Why Functional Pre-Erythrocytic and Bloodstage Malaria Vaccines Fail: A Meta-Analysis of Fully Protective Immunizations and Novel Immunological Model

Background: Clinically protective malaria vaccines consistently fail to protect adults and children in endemic settings, and at best only partially protect infants. Methodology/Principal Findings: We identify and evaluate 1916 immunization studies between 1965-February 2010, and exclude partially or nonprotective results to find 177 completely protective immunization experiments. Detailed reexamination reveals an unexpectedly mundane basis for selective vaccine failure: live malaria parasites in the skin inhibit vaccine function. We next show published molecular and cellular data support a testable, novel model where parasite-host interactions in the skin induce malaria-specific regulatory T cells, and subvert early antigen-specific immunity to parasite-specific immunotolerance. This ensures infection and tolerance to reinfection. Exposure to Plasmodium-infected mosquito bites therefore systematically triggers immunosuppression of endemic vaccine-elicited responses. The extensive vaccine trial data solidly substantiate this model experimentally. Conclusions/Significance: We conclude skinstage-initiated immunosuppression, unassociated with bloodstage parasites, systematically blocks vaccine function in the field. Our model exposes novel molecular and procedural strategies to significantly and quickly increase protective efficacy in both pipeline and currently ineffective malaria vaccines, and forces fundamental reassessment of central precepts determining vaccine development. This has major implications fo

The kiss I can't forget.

Gift of Dr. Mary Jane Esplen.Piano vocal [instrumentation]F major [key]Valse Lento [tempo]Waltz song [form/genre]Couple kissing under a tree [illustration]Publisher's advertisement on front inside cover and back cover [note

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases

Enhancement of Anthrax Lethal Toxin Cytotoxicity: a Subset of Monoclonal Antibodies against Protective Antigen Increases Lethal Toxin-Mediated Killing of Murine Macrophages

We investigated the ability of using monoclonal antibodies (MAbs) against anthrax protective antigen (PA), an anthrax exotoxin component, to modulate exotoxin cytotoxic activity on target macrophage cell lines. Anthrax PA plays a critical role in the pathogenesis of Bacillus anthracis infection. PA is the cell-binding component of the two anthrax exotoxins: lethal toxin (LeTx) and edema toxin. Several MAbs that bind the PA component of LeTx are known to neutralize LeTx-mediated killing of target macrophages. Here we describe for the first time an overlooked population of anti-PA MAbs that, in contrast, function to increase the potency of LeTx against murine macrophage cell lines. The results support a possible mechanism of enhancement: binding of MAb to PA on the macrophage cell surface stabilizes the PA by interaction of MAb with macrophage Fcγ receptors. This results in an increase in the amount of PA bound to the cell surface, which in turn leads to an enhancement in cell killing, most likely due to increased internalization of LF. Blocking of PA-receptor binding eliminates enhancement by MAb, demonstrating the importance of this step for the observed enhancement. The additional significance of these results is that, at least in mice, immunization with PA appears to elicit a poly-clonal response that has a significant prevalence of MAbs that enhance LeTx-mediated killing in macrophages

A High-Affinity Monoclonal Antibody to Anthrax Protective Antigen Passively Protects Rabbits before and after Aerosolized Bacillus anthracis Spore Challenge

We have developed a therapeutic for the treatment of anthrax using an affinity-enhanced monoclonal antibody (ETI-204) to protective antigen (PA), which is the central cell-binding component of the anthrax exotoxins. ETI-204 administered preexposure by a single intravenous injection of a dose of between 2.5 and 10 mg per animal significantly protected rabbits from a lethal aerosolized anthrax spore challenge (∼60 to 450 times the 50% lethal dose of Bacillus anthracis Ames). Against a similar challenge, ETI-204 administered intramuscularly at a 20-mg dose per animal completely protected rabbits from death (100% survival). In the postexposure setting, intravenous administration of ETI-204 provided protection 24 h (8 of 10) and 36 h (5 of 10) after spore challenge. Administration at 48 h postchallenge, when 3 of 10 animals had already succumbed to anthrax infection, resulted in the survival of 3 of 7 animals (43%) for the duration of the study (28 days). Importantly, surviving ETI-204-treated animals were free of bacteremia by day 10 and remained so until the end of the studies. Only 11 of 51 ETI-204-treated rabbits had positive lung cultures at the end of the studies. Also, rabbits that were protected from inhalational anthrax by administration of ETI-204 developed significant titers of PA-specific antibodies. Presently, the sole therapeutic regimen available to treat infection by inhalation of B. anthracis spores is a 60-day course of antibiotics that is effective only if administered prior to or shortly after exposure. Based upon results reported here, ETI-204 is an effective therapy for prevention and treatment of inhalational anthrax

VT increases cellular CFTR and F508del CFTR.

<p>HeLa cells transfected with wild type CFTR or F508del CFTR (were grown for 2hrs ± VT or A subunit inactive VT (VT0). Cells were solubilized in SDS, separated by PAGE and subject to western blot using anti CFTR; arrows = mature band c(upper) and immature band b (A). Ponceau S protein stain of the CFTR molecular weight range provides a loading control(B).</p

Catalytically inactive CT0 increases F508del CFTR Western blots were stained with anti CFTR.

<p><b>a</b>)HEK cells transfected with F508delCFTR were incubated for 4hrs ± 65- 260pM A subunit inactivated CT0; the arrow emphasizes the comparison of untreated baseline to maximal CT0 enhanced CFTR expression, <b>b</b>) quantitation of F508del CFTR rescue, <b>c</b>) F508delCFTR HEK cells were treated with 65pM CT0 containing a 9(CT9L) or 18(CT18L) leucine N-terminal A subunit extension for 4 hr, d) CFBE cell line was treated with increasing CT0 for 4hr and compared to cells rescued by 27°C culture-arrows = band c (upper) and band b, e) Quantitation of F508delCFTR increase in CFBE cells -relative scale ±SEM. Groups were compared using ANOVA followed by Tukey’s test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166948#pone.0166948.ref104" target="_blank">104</a>].</p

Effect of CTO on primary F508delCFTR bronchiolar epithelial cell monolayer chloride transport Triplicate F508delCFTR patient derived primary bronchiolar epithelial cell monolayers were treated with 13pM(-∙-∙-∙-∙), 130pM (^{__ __ __}), 13nM(- - - -) CT0 or 3μM VX809(∙∙∙∙) and chloride channel function measured by short circuit current (±SEM) determined by an Ussing epithelial voltage clamp apparatus.

<p>CFTR was activated by 10μM forskolin addition (at A), augmented by addition of potentiator 0.1μM VX 770 (at B) and inhibited by 20μM CFTRinh-172 (at C). 13nM, 130pM, but not 13pM, CT0 induced a significant difference from vehicle at peak F508delCFTR mediated chloride transport: P>0.001 by Dunnett’s test [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166948#pone.0166948.ref056" target="_blank">56</a>]. CT0 achieved a maximum increase of 20% that observed for corrector VX809. There were no toxic effects at these concentrations of CT0 as indicated by normal, uniform transepithelial resistance measurements and uniform response to BaCl₂ (not shown).</p

Cell surface expression of F508del CFTR rescued by CT18L F508delCFTR expressing HEK cells were treated with CT18lL (260pM) for 4hr at 37°C and compared to control untreated cells by double labeling with wheat germ agglutinin to label the cell surface, and MabM3A7 anti CFTR.

<p>Colabelling (yellow) was only seen for treated cells. This experiment was repeated four times. Bar = 20μm.</p