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.

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.

© 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.

A study carried out by teams from the Institut Pasteur, the Institut Cochin (Inserm, CNRS, Paris Descartes University), and the Wellcome Trust Centre for Molecular Parasitology at the University of Glasgow, may very well redefine current approaches to malaria and toxoplasmosis research in terms of treatment development. Their research which focuses on the role played by the protein AMA1 (present in both parasites) was published october 9, on the Nature Communications website. For many years AMA1 has been the focus of studies aiming to develop malaria treatments and vaccines. However, the authors of this study express their reservations about strategies that focus strictly on blocking AMA1 and show that malaria and toxoplasmosis parasites without AMA1 are still capable of developing normally. 

Stade mérozoïte de Plasmodium falciparum – Copyright Institut Pasteur

One million people die each year from malaria, making it the most widespread parasitic disease in the world today. Toxoplasmosis, which often presents no symptoms in healthy subjects, is a parasitic disease that mainly affects immunedeficient individuals. Pregnant women are also particularly at risk for this disease and if infected for the first time during their pregnancy could spread infection to the fetus.

Plasmodium and Toxoplasma, genera of apicomplexan parasites, are the agents responsible for these diseases. They both contain the same protein, AMA1, which many studies consider necessary for parasites to enter host cells and propagate infection. Because of this, numerous studies conducted since the discovery of the protein have made it their primary focus in the development of anti-parasitic treatments. However, this trend may soon change, thanks to the collaborative efforts of Robert Ménard (Institut Pasteur, Paris), Isabelle Tardieux (Institut Cochin, Paris), Markus Meissner (University of Glasgow), and their teams. These scientists recently showed that in the total absence of AMA1 Plasmodium berghei and Toxoplasma gondii are still capable of entering infected cells and multiplying. This discovery could have a significant impact on how research is conducted in the development of treatments for malaria and toxoplasmosis.

In 2011, the scientists had already shown that parasites genetically modified with very low levels of AMA1 were still able to infect host cells. In today’s study, parasites were created free of all AMA1 using “reverse genetics” (a technique never before used in this field). The scientists showed that in the absence of AMA1, during the blood and hepatic stages of Plasmodium berghei, as well as during the replication stage of Toxoplasma gondii (the stage where the disease spreads to humans), the parasites were still able to enter host cells. What was affected by the lack of AMA1, however, was the parasites’ ability to bind to host cells (this is the step that precedes cell entry). Because of this, the scientists concluded that AMA1 is not a requirement for cell entry but rather is used to bind parasites to the host cells. These observations have prompted the scientists to put forward new recommendations for optimizing research that focuses on AMA1 for the development of new treatments. In particular, they suggest that therapeutic strategies and vaccine development should focus on other proteins in addition to AMA1.

The ANRS-12126 “Bophelo Pele” Project implemented in the township of Orange Farm in South Africa has confirmed the effectiveness of a large-scale program of voluntary medical male circumcision in prevention of heterosexually acquired HIV infection. The follow-up of over 3300 men shows a 57% to 61% reduction in the rate of new HIV infections in circumcised men compared with uncircumcised men. This study, headed by Prof Bertran Auvert and his colleagues, also shows that a circumcision program can be rapidly and effectively implemented in African communities where circumcision is not a social norm. These results, published in PLoS Medicine, argue for accelerated roll-out of voluntary male circumcision programs on the African continent in order to improve prevention of HIV transmission.

Three randomized trials have shown that male circumcision has a protective effect on the risk of HIV infection in men. The first publication dates from 2005 (ANRS-1265 study in South Africa) and its results were subsequently confirmed in Kenya (2007) and Uganda (2007). These studies showed that the risk of circumcised men being infected by HIV was reduced by 50% to 60%. These results led UNAIDS/WHO to recommend in 2007 the circumcision of adult males as a strategy of additional HIV prevention in communities with a high prevalence of HIV and a low prevalence of circumcision.

There remained, however, a need to show that the roll-out of circumcision “in real life” reduces both the incidence (rate of occurrence of new infections) and the prevalence (proportion of people infected in the total population) of HIV infection in men. This had been suggested by preliminary results from the ANRS-12126 “Bophelo Pele” Project of Bertran Auvert and colleagues presented at the International AIDS Society Conference 2011. These results are now confirmed in the PLoS Medicine article, the first scientific publication to show that adult male circumcision “in real life” effectively reduces the risk of HIV infection in men (1).

Between 2007 and 2011, the ANRS-12126 “Bophelo Pele” Project was conducted by Prof Bertran Auvert (UMRS-1018 Inserm, Hôpital Ambroise Paré and Université de Versailles Saint-Quentin) and his colleagues from the National Institute for Communicable Diseases, the Social Sciences Faculty, and Progressus (Johannesburg, South Africa), and from Johns Hopkins University (Baltimore, USA) and the Bichat Hospital (Paris, France). Free, medical circumcision was offered to all male volunteers aged from 15 to 49 years in a population of 110 000 adults in the township of Orange Farm in South Africa. More than 20 000 circumcisions were performed, accompanied by a large information provision and prevention program.

An anonymous questionnaire on sexual practices was administered to 3338 men recruited from the township population who were invited to undergo HIV screening, which included a test to determine, in the event of seropositivity, whether the infection was recent.

The proportion of circumcised men in this sample of 3338 increased from 12% at the start to 53%, and was 58% in the 15- to 29-year-olds. Importantly, sexual behavior, in particular condom use, did not differ between circumcised and uncircumcised men. In contrast, the prevalence and incidence of HIV infection were much reduced in the circumcised men.

The researchers considered that without the voluntary circumcision program, HIV prevalence would have been 19% higher in the study population. This effect is more marked in the 15- to 29-year-olds, in whom the prevalence would have been 28% higher. Also apparent was a decrease in the number of recent infections among the circumcised men. Circumcision was therefore associated with a 57% to 61% reduction in the rate of new infections.

Prof Bertran Auvert considers that “these results are important in two ways. First, they confirm the efficacy of the circumcision practiced on a population scale in reducing HIV transmission appreciably among the men of this population. Second, they show that it is possible to achieve this result in just a few years, including in populations where circumcision is not a common practice.”

The ANRS-12126 Project provides an argument for speeding up the roll-out of voluntary circumcision programs, notably in sub-Saharan Africa, which is home to the vast majority of the 2.2 million people infected by HIV every year worldwide. Prof Jean-François Delfraissy, Director of the ANRS, considers that “given the impact observed in this study on limiting the risk of HIV acquisition in circumcised men, the scale-up of circumcision should more than ever be a public health priority in South and East Africa.”

The ANRS-12126 Project is continuing with the aim of elucidating the effect of circumcision on infection risk reduction in the general population, and in particular among women.

Scientists at the Institut Pasteur and Inserm have identified the group of cells within the immune system that make immunotherapy treatment (therapeutic antibodies) effective. Immunotherapy is frequently used to treat breast cancer. In animal models they showed that neutrophils, the most common white blood cells in the body, are not only necessary but suffice on their own to eliminate tumor cells. The scientists produced these results using both skin and breast cancer models. If confirmed in humans, these discoveries should lead to the optimization of current treatments for several cancers. This work is being published online August 26, 2013 by the medical journal Blood.

With its millions of victims each year, cancer remains one of the leading causes of death worldwide. In women, breast cancer is the most common offender. Nearly one in nine women will develop breast cancer in their lifetime. In France alone, over 50,000 new cases are diagnosed each year.

Between 20 and 30% of breast cancer cases are deemed eligible for immunotherapy treatments which involve the injection of antibodies. Until now, the effectiveness of this type of treatment was attributed to various immune system components such as Natural Killer cells and macrophages. However, work by Pierre Bruhns, head of the Antibodies in Therapy and Pathology Laboratory (Institut Pasteur/Inserm Unit 760), and his team, in collaboration with Professor Clifford A. Lowell of the University of California Department of Laboratory Medicine and two other teams from the Institut Pasteur, has cast serious doubt on this theory.

Neutrophile observed by optical microscopy ©Inserm/E.Cramer

The scientists have shown in an animal model that neutrophils alone are effective enough to induce the therapeutic effect of immunotherapy treatments often used to treat breast cancer. Neutrophils are drawn to the tumor after the injection of therapeutic antibodies and are activated after coming into contact with these antibodies. Once activated the neutrophils are capable of destroying the tumor cells. This discovery represents an important step towards the optimization and development of immunotherapy treatments used to treat these cancers.

The scientists made the following observations: the tumor mass did not regress in mice that were neutropenic (having a significantly low level or total lack of neutrophils) or in mice with neutrophils that could no longer be activated through contact with therapeutic antibodies. Then, scientists showed that the anti-tumor effect achieved via immunotherapy could be reproduced in deficient mice by administering them with neutrophils taken from healthy mice. These observations were made using models for both skin and breast cancer leaving us to assume that neutrophils play an important role in immunotherapy treatments used to fight various other cancers.

In their conclusion, the scientists highlighted current immunotherapy treatments used to fight cancer. These treatments are often coupled with other treatments. Because of this, a good strategy might be to favor treatment pairings that increase neutrophil count and activity.

This study stresses the need to monitor the development of the sensitivity of HIV -1 variants to the various neutralising antibodies. The work of the researchers opens up interesting prospects for teams involved in researching a vaccine to prevent HIV .