaccinated mice (white bars). IFN-γ concentrations (C) were measured in culture supernatants of splenocytes from BPZE1-vaccinated mice stimulated with total B. pertussis BPSM lysate (black bars) and compared with those from mock-vaccinated mice (white bars) 1 wk (w1) or 4 wk (w4) after vaccination. The limit of detection of the IFN-γ response was 30 pg/ml. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01 between vaccinated and nonvaccinated mice.
In addition, the B. pertussis–specific cellular responses induced by BPZE1 were assessed 1 and 4 wk after vaccination by measuring IFN-γ and IL-5 in the culture supernatants of spleen cells incubated with BPSM extracts. As shown in Fig. 2C, the B. pertussis–specific IFN-γ response was below detectable levels 1 wk after vaccination but was significant 4 wk after vaccination. Unlike IFN-γ, the production of IL-5 was not induced upon stimulation of splenocytes from BPZE1-vaccinated mice with BPSM extracts, neither at 1 wk nor at 4 wk after vaccination (data not shown), which is consistent with the fact that BPZE1 induces a Th1-type of immune response in mice (33). The early protection induced by BPZE1 already observed 1 wk after administration appears, thus, to be independent of significant Ab and Th1 or Th2 cytokine production.
BPZE1-induced protection during coadministration with virulent B. pertussis
To further examine the early protection provided by BPZE1, mice were simultaneously infected with BPZE1 and a virulent B. pertussis strain, and the colonization of the virulent strain was followed over time and compared with that of the same strain in mice infected with the virulent strain alone. To easily distinguish the vaccine strain from the virulent strain, a BPSM derivative that is resistant to gentamicin (BPBCTA1) was used. BALB/C mice were thus infected with a mixed inoculum of 1 × 104 CFU of virulent strain BPBCTA1 and 1 × 106 CFU of BPZE1. A control group received 1 × 104 CFU of BPBCTA1 alone. The colonization of BPBCTA1 was followed by counting gentamicin-resistant CFUs in the lungs 3, 7, 14, 21, and 28 d after administration. Compared with BPBCTA1 infection alone, the coadministration with BPZE1 resulted in substantially faster clearance of the virulent strain (Fig. 3A). A significant difference between the two groups was already evident 3 d after administration. This difference increased over time and resulted in total clearance of the BPBCTA1 strain after 21 d in the coadministered animals. In the mice infected with BPBCTA1 alone, ∼100 CFU were still detectable 28 d postinfection.
Coadministration experiments with BPZE1 and BPBCTA1. BALB/C (n = 5 per group and time point) (A) and SCID mice (n = 5 per group and time point) (B) were coadministered with 1 × 104 CFU of B. pertussis BPBCTA1 together with 1 × 106 CFU of BPZE1 (black squares) or were infected with 1 × 104 CFU of BPBCTA1 alone (black dots), and the BPBCTA1 load was measured in the lungs at the indicated time points by plating lung homogenates in serial 10-fold dilutions on gentamicin-containing BG blood agar plates. The bacteria were counted after 4 d of incubation at 37°C. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, when coadministered mice were compared with monoinfected mice.
BPZE1-induced early protection in SCID mice
As BPZE1-induced protection was observed as early as 3 d after vaccination, at a time when no B. pertussis–specific Ab nor cytokine response could be measured, we reasoned that this early protection may be independent of an adaptive immune response. To test this hypothesis, a coadministration experiment was performed using SCID mice, as they lack T and B lymphocytes and are therefore defective in adaptive immunity. As shown above for the BALB/C mice, a significant difference in colonization by BPBCTA1 was observed as early as a few days postinfection between the coadministered mice and the mice infected with the virulent strain alone (Fig. 3B). However, unlike the BALB/C mice, the SCID mice were not able to clear the BPBCTA1 infection, even when BPBCTA1 was coadministered with BPZE1, which is consistent with the notion that long-term clearance of B. pertussis is mediated by Ag-specific Ab and T cell responses (17). These results thus confirm that the early protection offered by BPZE1 is independent of adaptive immunity.
Role of MyD88 in early protection induced by BPZE1
The early protection may be mediated by bacterial competition between BPZE1 and virulent B. pertussis or by BPZE1 stimulation of innate immune defenses. To distinguish between these two possibilities, we performed the coadministration experiment in MyD88-deficient mice, which are severely affected in innate immune signaling (46). Given the lack of published information about the fate of MyD88-deficient mice postinfection with B. pertussis, we first infected Myd88−/− mice with 1 × 104 CFU of virulent B. pertussis BPBCTA1 or with 1 × 106 CFU of BPZE1. The mice infected with 1 × 104 CFU of the virulent strain lost weight (Fig. 4A) and died (Fig. 4B) within 10 d postinfection. In contrast, i.n. administration of BPZE1 to Myd88−/− mice did not induce weight loss, even with 100-fold higher dose, and all mice survived up to day 30 postinfection, when the experiment was terminated (Fig. 4A, 4B). Virulent B. pertussis induced strong inflammation in the lungs of mice, whereas BPZE1 did not (Fig. 4C). Furthermore, Myd88−/− mice were unable to control the B. pertussis growth in the lungs, and 1 wk postinfection, just before death occurred, Myd88−/− mice carried 100-fold more BPSM organisms than the MyD88-sufficient C57BL/6 mice (Fig. 4D).
Infection of Myd88−/− mice. Myd88−/− mice were infected with 1 × 104 CFU of B. pertussis BPBCTA1 [n = 9, black dots in (A); n = 5, full line in (B)] or with 1 × 106 CFU of BPZE1 [n = 10, black squares in (A); n = 5, dotted line in (B)]. The weight loss (A) and survival (B) of the mice were measured at the indicated time points postinfection. (C) Lung of Myd88−/− mice infected with 1 × 104 CFU of BPBCTA1 (left) or 1 × 106 CFU of BPZE1 (right). (D) The BPBCTA1 load was measured in the lungs of C57BL/6 (n = 5 per time point, white bars) and Myd88−/− mice (n = 5 per time point, black bars) at the indicated time points postinfection by plating lung homogenates in serial 10-fold dilutions on gentamicin-containing BG blood agar plates. The bacteria were counted after 4 d of incubation at 37°C. The results are expressed as mean ± SEM. **p < 0.01.
These observations allowed us to investigate whether coadministration of BPZE1 with virulent B. pertussis would prevent B. pertussis–induced death of the Myd88−/− mice. As shown in Fig. 5, Myd88−/− mice died rapidly postinfection with 1 × 104 CFUs of BPBCTA1, regardless of the coadministration of BPZE1, indicating that BPZE1 did not protect Myd88−/− mice from death induced by virulent B. pertussis.
Mortality of Myd88−/− mice after single or coadministration. Myd88−/− mice were infected with 1 × 104 CFU of B. pertussis BPBCTA1 together with 1 × 106 CFU of BPZE1 (n = 10 per group, dotted line) or with 1 × 104 CFU of BPBCTA1 alone (n = 10 per group, black line), and survival of the mice was monitored over time up to 15 d postinfection. ns, not significant.
Altogether, these results indicate that the early protection afforded by BPZE1 is not due to competition between BPZE1 and virulent B. pertussis but requires the innate immunity arm via MyD88-dependent signaling.
Role of TLR4 and TLR2 in early protection induced by BPZE1
MyD88 is a cytosolic adapter protein that plays a central role in transducing signals perceived by TLRs to activate transcription factors, such as NF-κB and AP-1, thereby leading to the induction of proinflammatory cytokine production. Among the TLRs that signal through MyD88, TLR2 (47) and TLR4 (48) have been reported to recognize B. pertussis agonists, respectively, lipoproteins and lipo-oligosaccharides (LOS). We therefore investigated the role of these TLRs in the early BPZE1-mediated protection. Coadministration of BPZE1 with BPBCTA1 resulted in a significant decrease in BPBCTA1 load in the lungs 1 wk postinfection, as compared with BPBCTA1 infection alone in both C3H/HeJ (harboring a mutation in Tlr4) and C3H/HeN control mice (Fig. 6A). However, the BPBCTA1 bacterial load in the lungs of the TLR4-defective mice was approximately two orders of magnitude higher than in the lungs of the C3H/HeN controls. C3H/HeJ and C3H/HeN mice are mouse strains that differ in a single-nucleotide polymorphism in the Tlr4 gene, which leads to the production of ineffective TLR4 signaling (49). Although C3H/HeJ mice respond much less to LPS than C3H/HeN mice, their response to LPS is not totally absent (50). In a repeat experiment, we therefore used Tlr4−/− mice and compared them to Tlr2−/− mice. Whereas a significant decrease in BPBCTA1 burden was observed in the lungs of BPZE1-BPBCTA1–coadministered Tlr2−/− mice 1 wk postinfection, as compared with mice infected with BPBCTA1 alone (Fig. 6B, left columns), this was not observed in the Tlr4−/− mice (Fig. 6B, right columns), indicating a role of the TLR4 rather than the TLR2 signaling pathway in the early protection induced by BPZE1.
Early BPZE1-mediated protection of TLR4- or TLR2-deficient mice. (A) C3H/HeN control mice (n = 4 per group and time point, white bars) or C3H/HeJ TLR4-deficient mice (n = 4 per group and time point, black bars) were infected with 1 × 104 CFU of BPBCTA1 together with 1 × 106 CFU of BPZE1 (BPBCTA1+BPZE1) or with 1 × 104 CFU of BPBCTA1 alone (BPBCTA1). (B) Tlr2−/− (n = 5 per group and time point, left bars) and Tlr4−/− (n = 5 per group and time point, right bars) were infected with 1 × 104 CFU of BPBCTA1 together with 1 × 106 CFU of BPZE1 (BPBCTA1+BPZE1, black bars) or with 1 × 104 CFU of BPBCTA1 alone (BPBCTA1, white bars). The BPBCTA1 load was measured in the lungs 1 wk postinfection by plating lung homogenates in serial 10-fold dilutions on gentamicin-containing BG blood agar plates. The bacteria were counted after 4 d of incubation at 37°C. The results are expressed as mean ± SEM. *p < 0.05, **p < 0.01.
We report in this study that the live attenuated nasal pertussis vaccine BPZE1 provides significant protection against virulent B. pertussis within days after vaccination, whereas Ab and T cell responses are detectable only after several weeks. This early protection is reminiscent of that induced by a live attenuated nasal Bordetella bronchiseptica vaccine, which protected dogs challenged with virulent B. bronchiseptica as early as 48 h after vaccination before Abs to B. bronchiseptica could be measured (51). After the initial phase, a further increase in protection correlated with an increase in IgA titers to B. bronchiseptica. Similarly, we found in this study that after the early stage, when vaccination reduced the bacterial burden by two orders of magnitude in the absence of detectable anti–B. pertussis Abs and T cell responses, further protection increased with increasing specific IgG and IgA. Ab- and T cell–dependent protection in the later stages was confirmed by coadministration of BPZE1 and virulent B. pertussis in SCID mice, lacking both T and B lymphocytes. In these mice, coadministration resulted in a significant protection against the virulent strain at day 7 postinfection, with an ∼100-fold difference over mice infected with the virulent strain alone. However, the SCID mice did not clear the infection, consistent with the role of T and B cells in clearance (17). Interestingly, however, the coadministered mice continued to carry ∼100-fold less virulent bacteria than the mice infected with the virulent strain alone over the entire course of the experiment (i.e., for at least 60 d), suggesting that the innate immune effects are relatively long-lasting. It is conceivable that BPZE1 induces epigenetic and metabolic reprogramming of innate immune cells via TLR signaling, which may lead to relatively long-lived trained immunity, as has been observed for monocytes incubated in the presence of bacillus Calmette-Guérin (52). However, this remains to be investigated.
That the early protection afforded by BPZE1 was due to innate immune stimulation and not to bacterial competition was demonstrated by the loss of protection in Myd88−/− and Tlr4−/− mice. TLR2 did not appear to be essential for early protection by BPZE1, as Tlr2−/− mice were still significantly protected by the coadministration. Myd88−/− mice are highly susceptible to B. pertussis infection and died within a few days postinfection, regardless of coadministration with BPZE1. Mortality was paralleled with weight loss, exacerbated bacterial burden, and very profound lung inflammation. Consistent with previous reports (53), the phenotype of Tlr4−/− mice was not as severe as that of Myd88−/−, as none of the Tlr4−/− mice died during the experiments. This is unlike high mortality observed when TLR4-defective mice are infected with an inoculum as low as 1000 CFU of B. bronchiseptica (54). As BPZE1 also protects mice against B. bronchiseptica (55), it would be interesting to determine whether coadministration of BPZE1 or live attenuated B. bronchiseptica vaccine together with virulent B. bronchiseptica would prevent mortality of the TLR4-defective mice.
Although protection of the TLR4-defective mice against mortality could thus not be investigated in the B. pertussis model, the effect of the coadministration on the bacterial burden was studied, and we found that the bacterial burden of Tlr4−/− mice was not significantly diminished by the coadministration, indicating that TLR4 signaling is an important factor for early BPZE1-mediated protection. However, unlike Tlr4−/− mice, C3H/HeJ mice expressing defective Tlr4 were protected to a certain degree by BPZE1, albeit not as well as the C3H/HeN control mice. This observation suggests that defective TLR4 in the C3H/HeJ mice may still allow residual protection, in line with the fact that C3H/HeJ mice are still able to respond to certain TLR4 agonists (50).
Our observations are consistent with several studies describing a protective role of TLR4 in the control of pertussis (56), including during the early stages of infection (48, 57). Higgins et al. (58) found that upon infection with B. pertussis, the initial inflammatory cytokine response and neutrophil infiltration into the lungs were much reduced in TLR4-deficient C3H/HeJ mice compared with C3H/HeN control mice. This was paralleled by a faster clearance of B. pertussis in the latter than in the former. When the neutrophils were depleted in the C3H/HeN mice early during infection, bacterial burden was increased (48), suggesting an important role of neutrophils in B. pertussis clearance. Transcriptomic studies have shown that during the first hours of B. pertussis infection, approximately140 genes are expressed in the pathways of immunity and cytokine/chemokine responses in C3H/HeN controls but not in TLR4-deficient C3H/HeJ mice (48). Later, during the infection, C3H/HeJ mice showed enhanced inflammation and a decrease in IL-10 production (58), arguing that one of the mechanisms of TLR4-dependent protection against B. pertussis involves innate IL-10 production limiting inflammatory pathology in the lungs.
TLR4 was also shown to be crucial for vaccine-induced immunity to B. pertussis. Vaccination with wP failed to protect TLR4-deficient C3H/HeJ mice from B. pertussis challenge, in contrast to C3H/HeN control mice (59). This difference was less marked with aP and was linked to an impaired Th17 response in C3H/HeJ mice upon wP vaccination. In humans, a single-nucleotide polymorphism in the Tlr4 promoter region has been associated with PT-specific IgG responses after wP vaccination (60), suggesting that TLR4 signaling may also be important in humans for the induction of optimal immune responses to wP. This notion is supported by the observation that single-nucleotide polymorphisms in other genes involved in TLR4 signaling, such as TOLLIP and IRAK1, were also associated with anti-PT IgG responses after wP vaccination (61). A polymorphism in the coding region of Tlr4 has also been associated with the strength and persistence of anti–B. pertussis Abs after aP boosting of wP-primed adolescents, whereas there was no effect of this polymorphism on Ab responses to tetanus and diphtheria toxoids, coadministered with aP (62).
Taken together, these studies thus show that TLR4 signaling plays a dual role in B. pertussis control by inducing an early inflammatory response, which leads to the recruitment of B. pertussis–clearing neutrophils, and by increasing B. pertussis–specific Ab (and possibly T cell) responses, aiding in the clearance at later stages. It is thus likely that the TLR4-dependent protective effect of BPZE1 relies on both.
The main B. pertussis agonist of TLR4 is the lipid A moiety of LOS (63). However, PT has also been described to signal through TLR4 (64). It remains to be established which TLR4 agonist plays the dominant role in the early innate immune protection against B. pertussis. PT is a multimeric protein composed of a receptor-binding moiety called the B oligomer (PTB) and an A subunit expressing ADP-ribosyltransferase activity (65). Both active PT and PTB have been shown to signal through TLR4 (66). However, whereas PT activates both the MyD88-dependent and the MyD88-independent TLR4 pathways, PTB, lacking the ADP-ribosyltransferase moiety, only activates the MyD88-independent pathway. In BPZE1, the enzymatic activity of the PT A subunit has been genetically abolished (29), yet we found in this study that the early protective effect induced by BPZE1 requires signaling through the MyD88-dependent pathway. Therefore, it is unlikely that PT plays a dominant role in BPZE1-mediated innate immune protection, leaving LOS as the prime candidate. Minor modifications of lipid A, naturally occurring in various B. pertussis strains, can have profound effects on TLR4 activation (67). It may therefore be interesting to investigate whether these changes, which could be genetically engineered in BPZE1, affect the early protection described in this study.
The fact that the pathologic condition induced by B. pertussis was much more severe in Myd88−/− than in Tlr4−/− mice suggests that in addition to the TLR4 signaling pathway, other MyD88-dependent pathways are also critical for the control of B. pertussis. MyD88 is not only an adaptor for TLR4 and TLR2 but also for other TLRs and for the IL-1βR (for recent review, see Ref. 68). Although so far not yet investigated, it is possible that some of these pathways may be involved as well.
Unlike other TLRs, such as TLR5, 7, 8, and 9, which bind directly to MyD88 via their cytoplasmic domain, bridging TLR4 and TLR2 to MyD88 requires an additional adaptor protein called Mal or TIRAP (69). Bernard et al. (70) have shown that the bacterial burden in the lungs of Mal−/− mice was 10- to 100-fold higher than in the lungs of control mice after B. pertussis infection with an initial load of ∼1 × 104 CFU, but the mice eventually cleared the infection. Approximately half of the mice died between day 14 and 21 postinfection, and the Mal−/− mice showed considerably more inflammation in their lungs than the Mal+/+ mice. Although not directly assessed by a head-to-head comparison, these findings indicate that the Mal−/− mice have an intermediate patho-phenotype upon B. pertussis infection between the Myd88−/− mice and the Tlr4−/− mice. This intermediate patho-phenotype suggests that, in addition to TLR4, Mal-dependent signaling may also be important via other pathways to control B. pertussis infection. Mal also bridges TLR2 to MyD88. However, we found that TLR2 was not required for protection by BPZE1. We cannot exclude that Tlr2−/−Tlr4−/− double knockout mutants may be more affected than the Tlr4−/− single mutant. Alternatively, Mal may play a role independent of MyD88 signaling, as in addition to proteins of the MyD88 interactome, Mal can also bind to a number of other proteins not involved in MyD88 signaling (71).
The work described in this study has important practical implications for the development of BPZE1 as a live vaccine. Although the severely immunocompromised Myd88−/− mice rapidly succumbed postinfection with as little as 1 × 104 CFU of virulent B. pertussis, even up to 100-fold more BPZE1 did not induce pathology, weight loss, and death in these mice, adding an important piece of information to the safety profile of BPZE1. A second important observation relevant to the BPZE1 safety profile is the fact that coadministration with virulent B. pertussis did not result in exacerbated outgrowth of the virulent strain, when compared with infection by virulent B. pertussis alone. This might have been a concern, as Foster et al. (72) have shown that this is the case with the live attenuated Salmonella vaccine. In this model, the numbers of virulent Salmonella enterica were increased in mice, and disease was exacerbated by the presence of a live attenuated Salmonella typhimurium vaccine strain. In this study, we show that, instead, BPZE1 protected against virulent B. pertussis in coadministration experiments. If these observations can be extrapolated to humans, the BPZE1 vaccine can thus be safely administered, even to subjects with already established B. pertussis infection. This appears to be particularly important, as several reports have shown that silent, asymptomatic B. pertussis infection is much more frequent in human populations than previously appreciated (73–77).
In conclusion, this study describes an additional mechanism by which the live attenuated BPZE1 provides protection against B. pertussis infection. In addition to already described CD4+ T cells and Abs (33) and mucosal IgA together with resident memory T cells (34) involved in clearance of B. pertussis in the lungs and nasal tissues of mice, this report identifies a TLR4-dependent innate immune mechanism in early protection by BPZE1. Protection by BPZE1 through the innate immune arm may potentially explain some of the off-target effects observed in BPZE1-vaccinated mice (78), as BPZE1 has been shown to offer protection against a variety of inflammatory diseases, such as influenza (79) and allergic asthma (31, 80). It will be interesting to investigate whether the protective mechanism against these diseases also involves TLR4 signaling pathway.
C.L. and N.M. are consultants for ILiAD Biotechnology, the company that develops the live attenuated B. pertussis vaccine BPZE1. Both are inventors of patents that have been licensed by ILiAD Biotechnology. The other authors have no financial conflicts of interest.
We thank Dr. Shizuo Akira for generously providing the Myd88−/− mouse strain.
This work was supported by a grant from the European Commission under Grant Agreement 201502 (Child-Innovac).
Abbreviations used in this article:
acellular vaccine
Bordet-Gengou
i.n.
intranasal(ly)
lipo-oligosaccharide
pertussis toxin
whole-cell vaccine.
Copyright © 2019 by The American Association of Immunologists, Inc.
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The Journal of Immunology December 15, 2019, 203 (12) 3293-3300; DOI: 10.4049/jimmunol.1901102
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Surging Need to Strengthen Data Security
Sam Masiello, CISO, TeleTech [NASDAQ: TTEC]
The threat landscape continues to evolve on seemingly a minute-by-minute basis. With cyber criminals looking to breach organizations both from the outside-in and from the inside-out, both companies and their employees need to continue raising their game relative to the technical controls being used to prevent and detect compromise as well as the awareness methods being used in order to ensure that the people within the company know what a potential threat looks and acts like. Threats such as Business Email Compromises (BECs), phishing, and ransomware continue to evolve and become more sinister resulting in companies scrambling to figure out how they are going to protect their employees, network, intellectual property, and bank accounts. Given the ongoing success of these threat vectors and the staggering numbers accumulated by these attacks both in numbers of victims and monetary losses measured in the billions, they will not be going away anytime soon. It is too large of a market for cyber criminals to ignore.
Significance of Cyber Security Awareness among Employees
It is important to maintain a delicate balance between technical controls used to prevent and detect cyber intrusion and carefully targeted awareness campaigns of how intrusions occur within the organization and how those intrusions manifest themselves within various parts of the business. For example, ensuring software developers understand the importance of a secure software development lifecycle, educating finance and treasury workers on BECs, and teaching people throughout the organization about the dangers and potential damages that can result from phishing and malware. Ultimately, everyone wants to do the right thing and does not want to be Patient Zero of how a malware infection or a system compromise brought about potentially thousands of hours of unproductive time or millions of dollars of fines upon the organization. To that end, however your employee population needs to be aware of how to identify when a threat is in front of them and know how to react and respond followed by the implementation of strong technical controls on the backend to support them if they make a mistake or can prevent the threat from ever appearing in front of the employee giving them the opportunity to make an incorrect decision in the first place.
If the security organization introduces too many obstacles to basic productivity, users will look for and invent ways to establish work-arounds
Understanding Requirements of Reliable Network
One of the most important things to remember is that your network is everywhere. You can no longer focus on the perimeter and believe that you will be protected enough because your network is borderless. You also cannot just focus on the endpoint because the company data that you are responsible for protecting also lives out in the cloud. Your network is wherever your employees are. This means that your network is in an office, in a hotel, on an airplane, in a taxi, on a boat, or everywhere in between. This makes the job of the cyber security professional more complex and difficult to control on a daily basis. One of the most important aspects of building a successful cyber security program is to ensure that it can incorporate itself into the business and protect the company's assets while at the same time not introducing too many barriers for the employee population to do their jobs. Such an approach to security can often lead to the opposite of the visibility that the security teams needs to be effective in their jobs. If the security organization introduces too many obstacles to basic productivity, users will look for and invent ways to establish work-arounds such that the security team may lose visibility into how data is traveling into, out of, and across the network. This situation makes it nearly impossible for the security team to be effective, but is an avoidable situation that they ultimately brought upon themselves. Security and productivity must maintain a balance. Skew too heavily towards one or the other and the results can be disastrous.
More regulation isn't the answer. In fact, as the government races to design and pass new regulations and if it does so without enough consultation with the private sector, the wrong regulations could actually hinder innovation. Such a result would be counterproductive to continued advancement in the space as cyber criminals and their tactics continue to evolve.
The issue that many companies struggle with is they equate compliance with security, or assume that compliance is a sufficient baseline for security. This couldn't be further from the truth. While there may be some sound security practices within the various regulatory frameworks that our companies are beholden to adhere to, compliance is a point in time snapshot of specific controls and neither represents ongoing operational nor organizational practice or maturity. Compliance should be a validation of the existence of controls already being in place and inherent in how the company operates, not a task list of items that are addressed in an adhoc fashion when it is time for the annual audit.
Compliance in Cloud
One thing that I have found myself repeating more times than I can count to folks who are more infrastructure or operations minded is that you cannot buy your way to security or compliance. All too often executives see terms like "FISMA Compliant" or "PCI Compliant" when looking to purchase solutions from IaaS providers believing that by partnering with these companies that they are magically compliant or that all they need to do is throw a few logical controls around the environment to logically separate it from the rest of your infrastructure and your job is done. While cloud computing may give you the ability to quickly and easily scale up and scale down the infrastructure as the needs of your business expand and contract, they merely provide the capability to make it easier to meet your control objectives, not necessarily as a fast track to compliance.
Another area that companies are currently struggling with as it relates to the migration to the cloud is how to protect and control data once it is off network. Traditional DLP solutions do a good job of tracking data around the network, on servers, and on endpoints. Where the existing paradigm falls short is how to do the same when that data is stored in Google Drive, Microsoft OneDrive, Dropbox, Box.com, or the myriad of other online file storage and collaboration tools available. How do you track who that data is being shared with? How do you know who is copying, downloading, or printing those documents? How do you track when that data might be moving from cloud to cloud? How do you control that data once it is accessible on a mobile device? While there are some burgeoning solutions in the space looking to tackle this problem, it is still something that many security teams are still trying to wrap their arms around, particularly since many organizations, whether they know it or endorse it or not, are using multiple file sharing applications today, even if most of them are falling into the Shadow IT bucket.
A Word for CIOs
The role of the CISO has changed dramatically over the past few years. When I was in my first senior security role the job was primarily technical. While there may have been exposure to executive management, the most successful security leaders were the ones that were less known. In other words, if executives weren't breathing down your neck, nothing was wrong and you were doing your job well.
Fast forward to today and the landscape has significantly changed. Today's cyber security leader not only is expected to have executive presence, but is a regular attendee in Board meetings, has control over a budget to control their program, and has a strong knowledge of both security and enterprise risk management. The job has moved from one that is primarily technical to one where the person in the role must have a good balance of technical savvy, business expertise, and the ability to build effective cross-functional relationships while making reasonable and balanced risk/security decisions.
New technologies and a constantly evolving threat landscape continue to make the CISO role both challenging and extremely rewarding. Ever evolving regulatory and privacy standards and the constantly changing threat landscape make the CISO role one where you are constantly learning and updating your vision and priorities of the types of problems you need to consider to keep your company, its users, and its clients safe. I wouldn't want it any other way!
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Movie, TV Reviews, Discussion, News
The Roth Project Online Comic Launched with Song Dedicated To Eddie Van Halen
November 2, 2020 November 1, 2020 ~ Todd Russell	~ Leave a comment
David Lee Roth's The Roth Project: weird, wonderful or …? https://therothproject.com/
Say what you want about the legendary frontman David Lee Roth. Many believe that he can't sing any more, but he sure is a way out there guy and wants to remains creative. I'm holding hope that Wolfgang Van Halen and Eddie's drummer brother Alex mine some musical gems out of Eddie's 5150 studio (we know the music is there!) and at some respectful time in the future and, as the Doobie Brothers would say, let us "Listen to the music!"
In the meantime, we've got whatever Sammy Hagar is doing in his various band projects and the industrious, fiercely unusual David Lee Roth.
Roth's newest creative project is — yes, totally serious — an online comic narrated by him, complete with music featuring music from John 5.
David Lee Roth has launched The Roth Project, a new online comic narrated by the legendary VAN HALEN singer, with music from John 5 on guitar and bass, Greg Bissonette on drums, Brett Tuggle on keyboards, and Luis Conte on percussion. The 17-chapter comic also includes five songs Roth co-wrote with John 5 and recorded several years ago at Henson Recording Studios in Hollywood, California: "Giddy Up", "Somewhere Over The Rainbow Bar And Grill", "Alligator Pants", "Lo-Rez Sunset" and "Manda Bala".
David Lee Roth Launches 'The Roth Project' Online Comic Featuring Five Songs Co Written With John 5 – Blabbermouth.net
Within this depth of music and story comes a track called "Somewhere Over The Rainbow Bar and Grill" dedicated to Eddie Van Halen. It's a curious track that you can listen to via YouTube:
Hmm, singing is definitely Dave, and the music is … well, this song does kind of grow on me after a few plays. Like what if Judy Garland and David Lee Roth had some fantasy night together and produced this for fun.
I'd rather hear him get back with Steve Vai and do that rock thing, but this is acoustic distraction will have to suffice. Again, even if it's not your cup of musical tea, it's great to see creative people doing something creative. We haven't seen DLR do anything new musically in a while.
There was once a time he was going to make a movie and that fell through. Perhaps this online comic will lead to something in the streaming world? I'd be interested in seeing DLR on some kind of screen — streaming, whatever. What do you think?
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THE RISE OF THE AMINO
FIRST WE HAD THE RINOS TO CONTEND WITH. NOW WE HAVE THE AMINOS.
Christie Wagner
Image courtesy: Fox News
We have more than enough RINOS (Republicans in name only) polluting the Party. One of the most disgraceful recidivists is from Utah, Senator Mitt Romney. Romney is like the wallflower at a dance. Nobody asked him to dance. Nobody wants him for president, and nobody wants him in the Cabinet.
He tapped President Trump on the shoulder hoping to dance as his Secretary of State, after repeatedly badmouthing him. Yet, he was left on the dance floor, all alone and embarrassed.
I have some advice for you, buddy. #1 You need a common sense transplant. You don't castigate somebody–repeatedly–then expect him to pat you on the head, say "Good boy," and appoint you as a Cabinet Secretary. #2 Just leave the Republican Party. You're not needed or wanted. You're like a would-be transgender who's too cowardly to make the commitment. Accept who you really are. Go get the surgery. Cut yourself off from the Republican Party. Have the balls, before they're cut off, to make the change. You vote with the Democrats all the time anyway.
And now, ladies and gentlemen, we have a new species which I have named the AMINO: AMERICAN IN NAME ONLY.
The three most dangerous specimens of this species are JoJo Demento Biden, the Chairman of the Joint Chiefs of Staff, General Mark Milley, and Secretary of Defense, Lloyd Austin. Collectively, these men hold all responsibility for the Afghan bloodbath.
All three are morally diseased. Two out of the three are incompetent with one being an intellectual flyweight, the other being cognitively impaired.
Traitorous Mark Milley is by no means intellectually inferior.
With two Ivy League degrees–a Princeton B.A. in Politics and a Columbia Master's in International Relations–and a second M.A. in National Security and Strategic Studies from the Naval War College, he knows better.
It's just that Milley has decided to get on all fours and spread his butt cheeks for the Left by quoting Chinese Communist Mao Zedong and destroying our military with Marxist-founded Black Lives Matter and Antifa wokeisms. All this is to ensure Milley a cushy job after he leaves the military. However, he could easily accomplish this without dishonoring the military and besmirching our country. Instead, he's a corrupt and evil mind in attractive physical giftwrap.
On the other hand Lloyd Austin, the in-over-his-head Secretary of Defense, is an altogether different story.
Austin is unqualified. Moreover he typifies the new, clueless type of black who thinks he's free. Yet, in reality Austin only transitioned from one plantation to another: from the agricultural to the political. Just as eager to please his new Massa, Lloyd Austin, like all the new black plantation slaves, is anxious. Much like a shaking, peeing Chihuahua, desperate to be patted on the head for going along with woke Massa's agenda.
A toady of the Left. Austin is too stupid to even recognize the Left. That's why he hired a perpetually offended black man, Bishop Garrison, to force (à la the Communist playbook), woke indoctrination programs down the throats of our soldiers. Further, Garrison aimed to hunt down and purge fantasized enemies from the ranks of the military whom he hallucinates to be white supremacists and domestic terrorists.
Garrison rabidly tweeted that the "eradication of white nationalism (translation: patriotism by white folks), and white supremacy must be a top priority." Garrison's idée fixe is that racism is the top problem facing America. Interestingly, scuttlebutt is that Garrison holds BLM connections.
All this hatred-saturated, hysterical, delusional, white-demonizing and witch hunting is notwithstanding that the Pentagon already has an office of equity, diversity and inclusion.
Senior advisor Bishop Garrison exerts such hypnotic sway over lamebrained, racism-obsessed Austin, that Garrison reports exclusively to Austin.
Talk about a surefire way to avoid transparency and accountability!
Furthermore, and unprecedentedly, intellectual flyweight Austin engaged in a hiring orgy of appointing "advisors" for himself. Obviously he needs to compensate for and cover up his cerebral and experiential deficiencies.
Kicked upstairs because of melanin. But of course, it was Obama who appointed Austin to CENTCOM, even though Austin's credentials seem barely a cut above a Girl Scout Brownie's. Wikipedia lists 5 "badges" of his, none of which are awards or decorations. They're just i.d's, like the work badge you wear on your lanyard to get into the building. They are the United States Central Command badge; the Office of the Joint Chiefs of Staff i.d. badge; the Army Staff i.d. badge; XVIII Airborne Combat Service i.d. badge; and the 505th Infantry Regiment Distinctive Unit Insignia.
By the way, while Austin was with CENTCOM, he came to be known as "the invisible general" because he hid from the press. He's also been described as "intensely private." Could these be euphemisms for the fact that he's frightened? Scared to reveal his incompetence to the world? Thus, Austin realized the less he says, the better off he is.
Just what are Lloyd Austin's credentials?
After West Point, (and I'm beginning to wonder if he got admitted there because of melanin), he earned a Master's in Counseling. So basically Austin's a glorified guidance counselor who studied hand-holding. Not too useful on the battlefield, but academically a breeze compared to the tough stuff real generals study.
As for his assignments? They were cupcake. Was he battle-tested? You be the judge.
Here's the full list:
After graduation from West Point (must have been by the skin of his teeth because Austin's class ranking is nowhere to be found), Germany was his initial assignment. (I hear they have lots of fun rathskellers there.)
Austin was stationed at his alma mater twice; the Pentagon twice. He was at Fort Leavenworth, Kansas once; ditto MacDill in Tampa, Florida and Fort Stewart, Georgia. Further, he was deployed to Fort Bragg, North Carolina four times. (Oo! There must have been some big, spooky wars at Fort Bragg.)
When he was a Director of the Joint Staff under Chairman Mike Mullen, Admiral Mullen told Austin to bring some "diversity" to the Joint staff. It wasn't even Austin's idea! Though race-obsessed, he's obviously not an innovator, even on that issue. But Austin considers it a landmark of his career: ""People who might not have known Lloyd Austin began to know him."
Yeah, yeah. So he sprinkled some colored M&Ms among the white ones. Were the appointments based on color or merit?
Austin spent a little time in Iraq. He spent a little time in Afghanistan. All the more proof that Austin learned nothing about the cultural complexities of these countries. Nor does he understand the modus operandi of the Taliban, ISIS and Al-Qaeda, knowledge of which is directly applicable to and imperative for understanding Afghanistan and avoiding the current debacle. Moreover, Austin should be able to explain such complexities to his Cretin-in-Chief.
If I may go a little argumentum ad hominem here, the guy doesn't even look like a general. Not a speck of strength or iron discipline in his eyes. He looks like an overinflated Uncle Ben on the rice box.
Compare that with the credentials of Stormin' Norman Schwarzkopf: a general who led from the front, who earned 2 Purple Hearts and who was unparalleled for forging rapport with world leaders and cooperation with Allied Commanders, along with establishing excellent relations with the news media.
Here's just a partial list of General Schwarzkopf's accomplishments in addition to his spectacular scholastic and athletic achievements from childhood on.
A member of MENSA, the high school valedictorian with an I.Q. of 168, Schwarzkopf was accepted at West Point. After graduation he earned a Master's degree in Mechanical and Aerospace Engineering, which involved the study of missile mechanics.
His outstanding active duty service included many commands, including CENTCOM, where he headed an international coalition of 750,000 troops. His U.S. military awards are numerous. He is the only person ever to have been awarded the Distinguished Service Medal by the Army, Navy, Air Force and Coast Guard. In addition he was honored with a colossal number of international military awards and an honorary knighthood from Queen Elizabeth.
General Schwarzkopf fought in 3 wars: Vietnam, the Invasion of Grenada, and the Gulf War, along with 13 battles, including the Highway of Death
By comparison, this Austin dude is known to refer to himself in the third person. Delusions of grandeur? A legend in his own mind?
And last, as well as least, we have AMINO Joe, who might or might not know that his diapered butt is sitting in the Oval Office.
Not only is Biden an AMINO but in addition to and before his severe cognitive impairment Joseph Biden was always stupid.
How do I know this? It's been an open secret since Biden first set foot in Washington. (His plagiarism was well known, too, starting in his very first year of law school and continuing on into his Senate speeches.)
As a child I lived on Capitol Hill. Daddy would often take me to the Senate gallery. At age five, I could recognize every single senator by sight, knew their names and their states. In high school many of my classmates and schoolmates were the daughters of senators; congressmen; members of the cabinet; ambassadors; presidents and vice presidents of foreign countries; and one whose father was Chairman of the Democrat Party. We kids knew scuttlebutt long before the press got wind of it. Later as an adult I worked on the Hill, then on the White House staff.
Biden's only claim to fame is that he became a young widower when his first wife and infant daughter were killed in a car crash. All the gold-diggers swarmed him because he was one of very few bachelors on the Hill and at just 30 years of age, a lot younger, and much better looking, than most of the Senators and Congressmen.
Not only is Biden stupid but he is lawless and treasonous.
Just three of many examples are the following.
Biden admitted in a White House press conference that he knew his blocking of landlord evictions was illegal and unconstitutional.
He and his puppeteer AMINO henchmen are responsible for the slaughter, torture and beatings by the Taliban of thousands of Americans and our Afghan allies. Most of them will never be able to escape from Afghanistan.
He armed the Taliban with $82 million dollars' worth of weaponry, including 2,000 planes, plus military uniforms left behind in his unconscionable, stupid mode of withdrawal.
The Taliban are beating Americans and Afghans–even little children–at the Kabul airport. They confiscate passports and driver's licenses. Away from the airport, the Taliban hunt down our citizens and our Afghan allies, going door-to-door torturing and executing adults and children.
Biden is officially a global embarrassment.
Foreign heads of state are askance. Former UK Prime Minister, Tony Blair, decried Biden's withdrawal as "imbecilic". French president Emmanuel Macron told Biden in a phone call that it is his "moral obligation" to rescue his own countrymen and our Afghan allies. Colonel Richard Kemp, former head of all UK troops in Afghanistan, angrily speaks out that Biden should be court-martialed. Let me add to that- and tried for treason.
Biden disgraced the United States of America and made it both the world's laughingstock and object of contempt.
He is a non-stop danger to our national security. A dishonorable, stupid opportunist from the 1972 beginning of his political career; and now a mental defective. Thus, Biden must be removed from office immediately.
Nikita Kruschev prophetically said "We will take America without firing a shot. We do not have to invade the U.S. We will destroy you from within…." And the AMINOs are making Kruschev's prophecy come true.
Make your voice heard on Twitter: Milley's handle is @theJointstaff; Austin's is @SecDef; and Biden's is @POTUS.


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NanoBNK and Anglo African nominated for Finance for the Future 2018 Awards
NanoBNK CEO Jessen Valaythen says that the company, as well as Anglo African Investments, take pride in being nominated for the ICAEW Finance for the Future 2018 Awards alongside international players like HSBC, Salesforce and Coca-Cola. Beside competing with world-class groups, Valaythen says that "being recognised internationally in the financial inclusion field" is already an achievement.
Mauritian Anglo African Investments and its subsidiary NanoBNK are respectively nominated for the "Embedding an integrated approach Award" and the "Innovative Project Award". The award ceremony will take place in London, this October 16th.
The Finance for the Future Awards were developed in 2012 by the Institute of Chartered Accountants in England and Wales (ICAEW) and The Prince's Accounting for Sustainability Project (A4S) under the patronage of Prince Charles. The 24-member judging panel for the 2018 Awards comprises CEOs of international players like VEOLIA and Wates group as well as academics and senior executives from ICAEW.
Copyright 2018 Anglo African Group. All rights reserved.
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MALIPO POLEPOLE mazda demio 2012 with skyactive technology. hire purchase accepted with 50-60 percent deposit and repayment upto 12 months.
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SWAT Literature Database | Streamflow modeling in a fluctuant climate using SWAT: Yass River catchment in south eastern Australia.
Title: Streamflow modeling in a fluctuant climate using SWAT: Yass River catchment in south eastern Australia.
Authors: Saha, P. P., Zeleke, K. and Hafeez, M.


Malik related to me that Rabia ibn Abi Abd ar-Rahman said, "An Iraqi man came before Umar ibn al-Khattab and said, 'I have come to you because of a matter which has no beginning and no end.' Umar said, 'What is it?' The man said, 'False testimony has appeared in our land.' Umar said, 'Is that so?' He said, 'Yes.' Umar said, 'By Allah! A man is not detained in Islam without just witnesses.' " Malik related to me that Umar ibn al-Khattab said, "The testimony of someone known to bear a grudge or to be unreliable is not accepted."
From Malik from Abuz-Zinad that Umar ibn Abd al-Aziz wrote to Abd al-Hamid ibn Abd ar-Rahman ibn Zayd ibn al-Khattab who was the governor of Kufa, "Pronounce judgement on the basis of an oath with one witness."
Yahya said, "Malik said about Jamil ibn Abd ar-Rahman al-Muadhdin that he was present with Umar ibn Abdul Aziz when he was judging between people. If a man came to him with a claim against a man, he examined whether or not there were frequent transactions and dealings between them. If there were, the defendant could make an oath. If there was nothing of that nature he did not accept an oath from him." Malik summed up, "What is done in our community is that if someone makes a claim against a man, it is examined. If there are frequent transactions and dealings between them, the defendant is made to take an oath. If he takes an oath, the claim against him is dropped. If the defendant refuses to take an oath, and returns the oath to the claimant, the one claiming his right takes an oath and takes his due."


Railway Jobs KRCL Recruitment 2017, Konkan Railway Corporation Ltd released notification for recruitment of 12 Graduate Engineers based on their GATE score.Inter5ested & Eigible candidates can apply online from 13-7-2017 to 14-8-2017.
Educational Qualification : – Candidates must have completed their Bachelors Degree in Engineering/Technology with minimum of 65% of marks from any recognized university.
Age Limit : – Maximum age not to exceed 25 years as on 01-01-2017.
Application Fee : – All candidates need to pay Rs 500/- as application fee through DD in favor of FA & CAO, KRCL payable at Mumbai.No Fess for Sc & St Candidates.
Selection Process : – Candidates selection will be mainly based on their GATE Score, GD & Interview.
Go through the official notification carefully before applying for this Railway Jobs KRCL Recruitment 2017.
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4.Establishment of Canada-Africa Women Scientists Network.
The Challenges and Opportunities for women in STEM (Science Technology Engineering & Mathematics ) event is being organized within the broader framework of the International Day of Women and Girls in Science and the International Women's day celebrations. The women and girls in Science day is celebrated each year on February 11 whereas the international women's day is commemorated each year on 8th March. Both days are a reminder that women and girls play a critical role in science and technology and that their participation must be strengthened, especially in Africa.
We will be showcasing 4 to 5 African women scientist based in Africa and Canada while providing a platform for these women scientists to share and exchange knowledge and ideas on how to enhance the participation of women in STEM and help us to celebrate the successes registered on the African continent. The output will be the establishment of networks and collaboration between African and Canadian Women Scientists.
On the morning of March 25 at the office of IDRC, 150 Kent Street by invitation only, a roundtable event will be held to establish some frameworks on how best to improve the participation of women in the STEM sector. A formal proposal will be drawn up.
In the afternoon the event will be open to the public and moved to the The Delegation of the Ismaili Imamat at 199 Sussex Drive where there will be a high-level panel discussion on women in Science, Technology, Engineering and Mathematics (STEM). The panel will be made of women leaders in STEM and will be preceded by a Key Note address given by a special guest speaker.
We welcome this event in Canada as a huge step forward to strengthen our ties and build stronger bridges for African and Canadian women in STEM and hope you can join us.
Women in STEM in Africa: Where are we?
Where are the most successes and why?


This week's article is my response to a question by Lisa Bruce.
"I cold call into Fortune 1000 companies, often times the admin will tell me to send the CIO, or whoever I'm calling, an email about who we are and what we do. I tend to think of emails as a last ditch effort, but others think they are a good follow up to a live conversation. I'd be interested in what you think! - Lisa Boudreau, Sales Rep, ePresence Inc.
Thanks for writing in Lisa. The title I gave this article pretty much sums up what I think. When making cold calls, administrative assistants are instructed to get rid of salespeople as nicely as possible. One of the easiest ways to blow off a sales rep is to ask you to send some information, literature, or an email.
Many sales reps then think "I got one!" They believe that they have a prospect, and they put it onto their follow-up list, and they call and call again until they get through, give up, or are told to get lost. The reality is you were just told that your sales pitch didn't work. Your email, or brochure or whatever, goes into a huge pile of other solicitations, that may never get reviewed.
It's useful to think of cold-calling as advertising, only one at a time. In advertising, you are "interrupting" people with your message. By delivering it to hundreds, thousands, or millions at the same time, you can persuade a certain percentage of the people who view the ad. The people who are persuaded are those who fit the target profile that the advertiser is looking for.
Cold-calling is just like shoving a billboard in one person's face, or running a commercial for one prospect, and then asking them if they want the product. How well you do at this depends on two things. First the quality of the target list of prospects you are calling on is very important (this is akin to which freeway you post your billboard on, or which TV show you run your commercial on). When you have the right target list, the next element under your control is the effectiveness of your message at getting the prospect's attention.
The reason why I use the advertising analogy is that when cold-calling we are an interruption, until we have the prospect's attention and the permission to probe further and ask questions. When we fail at this, prospect's nicely ask us to "send info please".
To improve the effectiveness of your message, I advise you to focus on using prominent customer references with benefits centered around revenue increases, cost reductions, or efficiency gains in your cold-calling approach. Combine this with effective prospect targeting and some persuasive questioning techniques and you will get through to more decision-makers.


Bokeh is the filtering of light behind the subject to form appealing patterns of light. In this image, the sunlight behind Louis is being filtered by the roses and shrubs in the garden to create beautiful circular areas.
Next, check out Sam's Bokeh at Pet&Equine Photography, covering the Lancaster UK area.">SamAdele Pet&Equine Photography, covering the Lancaster UK area. Keep going around the blog ring until you end up back here. See you next Friday!
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This Christmas Season, TFC Angel Wreath Project wants to bless the children of our single parent volunteers. Looking for a way to bring a smile to a child's face this Christmas? Help our Angel Wreath Project by sponsoring a child. Single Parent Volunteers, please register.
Stop by our table at the Main Lobby beginning November 18th at each campus to pick up a tag that indicates a child's age and gender, so you can bless them with a $25 age appropriate gift.
• Cash or Check - Write Angel Wreath on your tithe envelope.
• Online - Select the Outreach - Angel Wreath Project fund from the drop down menu.
• Text Giving - Text the amount, then the word ANGEL to 956-272-1818.
Gifts and monetary donations are due Sunday, December 2nd.
This Christmas Season, TFC Angel Wreath Project wants to bless the children of our single parent volunteers. Help us to bring a smile to your child's face by allowing our TFC Family a chance to bless them with a gift and some goodies.
If you are a Single Parent or Guardian, and actively serving at TFC, please register here.
*Select yourself, not your children on Step 1 when asked to Choose Registrants.
Registration is available through November 11th.
Angel Wreath Project will take place on Saturday, December 8th, and we need volunteers to help organize and distribute gifts on the day of the event.
Volunteers need to arrive by 5:00 pm for setup, and help with cleanup until 8:30 pm. We are meeting at the TFC McAllen Campus Annex Auditorium.
You are encouraged to wear Christmas attire to this event.
Please be aware, space is limited.
Registration available through December 5th.
Volunteers need to arrive by 5:00 pm for setup, and help with cleanup until 8:30 pm. We are meeting at the TFC Weslaco Campus Kids Auditorium.


Sofia, the wonderful Portuguese model and trapeze artiste, stretching in the Japanese raincoat. Such fun to paint.
Acrylic on Canvas, 20 x 50 cm, 2014. This is a 3.5cm 'deep' canvas which does not need a frame and is ideal for a contemporary space.


Phase 2 construction of Miramar Parkway is currently underway as crews continue work in the middle bridge section. This phase is anticipated to be completed this summer.
Phase 3 construction will begin in the next few months. As part of this final phase, westbound traffic will be shifted onto the newly constructed middle bridge section. Crews will then demolish the westbound section of the existing bridge and will begin construction of the final section of the new bridge.
The final configuration will include three through lanes in the westbound and eastbound direction. In addition, there will be triple turning lanes from eastbound Miramar Parkway to I-75 northbound. Motorists on westbound Miramar Parkway will have triple turning lanes to I-75 southbound as well. The Miramar Parkway interchange is anticipated to be completed in early 2017.
This entry was posted in News on May 26, 2016 by Realty Services Florida.
The ramp from I-595 westbound to southbound I-75 (to Miami) will be closed nightly from 10:00 p.m. to 6:00 a.m. on Wednesday, May 11 and Thursday, May 12. Motorists will be detoured to exit at Glades Parkway (Exit 22), go south on Glades Parkway, east on Royal Palm Boulevard and then take the entrance ramp to enter southbound I-75.
All southbound lanes of the Sawgrass Expressway at the I-595 interchange will be closed nightly from 10:00 p.m. to 6:00 a.m. on Wednesday, May 11 and Thursday, May 12. Motorists will be detoured to take the entrance ramp to northbound I-75 to Naples, exit Glades Parkway (Exit 22), go south on Glades Parkway, east on Royal Palm Boulevard and then take the entrance ramp to enter southbound I-75. *Please note the ramp from southbound Sawgrass Expressway to eastbound I-595 will remain open.
The ramp from southbound I-75 (from Naples) at the I-595 interchange will be closed nightly from10:00 p.m. to 6:00 a.m. on Wednesday, May 11 and Thursday, May 12. Motorists will be detoured to continue to eastbound I-595, exit on Flamingo Road (Exit 2), go east on SR 84, make a U-turn on Flamingo road, west on SR 84 to Weston Road, south on Weston Road to continue east on Royal Palm Boulevard and then take the entrance ramp to enter southbound I-75.
This entry was posted in News on May 11, 2016 by Realty Services Florida.
All lanes of northbound Florida's Turnpike at the I-75 interchange will be closed from 11 p.m.Monday, May 2 to 5 a.m. Tuesday, May 3. Motorists will be able to take the entrance ramp to northbound I-75 (Exit 39), exit at Miramar Parkway East (Exit 7A), continue east on Miramar Parkway to NW 57 Avenue / Red Road and turn south on NW 57 Avenue / Red Road to re-enter northbound Florida's Turnpike. Please follow the posted detour.
All lanes of southbound Florida's Turnpike at the I-75 interchange will be closed from 11 p.m.Thursday, May 5 to 5 a.m. Friday, May 6. Motorists will be able to take the exit ramp to NW 57th Avenue / Red Road (Exit 43), continue north on NW 57th Avenue / Red Road and turn west on Miramar Parkway to enter southbound I-75 and then take the entrance ramp to re-enter southbound Florida's Turnpike (Exit 39). Please follow the posted detour.
This entry was posted in News on May 2, 2016 by Realty Services Florida.


With over a decade of experience in solar, SCS is the right partner for commercial solar in Melbourne. Our focus on commercial solar and technical advisory will prove to be the best suited setup to assist your company.
Take advantage of the benefits of solar modules for your company. We provide services in commercial solar panels in the Melbourne metropolitan area. We always carefully consider each individual client profile, to ensure that the best service is delivered. We take into account your site and location specifications, your roof to analyze if there are any shading issues and your financial needs.
Your company may even be eligible to receive a a solar system for free! By qualifying for our commercial solar sponsoring, your business can enjoy certain financial benefits that will provide an advantage for you. Whether or not the size of your business is small, medium or large, our commercial solar systems are tailored for each different business, be it small restaurants, schools, or even large manufacturing firms and factories. Our commercial solar products have sizes which range up to 100kW.
If your company is not eligible for our commercial solar sponsoring, then you can have an option to lease solar panels. Under this option, there won't be any worries of investing into the solar system nor maintaining it. There are many other financial benefits under the commercial solar leasing scheme and you can read more about it in our financial services section.
For your business, we can also provide a Power Purchase Agreement, known as PPA. This never generates any negative cash flow. You only pay for what you consume and don't have to invest anything. This is a very recent business concept which was first introduced in the United States. You can learn more about PPA within our financial services section.
We also have a solar calculator specifically prepared for a commercial setting, so you can exactly find out how much your business' energy bill could be reduced by with solar energy! The outlook for solar power in Australia is looking better after every passing year!