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Background The need for economical rabies post-exposure prophylaxis (PEP) is increasing in developing countries. Implementation of the two currently approved economical intradermal (ID) vaccine regimens is restricted due to confusion over different vaccines, regimens and dosages, lack of confidence in intradermal technique, and pharmaceutical regulations. We therefore compared a simplified 4-site economical PEP regimen with standard methods. Methods Two hundred and fifty-four volunteers were randomly allocated to a single blind controlled trial. Each received purified vero cell rabies vaccine by one of four PEP regimens: the currently accepted 2-site ID; the 8-site regimen using 0.05 ml per ID site; a new 4-site ID regimen (on day 0, approximately 0.1 ml at 4 ID sites, using the whole 0.5 ml ampoule of vaccine; on day 7, 0.1 ml ID at 2 sites and at one site on days 28 and 90); or the standard 5-dose intramuscular regimen. All ID regimens required the same total amount of vaccine, 60% less than the intramuscular method. Neutralising antibody responses were measured five times over a year in 229 people, for whom complete data were available. Findings All ID regimens showed similar immunogenicity. The intramuscular regimen gave the lowest geometric mean antibody titres. Using the rapid fluorescent focus inhibition test, some sera had unexpectedly high antibody levels that were not attributable to previous vaccination. The results were confirmed using the fluorescent antibody virus neutralisation method. Conclusions This 4-site PEP regimen proved as immunogenic as current regimens, and has the advantages of requiring fewer clinic visits, being more practicable, and having a wider margin of safety, especially in inexperienced hands, than the 2-site regimen. It is more convenient than the 8-site method, and can be used economically with vaccines formulated in 1.0 or 0.5 ml ampoules. The 4-site regimen now meets all requirements of immunogenicity for PEP and can be introduced without further studies. Trial Registration Controlled-Trials.com ISRCTN 30087513, Author Summary All human deaths from rabies result from failure to give adequate prophylaxis. After a rabid animal bite, immediate wound cleaning, rabies vaccine and immunoglobulin injections effectively prevent fatal infection. Immunoglobulin is very rarely available in developing countries, where prevention relies on efficacious vaccine. WHO approved vaccines are prohibitively expensive, but 2 economical regimens (injecting small amounts of vaccine intradermally, into the skin, at 2 or 8 sites on the first day of the course) have been used for many years in a few places. Practical or perceived difficulties have restricted widespread uptake of economical methods. These could largely be overcome by introducing a new, simpler regimen, involving 4 site injections on the first day. We vaccinated volunteers to compare the antibody levels induced by the 4-site intradermal regimen with those induced by the current 2-site and 8-site regimens and the “gold standard” intramuscular regimen favoured internationally. All the economical intradermal regimens were at least as immunogenic as the intramuscular method. The results provide sufficient evidence that the 4-site regimen meets the criteria necessary for its recommendation for use wherever the cost of vaccine is prohibitive and especially where 2 or more patients are treated on the same day.

Bacterial type III secretion system (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation, and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bacterial effector fusion Tir-LOV was functional following its translocation and localized to the host cell membrane in discrete foci, demonstrating that LOV-based reporters can be used to visualize the effector translocation with minimal manipulation and interference. Further evidence for the versatility of the reporter was demonstrated by fusing LOV to the C terminus of the Shigella flexneri effector IpaB. IpaB-LOV localized preferentially at bacterial poles before translocation. We observed the rapid translocation of IpaB-LOV in a T3SS-dependent manner into host cells, where it localized at the bacterial entry site within membrane ruffles.

International audience; In Cambodia, 1,303 bats of 16 species were tested for lyssavirus. No lyssavirus nucleocapsid was detected in 1,283 brains tested by immunofluorescence assay. Antibodies against lyssaviruses were detected by enzyme-linked immunosorbent assay in 144 (14.7%) of 981 serum samples. Thirty of 187 serum samples contained neutralizing antibodies against different lyssaviruses.

Shigella flexneri , the causative agent of bacillary dysentery, induces massive cytoskeletal rearrangement, resulting in its entry into nonphagocytic epithelial cells. The bacterium-engulfing membrane ruffles are formed by polymerizing actin, a process activated through injected bacterial effectors that target host small GTPases and tyrosine kinases. Once inside the host cell, S. flexneri escapes from the endocytic vacuole within minutes to move intra- and intercellularly. We quantified the fluorescence signals from fluorescently tagged host factors that are recruited to the site of pathogen entry and vacuolar escape. Quantitative time lapse fluorescence imaging revealed simultaneous recruitment of polymerizing actin, small GTPases of the Rho family, and tyrosine kinases. In contrast, we found that actin surrounding the vacuole containing bacteria dispersed first from the disassembling membranes, whereas other host factors remained colocalized with the membrane remnants. Furthermore, we found that the disassembly of the membrane remnants took place rapidly, within minutes after bacterial release into the cytoplasm. Superresolution visualization of galectin 3 through photoactivated localization microscopy characterized these remnants as small, specular, patchy structures between 30 and 300 nm in diameter. Using our experimental setup to track the time course of infection, we identified the S. flexneri effector IpgB1 as an accelerator of the infection pace, specifically targeting the entry step, but not vacuolar progression or escape. Together, our studies show that bacterial entry into host cells follows precise kinetics and that this time course can be targeted by the pathogen.

International audience; A large number of Gram negative pathogens use a specialized needle-like molecular machine known as Type III Secretion (T3S) system. This highly sophisticated molecular device consists of a basal body spanning the two bacterial membranes and a protruding needle structure that is connected to a distal translocator complex. The main features of the T3S system are (i) activation after host cellular membrane contact and (ii) the ability to "inject" effectors into host cells through the needle apparatus across three membranous structures--two bacterial and one host cellular--without effector leakage into the exterior space. The effector proteins execute multiple roles upon translocation including re-arranging the host cytoskeleton, manipulating signaling pathways and reprogramming the host immune response. We have established a novel approach to monitor the secretion of fluorescently labeled effectors through the T3S system of single living bacteria in real time. Our approach uses the tetracysteine-FlAsH labeling procedure. Here, we provide a detailed protocol and advice on its potential and experimental pitfalls. Using the entero-invasive pathogen Shigella flexneri for assay development, we have also successfully adapted our approach and developed procedures for T3S effector tracking for other pathogens such as Enteropathogenic Escherichia coli (EPEC).

Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri’s early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens., Author Summary Shigella flexneri is an intracellular bacterial pathogen and the causative agent of bacillary dysentery. It possesses the ability to invade and propagate within human cells by injecting bacterial effector proteins directly into host cells. Shortly after entry within a vacuole, S. flexneri induces vacuolar rupture and escapes into the host cytosol via an unknown mechanism. Using large volume correlative light electron microscopy (CLEM) and dynamic microscopy we studied discrete and highly transient steps of S. flexneri early invasion in detail. We provide the first 3D high resolution view of the S. flexneri invasion site and of vacuolar rupture itself. We find that vesicles formed at the invasion site due to injected bacterial effectors, termed macropinosomes, are functionally involved in vacuolar rupture and come into direct contact with the bacterial containing vacuole during this process. This unique and surprising pathogenic strategy stands in stark contrast to other invasive pathogens that induce direct lysis of their surrounding vacuole via the action of destabilizing bacterial proteins.