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Can injuries to the skin be painless?

When the body receives an injury to the skin, a signal is sent to the brain, which generates a sensation of pain. Teams led by Priscille Brodin in Lille[1] and Laurent Marsollier in Angers[2] have studied lesions in patients with Buruli ulcer, a tropical disease. In an article published in the journal Cell, they show that, despite the extent and severity of these wounds, they are less painful than others that seem relatively minor (e.g. scratches, low-degree burns). They discovered an analgesic mechanism that limits the transmission of pain signals to the brain. An understanding of this mechanism may be useful in developing new drugs for pain relief.

Buruli ulcer (caused by infection with Mycobacterium ulcerans) is the third most prevalent mycobacterial disease, after tuberculosis and leprosy. This tropical disease, which mainly affects children, causes ulcerative cutaneous lesions. The destruction of skin tissue is caused by mycolactone, a toxin secreted by the bacterium. Despite their size, the lesions are not especially painful in the early stages of the disease, explaining why patients are slow to seek medical help. The researchers explored the mechanism that causes these lesions to be painless.

schéma ulcère buruli

© Inserm / Conception Clerc-com – Simar Thibault

Until then, it had been thought that the lack of pain in the early stages of the disease was related to the destruction of nervous tissue. In the present study, the researchers show using infected mice that this hypothesis is not supported by nerve degeneration, which occurs only in the advanced stages of the disease. They also injected the toxin into mice to observe its effects on the animals’ sensitivity. The researchers show that the presence of the toxin can inhibit pain on its own, with no effect on the nerves.

“The bacterium, or more specifically its toxin, mycolactone, can interact with neurons and prevent the transmission of nerve signals, explaining the painless nature of the lesions,” explains Laurent Marsollier, a research fellow at Inserm.

A state-of-the-art imaging technique was used to demonstrate that mycolactone interacts with a neuronal receptor (angiotensin receptor 2), causing leakage of potassium. The potassium efflux causes neuronal hyperpolarisation, limiting the transmission of nerve impulses—which carry the pain signal—at local level.

The researchers then blocked the expression of this neuronal receptor in mice infected with the bacterium. Blocking the receptor prevented it from interacting with the mycolactone toxin, which re-established the animals’ sensitivity to pain, thus providing in vivo confirmation of the mechanism identified.

The Mycobacterium ulcerans bacterium employs a novel infection strategy by using the toxin it secretes to prevent the pain associated with the lesions it causes.

“The discovery of this mechanism, which limits pain in the cutaneous lesions during the early stages of the disease, opens up new possibilities in the search for new drugs to prevent pain,” says Priscille Brodin, Inserm Research Director and co-author of this study.

Indeed, the molecule that can block the action of the receptor does not belong to the class of analgesics in current use, such as paracetamol or opiates such as morphine. Generally speaking, clinicians are hoping for new drugs to fight pain, since the existing drugs all have limitations of greater or lesser importance in the context of personalised medicine.

Finally, according to the researchers, the receptor identified may be a target of choice, since another study[3] showed that blocking it led to the reduction of pain in patients with herpes infections.

Environment_Lake_(Large)

© OMS / Dr A. Chauty, AFRF, Benin


[1] Joint Research Unit 1019, “Center for Infection and Immunity of Lille” (Inserm – CNRS – Institut Pasteur Lille – University of Lille Nord de France), and formerly Inserm Avenir Team Institut Pasteur Korea

[2] Inserm Unit 892 – Inserm Avenir “ATOMycA” Team (Inserm – CNRS – University of Angers)

[3] Rice, A.S., Dworkin, R.H., McCarthy, T.D., Anand, P., Bountra, C., McCloud, P.I., Hill, J., Cutter, G., Kitson, G., Desem, N., et al. (2014). EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet.

H1N1 influenza: Vaccination induces an immune memory response comparable to that of a moderate infection

How long does the immune memory response produced by vaccination last? Is it similar to that induced by the infection itself? New information on the A(H1N1) pandemic influenza virus has just been brought to light by researchers at Joint Research Unit 1135, Cimi-Paris (Centre for Immunology and Infectious Diseases – Inserm – Pierre and Marie Curie University). They have shown that the immune response induced by vaccination is still strong one year later, and that it is similar to that produced by a moderate infection. Their findings have been published in the Journal of Clinical Investigation.

Influenza A virus subtype H1N1, originating in pigs, birds and humans, swept across the world between June 2009 and August 2010. While this pandemic was smaller and less severe than that of 1918, it affected young, healthy people too, in some cases causing very serious illness and death. These people were not naturally immune to the new virus strain, which was different from those that cause influenza epidemics every winter. However, some people had been vaccinated and it would be interesting to know how long their immune response to the pandemic A(H1N1) strain lasted, and if it was comparable to that induced by the infection itself.

The researchers sought to answer these two questions by conducting

the first ever study to compare vaccinated subjects with infected patients on the basis of so many immune response parameters

says the principal author Béhazine Combadière, Inserm research director at Cimi-Paris. At present, there is only one standard criterion for evaluating the efficacy of an influenza vaccine: the level of antibodies in the blood, which is correlated with the level of protection afforded. “We pushed our scientific assessment as far as possible, taking into consideration no less than eight parameters, adds Béhazine Combadière. These eight parameters covered both humoral immunity (where the antibodies bind to the virus and neutralise it) and cell-mediated immunity (where the white blood cells or T cells kill the cells infected by the virus). Thus, for example, in regard to humoral immunity the researchers focused not only on the level of antibodies in the blood, but also on serum avidity (i.e. the strength or affinity with which antibodies bind to virus antigens).

The study was also original in that the researchers compared vaccinated subjects with infected patients. It included 50 volunteers who received a monovalent vaccine that targeted the pandemic H1N1 strain only and contained an adjuvant (designed to boost the effect of the vaccine, which had a low viral strain content). The vaccinated subjects were compared with 61 patients infected with A(H1N1) influenza. The infection was mild to moderate in 48 of the patients and severe in the remaining 13. The latter had to be admitted to hospital and treated with antiviral therapy after developing acute respiratory distress syndrome. The assessment was carried out one year after exposure to the virus or one year after vaccination.

The researchers showed first of all that the immune response induced by the vaccine was still strong one year later. They also identified similarities and differences between the three groups of people, depending on the immune response parameters. Thus, the effect of vaccination was similar to that of a moderate infection on several immune parameters. Both vaccination and moderate infection also caused a more significant migration of T cells to the mucosa.

The researchers then decided to investigate whether different immune response profiles existed, regardless of vaccination or infection status. They identified three profiles. The first one included a majority of vaccinated and moderately-infected subjects. Therefore, this research shows that different people have a different immune response capacity. It also confirms that influenza vaccination can induce an immune memory response of similar strength and quality to that produced by a moderate infection.

This study may have implications in terms of adjusting or optimising vaccination strategies. It could be used to improve vaccine efficacy assessment protocols. As a result, two parameters could be added to the antibody level: markers of T cell migration to the mucosa and the potential cytotoxic activity of one category of T cells (CD8).

Elucidating the pathogenic mechanism of meningococcal meningitis

Neisseria meningitidis, also called meningococcus, is a bacterium responsible for meningitis and septicemia[1]. Its most serious form, purpura fulminans, is often fatal. This bacterium, which is naturally present in humans in the nasopharynx, is pathogenic if it reaches the blood stream. Teams led by Dr. Sandrine Bourdoulous, CNRS senior researcher at the Institut Cochin (CNRS/Inserm/Université Paris Descartes), and Professor Xavier Nassif, Institut Necker Enfants Malades (CNRS/INSERM/Université Paris Descartes/Assistance Publique – Hôpitaux de Paris), have deciphered the molecular events through which meningococci target blood vessels and colonize them. This work opens a path to new therapeutic perspectives for treating vascular problems caused by this type of invasive infection. The study was published on June 1, 2014 in Nature Medicine.

image_Nassif

Colonization of brain vessels by N. meningitidis Immunofluorescence analysis of a human brain section infected by N. meningitidis. The bacteria (red) have colonized the brain’s endothelial cells that express CD147 (green). (Cell nuclei are blue) © Nature Medicine

When the bacterium Neisseria meningitidis multiplies in the blood, it interacts with the endothelial cells that line the inside of blood vessels and adheres to their walls. In the skin and mucous membranes, meningococcal infection in the vessels creates hemorrhagic skin lesions (called purpura) due to bleeding in the tissues. Those can rapidly progress to a serious and often fatal form of the disease (purpura fulminans). In the brain, when meningococci adhere to the vessels they can pass through the blood-brain barrier[2], and cause meningitis when they invade the meninges[3].

Teams of researchers have deciphered how Neisseria meningitidis adheres to blood vessels, a step that underpins the bacterium’s pathogenicity. In blood vessels they have identified receptor[4] CD147, whose expression is essential for initial meningococcal adherence to endothelial cells. If this receptor is absent, N. meningitidis cannot implant in blood vessels and colonize them.

It is a well-known fact that the adherence process of meningococcal bacteria to human cells relies on pili, long filaments that are expressed by the bacterium and composed of different sub-units (pilins). However, the pilins specifically involved in N. meningitidis’ adherence to blood vessels had never been identified. The researchers have determined that two pilins, PilE and PilV, interact directly with the CD147 receptor. Without them, meningococci cannot adhere to endothelial cells.

Humans are the only species that can be infected by meningococci. To show in vivo that pilins PilE and PilV are essential for N. meningitidis to colonize the vascular network, the researchers used a mouse model, where the mice were immunodeficient and grafted with human skin, keeping the functional human vessels within the graft to reproduce in mice the infection stages as observed in human skin. These mice were then infected by meningococci naturally having pilins PilE and PilV, or meningococci in which the expression of these pilins had been artificially suppressed. The human blood vessels were only infected by meningococci displaying PilE and PilV, which confirms that these two pilins are essential to the bacterial colonization process.

The researchers also showed in an ex vivo[5] infection model that cerebral vessels and meninges, particularly rich in CD147 receptors, allow colonization by meningococci, unlike other parts of the brain.

The scientists now wish to develop a new type of vaccine (to complement those already available) that would block the interaction between N. meningitidis and the CD147 receptors, thereby stopping the bacterium from colonizing the vessels.

This study was made possible by support from the teams of Dr. Frank Lafont at the Centre d’Infection et d’Immunité in Lille (CNRS/INSERM/Institut Pasteur de Lille/Université Lille 1/Université Lille 2), Professor Fabrice Chrétien at the Unité Histopathologie Humaine et Modèles Animaux at the Institut Pasteur in Paris, and Dr. Eric Chevet in the Groupe de Recherches pour l’Etude du Foie (INSERM/Université de Bordeaux).


[1] Systemic infections

[2] The endothelial cells in the brain’s capillaries are a physiological barrier at the interface between the blood and the brain (the blood-brain barrier). These cells, which have unique properties, act as a selective filter through which the necessary energy sources are transmitted to the brain and the waste is removed. Therefore they protect the brain from the external environment, including pathogens.

[3] Envelopes that protect the central nervous system.

[4] A receptor is a protein in the cell membrane onto which a specific factor (a ligand) can bind, triggering a response in the cell.

[5] This expression refers to culture tissues and live cells made in the laboratory, outside the organism they came from.

Inserm and the Institut Pasteur identify a new variant of Ebola virus in Guinea

In an article which appeared in The New England journal of Medicine on 16 April, researchers from Inserm (Jean Mérieux-Inserm BSL-4 Laboratory, Lyon) and the Institut Pasteur have published their initial findings on the characteristics of the Ebola virus discovered in Guinea. Initial virological investigations enabled them to identify Zaire ebolavirus as the pathogen responsible for this epidemic. Performed in less than a month, sequencing of the complete genome and subsequent phylogenetic analysis show that the virus present in Guinea forms a clade (variant) that is distinct from strains previously identified in the Democratic Republic of Congo and in Gabon. Epidemiological investigations also linked the laboratory confirmed cases with the initial deaths recorded during the December 2013 outbreak.

Laboratoire P4 Jean Mérieux/Inserm.©Inserm/ Guénet François

Ebola virus is a lethal, highly contagious virus for which there is presently no treatment. The symptoms are somewhat non-specific, and include fever, severe diarrhoea and vomiting. Between 30 and 90% of those infected with this organism die as a result.

On 2 April 2014, the World Health Organisation (WHO), in a communiqué published by the UN, reported that it had recorded 5 new cases of Ebola fever in Guinea. Since January, the total number of suspected and confirmed cases of Ebola fever in the present outbreak in Guinea is 127, with 83 deaths, according to WHO, which states that 35 cases were confirmed by laboratory testing.

The initial samples were analysed in Lyon in the Jean Mérieux-Inserm BSL-4 Laboratory directed by Hervé Raoul, Inserm Research Director, by the French National Reference Centre for Viral Haemorrhagic Fevers (attached to the Biology of Viral Emerging Infections Unit at the Institut Pasteur, directed by Sylvain Baize). A positive diagnosis was made.

A mobile BSL-4 laboratory was deployed in Guinea to provide assistance with diagnosis in the field. This mobile laboratory was developed as part of a European project, “EMP4,” coordinated by German researchers, and in which the Jean Mérieux-Inserm BSL-4 Laboratory is the French partner.

The researchers were able to analyse blood samples from 20 patients. Various tests were conducted by the scientists in order to establish a specific identity card for the virus.

Viral RNA was extracted from the blood samples, and then amplified and sequenced. These sequences were finally compared to 48 already known complete Ebola virus genomes. According to results, the analysis showed 97% identity with strains identified in the Democratic Republic of Congo in 1976 and 2007, and in Gabon in 1994 and 1996.

“These results demonstrate that we are facing the emergence of a new “form” of this virus in Guinea,” explains Hervé Raoul, Director of the BSL-4 Laboratory. This form is common to cases discovered since the month of December.

It would appear that the epidemic originated from a single introduction from animal to human.

Apart from the present epidemic, these results show that the endemic area for Ebola virus is greater than previously known, and that as a consequence, West Africa should henceforth be considered an area of risk for Ebola virus. Measures aimed at preventing transmission from wild fauna to humans and quickly identifying such events if there is a recurrence must be put in place in Guinea, as well as in neighbouring countries.

carte guinée


Further information on the BSL-4 laboratory

The Jean Mérieux BSL-4 Laboratory is a high-level containment laboratory dedicated to the study of Class 4 pathogens. The biological safety level applied is 4, the highest possible level. The researchers working there wear a full body, air-supplied, positive pressure suit to protect them from all contamination. The laboratory is itself maintained under negative pressure in order to protect the environment. Moreover, all wastes produced are completely inactivated, and the exhaust air is purified by a double absolute filtration system. This laboratory currently remains the structure offering the largest experimental capacity in Europe for this containment level.

Highly pathogenic agents:

Class 4 pathogens (or risk group 4) are highly pathogenic microorganisms characterised by a very high mortality rate, a lack of prophylactic or therapeutic measures to provide protection, and ready transmissibility. All class 4 pathogens currently known are viruses, and include viruses that cause haemorrhagic fevers or encephalitis. They include the Ebola, Marburg, Lassa, Junin, Machupo, Guanarito, Sabia, Crimean-Congo, Nipah and Hendra viruses.

To obtain photographs of the BSL-4 laboratory

Ribavirin – an effective and safe treatment for hepatitis E

Hepatitis E causes acute or chronic inflammation of the liver. It is an emerging disease which can be fatal and has no known treatment. Vincent Mallet, Stanislas Pol and their team at the Institut Cochin (Paris Descartes University, Assistance Publique – Hôpitaux de Paris, CNRS, Inserm) and French hospital-based teams* have proved the efficacy of a treatment for patients suffering from chronic hepatitis E virus infection. Most patients can be cured when treated with ribavirin in monotherapy for 3 months. This work was published in The New England Journal of Medicine of 20 March 2014.

The hepatitis E virus is the biggest cause of viral hepatitis in the world and it is estimated that a third of the global population is at risk of being infected with this virus. Although most cases occur in developing countries, there has been an increase in the number of cases reported in France and other industrialised countries where the virus is spread to humans by contaminated, undercooked food and blood. Until now there has been no treatment for patients suffering from hepatitis E.

Like the other hepatitis viruses, hepatitis E causes inflammation of the liver. In its acute form, the infection can be fatal for elderly people, pregnant women and people with liver disease. In immunosuppressed people (transplant patients, patients on chemotherapy or people living with HIV), the hepatitis E virus infection can progress to chronic hepatitis and cause cirrhosis.

Developing a treatment for hepatitis E

Ribavirin is a drug prescribed for some types of viral respiratory infections in children and some forms of haemorrhagic fever. It is also used to treat hepatitis C. Vincent Mallet, professor at Paris Descartes University and hospital practitioner at the Cochin teaching hospital (AP-HP) and Nassim Kamar, professor at Paul Sabatier University and hospital practitioner at the Rangueil teaching hospital previously proved its efficacy for immunosuppressed patients suffering from a chronic hepatitis E virus infection. **

In this new study, data from 59 ribavirin-treated transplant patients suffering from hepatitis E was collected at 13 French transplant centres by Nassim Kamar (from the Rangueil teaching hospital in Toulouse) and Vincent Mallet. The researchers confirmed that “ribavirin prescribed as monotherapy for 3 months is an effective treatment for chronic hepatitis E virus infection”. For most of the patients (46 out of 59 patients), the virus remained undetectable 6 months after treatment was discontinued. A longer treatment period appears desirable for highly immunosuppressed patients or those with detectable traces of the virus in the blood after a month of treatment. The only identified and foreseeable side effect of ribavirin is anaemia which was managed in most patients without any difficulty.

“This work is the result of a genuine partnership between a large number of centres in France. It represents a major advance in this area” states Vincent Mallet. We hope our results will pave the way for further prospective studies designed to assess the efficacy of ribavirin for severe forms of hepatitis E virus infection, especially in countries in the southern hemisphere where the disease is a serious problem”.

* CHU Rangueil et CHU Purpan  Toulouse –  Hôpital Cochin, AP-HP), Pitié Salpêtrière (AP-HP) – Hôpital Saint Eloi de Montpellier – Hôpital Foch de Suresnes – CHU Lyon et CHU de la Croix Rousse de Lyon – Hôpital Paul Brousse (Villejuif, AP-HP) – Hôpital Lapeyronie de Montpellier – Hôpital Bretonneau et CHU Trousseau de Tours – CHU Bordeaux – Hôpital Claude Huriez et CHU de Lille – CHU Le Bocage de Dijon – CHU de Nantes

** Mallet V, Nicand E, Sultanik P, Chakvetadze C, Tesse S, Thervet E, Mouthon L, Sogni P, Pol S. Brief communication: case reports of ribavirin treatment for chronic hepatitis E. Ann Intern Med. 2010 Jul 20;153(2):85-9 et Kamar N, Rostaing L, Abravanel F, Garrouste C, Lhomme S, Esposito L, Basse G, Cointault O, Ribes D, Nogier MB, Alric L, Peron JM, Izopet J. Ribavirin Therapy Inhibits Viral Replication on Patients With Chronic Hepatitis E Virus Infection. Gastroenterology. 2010 Nov;139(5):1612-8).

Europe is joining forces against neglected parasitic diseases

The international consortium A-PARADDISE (Anti-Parasitic Drug Discovery in Epigenetics), coordinated by Inserm, has just obtained funds of €6 million from the European Commission to conduct large-scale testing of innovative therapies against four neglected parasitic diseases: schistosomiasis, leishmaniasis, Chagas disease and malaria. The researchers have a common objective: to develop new drugs against the parasites that cause these diseases. The project involves 10 European partners, 5 Brazilian partners (who operate in the region where the diseases are endemic) and 2 Australian partners. They will all be meeting on 17 and 18 March at the Institute of Genetics and Molecular and Cellular Biology (Inserm / CNRS / University of Strasbourg Joint Research Unit), to get the project started.

Schistosomiasis, leishmaniasis, Chagas disease and malaria are regarded as neglected diseases because the effort and funds put into developing new treatment and control methods have not been commensurate with their catastrophic human impact. They affect people in developing countries, essentially in Africa, the Middle East, South America and eastern Asia, in tropical and sub-tropical regions. Around one billion people are regularly exposed to these diseases, which cause almost one million deaths every year.

At present, there is no vaccine against these parasites. Furthermore, the efficacy of existing treatments is limited, either by the side effects or by the current or potential development of resistance. Consequently, the A-PARADDISE consortium, which is coordinated by Inserm and headed by Raymond Pierce – Director of Research at the Centre for Infection and Immunity in Lille – is focusing on developing new drugs against these parasitoses.

The A-PARADDISE project will use a methodology that has already been tried and tested during a previous project of a similar scale (SEtTReND), which aimed to develop drugs against schistosomiasis. The researchers investigated histone-modifying enzymes (HME), which determine the structure of the parasite’s chromosomes. It was demonstrated that some HME inhibitors induce cell death, which makes them toxic to this parasite. This research provided the proof of concept that HMEs act on the schistosomiasis parasite, and has led to the development of a bank of candidate compounds which can rapidly be tested against other human parasites.

Thanks to the new project A-PARADDISE, researchers will be able to put the basic principle into practice and build on it by creating a unique platform for testing anti-parasitic drugs targeting HMEs, with a view to incorporating them into a clinical development programme.

The experimental method consists in physically and virtually testing the efficacy and the toxicity of the compounds, in vitro and in vivo.

The ultimate objective of the A-PARADDISE project is to provide several candidate treatments against the four parasites and to pave the way for clinical trials in the near future.

To ensure the success of the project, the participants were all selected for their high level of expertise in their respective fields: high-throughput screening, computer-aided screening, the production of recombinant proteins, next generation sequencing, phenotypic tests, toxicology and pharmacology.

A-PARADDISE: Anti-Parasitic Drug Discovery in Epigenetics

The A-PARADDISE project began on 1 February 2014 and will be backed by the European Union for three years (FP7, grant agreement no. 602080). It is coordinated by Inserm and includes 17 partners based in 5 European countries, Brazil and Australia:

Institut National de la Santé et de la Recherche Médicale, Group Avenir, Paris, France
Centre Européen de Recherche en Biologie et Médecine (CERBM*), France
Martin Luther Universität Halle- Wittenberg (MLU), Germany
Universidade Federal do Rio de Janeiro (UFRJ), Brazil
Universidade de Sao Paulo (USP), Brazil
Albert Ludwigs Universität Freiburg  (ALU-FR), Germany
Fundação Oswaldo Cruz, Centro de Pesquisas René Rachou (Fiocruz), Brazil
Fundação Oswaldo Cruz, Instituto Carlos Chagas (Fiocruz), Brazil
Inserm Transfert SA (IT), France
KANCERA AB (KAN), Sweden
Adlego Biomedical AB (Adlego), Sweden
GriffithUniversity (GU), Australia
University of Queensland (UQ), Australia
Università degli Studi di Roma La Sapienza  (UNIROMA1), Italy
University of East Anglia (UEA), Great Britain
Fundação Arthur Bernardes – Universidade Federal de Viçosa (UFV), Brazil
Institut Pasteur Paris (IPP), France

* The CERBM is the European branch of the Institute of Genetics and Molecular and Cellular Biology (IGBMC, Inserm/CNRS/Université de Strasbourg)

Malaria – “Wake and kill”: a new concept for the elimination of relapse

A team of researchers coordinated by Prof Dominique Mazier (AP-HP, UPMC, Inserm Unit 1135, CNRS ERL 8255) and Dr Georges Snounou, Research Director at CNRS (UPMC, Inserm Unit 1135, CNRS ERL 8255) has succeeded in culturing the dormant hepatic stage of the malaria parasite, previously inaccessible to researchers. The initial results from this technical breakthrough have enabled the development of a new concept for the elimination of malaria relapse due to the activation of these dormant forms. It should enable the establishment of a new strategy for the management of this illness, which would involve combining a drug capable of activating the dormant parasite with one of the many drugs effective against the parasite.
These results have just been published in the journal Nature Medicine.

Present-day management of malaria

After the bite from an infected mosquito, the parasite that causes malaria reaches the liver, where it multiplies. It then propagates in the bloodstream, where its proliferation causes a potentially fatal illness. In some cases, including that of the parasite Plasmodium vivax in humans, a fraction of hepatic parasites may remain “dormant” for a year or more, hence their name, hypnozoites. These subsequently “wake,” or reactivate, over time, and give rise to a bloodstream infection. This feature is probably the source of the belief that malaria persists for life.

The hypnozoite constitutes a two-fold problem in terms of controlling/eliminating malaria—a greater number of cases needing treatment, and increased transmission. Unfortunately, primaquine, and a recently developed analogue, tafenoquine, the only drugs capable of killing hypnozoites, have adverse effects on the body that are sometimes serious. The identification of reliable alternative drugs therefore constitutes a public health emergency. Until now, the search for new anti-hypnozoite drugs has been based on observations made in humans infected with P. vivax, or in monkeys infected with a parasite related to P. vivax, Plasmodium cynomolgi.

Methodology

Through a collaboration with teams from the national IDMIT Center[1] at the French Atomic Energy and Alternative Energies Commission (CEA), and those of the Biomedical Primate Research Centre(BPRC) in the Netherlands, the team led by Prof Dominique Mazier and Dr Georges Snounou first succeeded in maintaining cultures of infected hepatic cells for 40 days, i.e. nearly four times longer than is usually achieved. The team then demonstrated the persistence of dormant stages throughout the duration of culture, with some reactivating over the time, thus mimicking what happens in humans. It also tested new drugs (discovered at the Institut Pasteur in Paris), which inhibit epigenetic factors, on these hypnozoites. These drugs act by targeting histone methyltransferases, and are able to kill the blood stage of the parasite. Paradoxically, one of them activated the hypnozoites. This unexpected result led the team to formulate a new strategy, “Wake and Kill,” which involves combining a drug that activates the dormant parasite with one of the many available treatments known to be effective against the multiplying parasite.

Results provide hope for the management of malaria 

Thanks to this methodology, developed via an international and multi-institute collaboration (Inserm, CNRS, CIMI, CEA, UPMC, AP-HP, Institut Pasteur Paris), it will now be possible to screen drugs in vitro for their anti-hypnozoite effect, thus limiting the need for animals. The challenge is to adapt this technique to screening a large number of compounds. In addition, the possibility of growing hypnozoites in culture will finally allow scientists to study this enigmatic parasite stage, described 100 years after the discovery of the causative agent of malaria by Laveran in 1880.



[1] Infectious Diseases Models For Innovative Therapies

A good outcome for the CHILD-INNOVAC project: successful test in humans of a nasal vaccine against pertussis

The CHILD-INNOVAC European research programme, coordinated by Inserm, has enabled the development of an innovative vaccine that can be administered intranasally, to combat pertussis, which has shown a resurgence in developed countries in recent years. The research consortium, headed by Camille Locht, Director of the Centre for Infection and Immunity of Lille (a joint Unit involving Inserm, CNRS, Institut Pasteur de Lille and University of Lille Nord de France), today published promising results from Phase I clinical trials of the vaccine in human subjects in the online journal PLOS ONE

Researchers from the CHILD-INNOVAC European project, which brought together 10 European partners*, evaluated the efficacy and safety of a new concept in intranasal vaccination against pertussis. They also carried out clinical trials in humans, which provided conclusive results. 

Pertussis is a wrongly “forgotten” disease, according to Camille Locht, a Research Director at Inserm and Director of Scientific Affairs of the Institut Pasteur de Lille. The disease currently affects several tens of millions of individuals, and kills approximately 300,000 children annually worldwide. The associated morbidity and mortality are increasing throughout the world. Its resurgence has become a matter for major concern since 2010 in some developed countries, such as the USA, Australia, the UK, the Netherlands and France. 

The CHILD-INNOVAC project is more specifically focused on combating two major respiratory pathogens: Bordetella pertussis (the bacterium that causes whooping cough) and respiratory syncytial virus (which causes bronchiolitis in infants). These pathogens mainly affect infants aged 0 to 6 months, who are poorly protected by the vaccines presently available. The project also provides proof of concept that this vaccine may be applied to other respiratory infections.

The researchers from the CHILD-INNOVAC project have succeeded in testing in humans, for the first time, a live bacterial vaccine, genetically attenuated and specially designed for intranasal administration to combat major respiratory pathogens. “This original method of administration will make the vaccine accessible to greater numbers of people at a smaller cost,” explains the project coordinator, Camille Locht.

CHILD-INNOVAC: a European success
The main success of this European project is the achievement of a vaccine for which the immunogenicity and safety could be tested in humans in only two and a half years (compared with 5-7 years for most projects of this type). This is a very short time, which Camille Locht explains is due to “the skills and motivation of the consortium, which brought together experts in their respective areas of specialisation from seven European countries. It was possible to relay the data in a flexible and efficient manner at the different stages of the project.” The project received a budget of €5 million, awarded by the European Commission under FP7. 

The Phase I trials in humans allowed the immunogenicity and safety of the vaccine to be measured in comparison with a placebo, under double-blind conditions. They took place in Sweden, which has the most “naive” population with respect to vaccination against pertussis, given that vaccination was abandoned in that country for several years, for reasons of inefficacy. 
The main objective of these trials was to record all possible adverse events, namely cough, sneezing, nasal discharge, effects on general health, etc. These measurements were examined by an independent data monitoring committee.
The second objective was to assess colonisation of the nasal mucosa by the vaccine, and the triggering of an immune response. 
It was possible to test three different doses of the vaccines: a low, intermediate and high dose. 
Results obtained after monitoring the vaccinated subjects for 6 months, and analysing 60,000 data points, showed that the vaccine induced no adverse events compared with the placebo, even at the high dose. The vaccine colonised the nasal mucosa best at the high dose. Moreover, immune responses were triggered in all subjects whom the vaccine had colonised. “It is of special interest that a single nasal administration was able to induce an immune response that was maintained for at least 6 months, i.e. for the duration of the study,” comments Camille Locht. 

The next step will involve administering higher volumes in an effort to increase the level of colonisation of the nasal mucosa by the vaccine. Camille Locht and his collaborators also hope to improve the stability of vaccine over time, with a view to industrial development in the near future. 

Inserm Transfert, in charge of the valorization of the IP portfolio related to the BPZE-based technology, recently entered into an agreement with a biotech partner to further develop the technology.

Further information

CHILD-INNOVAC

The CHILD-INNOVAC project was aimed at developing innovative intranasal vaccines against the two main respiratory pathogens, pertussis and respiratory syncytial virus (RSV). The project has provided prototypes for polyvalent vaccines that can be administered intranasally, based on attenuated B. pertussis. The immunity induced by the BPZE1 vaccine was studied in detail, together with its genetic and biological stability and safety.
CHILD-INNOVAC began in 2008, and was supported by the EU (FP7) for 4 years. It was coordinated by Inserm, along with 27 other European projects. The project involved 10 partners, including 2 private companies and 8 laboratories, based in 7 European countries:

Inserm (coordinator), France: http://www.inserm.fr/
Inserm Transfert, France: http://www.inserm-transfert.fr/
Université Libre de Bruxelles, Belgium: http://www.ulb.be/
Innogenetics, Belgium: http://www.innogenetics.com
National University of Ireland, Maynooth: www.immunology.nuim.ie
Istituto Superiore Di Sanità, Italy: http://www.iss.it/
Swedish Institute for Communicable Disease Control: http://www.smittskyddsinstitutet.se/in-english/
Netherlands Vaccine Institute
National Institute for Public Health and the Environment, the Netherlands: http://www.rivm.nl/
Imperial College of Science, Technology and Medicine, England: http:/www3.imperial.ac.uk/

A prime target for the development of anti-inflammatories

For the first time, scientists from the Institut Pasteur and Inserm have demonstrated the key role played by a particular molecule in intestinal infection. The study was published online in Immunity on December 12, 2013. The molecule, known as ATP, serves as a trigger signal for the inflammatory response targeting pathogenic agents. Using the Shigella flexneri model, the scientists have also shown how this bacterium is able to block the release of ATP in order to escape this defense reaction.

Discovery of this blocking mechanism could be a milestone in therapeutics: the development of new drugs that mimic this process could open up new possibilities for the treatment of chronic inflammatory diseases, such as Crohn’s disease.

shigella

© Inserm/Tran Van Nhieu, Guy

It has been known for some years that the extracellular presence of ATP – a molecule normally found inside cells – is able to alert the body to danger and trigger an inflammatory defense response. Such may be the case when tissue is damaged and cell contents are released. Yet, whether an infectious agent would be capable of triggering the same mechanism remained unclear.

The team led by Philippe Sansonetti, who heads the Molecular Microbial Pathogenesis Unit (Inserm U786 / Institut Pasteur), in association with scientists from the University of Toulouse and the Collège de France have recently shown in vitro and in vivo that infection with enteric pathogenic bacteria – in this study Shigella, Salmonella and enteropathogenic E. coli – induces the cell to actively release ATP into the extracellular environment: in the presence of the bacterium, intestinal epithelial cells open cell surface channels, allowing ATP molecules to escape. By binding to extracellular receptors, ATP triggers a chain of reactions that drive the inflammatory immune response designed to eliminate the threat of infection.

The scientists also proved that the Shigella flexneri bacterium is capable of blocking this release of ATP by injecting an enzyme directly into the infected cell. The enzyme acts by closing the channels. This is the first time a pathogen has been observed to have the ability to suppress this mechanism, thus allowing it to escape defenses put in place by the body. This discovery underlines the importance of ATP as a key regulator of intestinal inflammation.

Although inflammation is a natural defense mechanism that plays an essential role in tissue response to attack, it persists abnormally in some cases. It then becomes chronic, and can lead to inflammatory diseases. These diseases result in a malfunction of the immune system, which attacks the body’s normal components. By showing this potential to halt the inflammatory process, the study highlights this process as a new, prime therapeutic target for the development of anti-inflammatory drugs.

Currently, there are no anti-inflammatory drugs on the market that targets the release of ATP. However, this release mechanism could have an important, as yet undefined part to play in the future treatment not only of certain chronic inflammatory diseases of the intestine, such as Crohn’s disease, but also other pathologies such as cancer, obesity, type 2 diabetes or arteriosclerosis.

Chemotherapy: when our intestinal bacteria provide reinforcement

Research jointly conducted by investigators at Institut Gustave Roussy, Inserm, Institut Pasteur and INRA (French National Agronomic Research Institute) has led to a rather surprising discovery on the manner in which cancer chemotherapy treatments act more effectively with the help of the intestinal flora (also known as the intestinal microbiota). Indeed, the researchers have just shown that the efficacy of one of the molecules most often used in chemotherapy relies to an extent on its capacity to mobilise certain bacteria from the intestinal flora toward the bloodstream and lymph nodes. Once inside the lymph nodes, these bacteria stimulate fresh immune defences which then enhance the body’s ability to fight the malignant tumour.

Results of this work are published in the journal Science on 22 November 2013

bacteria - blue version

©Fotolia

The intestinal microbiota is made up of 100,000 billion bacteria. It is a genuine organ, since the bacterial species that comprise it carry out functions crucial to our health, such as the elimination of substances that are foreign to the body (and potentially toxic), or keeping the pathogens that contaminate us at bay. They also ensure the degradation of ingested food, for better intestinal absorption and optimal metabolism. These millions of bacteria colonise the intestine from birth, and play a key role in the maturation of the immune defences.

However, the bacterial species that make up the intestinal microbiota vary from one individual to another, and the presence or absence of one or another bacterial species seems to influence the occurrence of some diseases, or, conversely, may protect us.

In the cancer area, the French team directed by Prof Laurence Zitvogel, Director of Inserm Unit 1015,Tumour Immunology and Immunotherapy,” at Institut Gustave Roussy, in close collaboration with Institut Pasteur (Dr Ivo Gomperts Boneca, “Biology and Genetics of the Bacterial Cell Wall” Unit) and researchers at INRA (Drs Patricia Lepage and Joël Doré, Micalis Unit, “Food Microbiology in the Service of Health”), has just provided evidence that the intestinal flora stimulates an individual’s immune responses to combat cancer during chemotherapy.

Cyclophosphamide is one of the most widely used drugs in chemotherapy. However, like any treatment, it involves side effects (inflammation of the mucosa etc.), and disrupts the normal balance of the intestinal microbiota. Certain bacteria (of the Gram+ group of bacteria) can pass the intestinal barrier and enter the bloodstream and lymph nodes.

These bacteria, once in the general circulation of the body, may be considered harmful, and the body generates an immune response.

“This chain reaction, a side effect of the treatment, actually turns out to be very useful,” explains Laurence Zitvogel. “Surprisingly, the immune response directed against these bacteria helps the patient to better fight his/her tumour, by stimulating fresh immune defence mechanisms.”

More specifically, immunisation against bacteria leads to the recruitment of effector lymphocytes different to those mobilised by chemotherapy. Their role consists of helping anti-tumour lymphocytes to stem the growth of tumours.

To verify these observations in mice, researchers suppressed all Gram+ bacteria from their intestinal microbiota. Results showed that the efficacy of the chemotherapy was reduced. The researchers also suggest that some antibiotics used during chemotherapy may destroy these Gram+ bacteria, and thus negate their beneficial effect.

“Now that these “beneficial” bacteria that potentiate the anti-tumour immune response have been identified, we should soon succeed in supplying more to the body, especially via pro- or prebiotics and/or a specific diet,” the researcher concludes.

This work has received support from the French National Cancer League, the French National Cancer Institute (lNCa; SIRIC SOCRATES) and from LABEX Onco-Immunology
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