Identification of a new mechanism in the most commonly used immunotherapy for lymphoma

Using innovative dynamic imaging technique, scientists at the Institut Pasteur, Inserm and the VU Medical Center in Amsterdam have uncovered the mode of action of anti-CD20, an antibody therapy frequently used in the treatment of lymphomas (cancers of the immune system) as well as some auto-immune diseases. In a lymphoma model, the scientists have been able to carry out real time in vivo imaging of the cellular events activated by the treatment and resulting in the destruction of tumor cells. These discoveries should help optimize the efficacy of future therapies involving anti-CD20 antibodies. This work is the subject of an article published online November 1 on the Journal of Clinical Investigation website.

A lymphoma usually develops as a result of abnormal proliferation of one of two types of immune cells:  B lymphocytes (in the vast majority of cases) or T lymphocytes. For the last fifteen years or so, anti-CD20 antibody therapy has frequently been used in the treatment of B-cell lymphomas (in particular those known as non-Hodgkin lymphomas), in combination with conventional chemotherapy. These antibodies are directed against B lymphocytes, bind to cancer cells and mark them for depletion by other immune cells. Anti-CD20 antibody therapy also triggers a decrease in the normal B lymphocyte population, dampening immune responses. For this reason it is also used to treat autoimmune diseases. However, how anti-CD20 antibody therapy works in vivo was not fully understood.

A study led by Philippe Bousso, head of the Dynamics of Immune Responses Unit (Institut Pasteur / Inserm U668), along with researchers at Inserm and the VU Medical Center in Amsterdam, has provided the first conclusive answers. Using dynamic imaging techniques developed at the Institut Pasteur, the scientists have carried out real time in vivo imaging of the destruction of cancerous and normal B lymphocytes during anti-CD20 antibody treatment. The scientists noticed that the phenomenon of B lymphocyte depletion resulting from anti-CD20 antibody therapy primarily takes place in the liver and involves a specific cell type, known as Kupffer cells. The images produced by the scientists clearly show Kupffer cells (in green) capturing cancerous B lymphocytes (in orange) and preventing their circulation before destroying them.

These discoveries provide important insight for optimizing the efficacy of future treatments using anti-CD20 antibodies. Non-Hodgkin lymphomas affect 10,000 people per year in France, and account for 10% of pediatric cancers.

Fine-tuning the approach to malaria and toxoplasmosis research

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. 

photo CP toxoplasmose

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.

Circumcision effectively reduces the risk of HIV infection “in real life”

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.

Neutrophils: the Unsung Heroes of Immunotherapy Cancer Treatment

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.

Etude de la moelle osseuse et de l'hématopoièse

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.

The way the virus continually changes complicates the development of a vaccine against HIV

A team from INSERM Mixed Research Unit 966 Morphogenesis and Antigenicity of HI and Hepatitis Viruses” headed by Martine Braibant and Francis Barin in Tours, has confirmed, with the support of the ANRS, that the AIDS virus has gradually adapted itself to the immune response of the human population during the course of the epidemic. Starting from the time it was isolated in the 1980s to the present day, the virus has become less and less sensitive to neutralising antibodies, complicating the creation of an effective preventive vaccine. Researchers have also shown that the combination of two powerful neutralising antibodies remains capable of neutralising in vitro the most recent HIV variants. Details of the research are published in the journal PLoS Pathogens.

The development of a vaccine that is effective against HIV remains one of the major challenges in the fight against AIDS. Considerable progress has been made in recent years with the identification of human neutralising antibodies capable of blocking in vitro infection caused by the very numerous variants of HIV -1. The scientific community is considering performing clinical trials in humans, using some of these antibodies. One of the issues that remains to be resolved is that of the choice of antibodies and the best combination of antibodies to be used in order to afford the widest possible protection in view of the huge variability of possible forms of HIV -1 currently circulating in the world.

Particules virales du virus de l'immunodéficience humaine (VIH)


© Inserm /  Philippe Roingeard

The research, headed by Martine Braibant and Francis Barin (INSERM-Université de Tours Mixed Unit) with the support of the ANRS, confirms that during the course of the epidemic HIV-1 has gradually adapted itself to the immune response of the human population by becoming less and less sensitive to certain neutralising antibodies. In order to arrive at these results, the researchers were able to analyse viruses taken from samples in patients infected between the late 1980s and the year 2010[1].

“Although it was known that, at an individual level, the virus was able to adapt and circumvent the individual’s own means of defence, our work confirms that the pressure of selection exercised on the virus has had repercussions on a population-wide scale”, explains Martine Braibant.

Despite this depressing prospect, in the same publication, researchers identify a combination of two powerful neutralising monoclonal antibodies developed by Caltech and the Scripps Research Institute (NIH45-46G54W and PGT128) that still remain capable of neutralising in vitro the most recent variants and do so at a concentration compatible with their use in humans.

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 .

Neutralising antibodies: one of the research pathways for developing an anti-HIV vaccine

Current anti-viral vaccination concepts are largely based on the fact that certain so-called neutralising antibodies, by attaching themselves to viral proteins, inhibit the early stages of HIV infection. When blocked in this way, the virus cannot reproduce and is eliminated.

Scientists have currently identified the immune response to be induced via the identification and characterisation of wide-spectrum neutralisation antibodies. They have not yet succeeded in enabling this type of antibody to be produced in the human body through a standard vaccination. One of the research pathways being pursued is the introduction of gene coding for these antibodies in viral expression vectors so as to cause them to be secreted directly into the circulation. This antibody approach has been tested in France as part of the ANRS/VRI vaccine research programme (Vaccine Research Institute)

[1] PRIMO and SEROCO ANRS cohorts, collaboration with Laurence Meyer and Cécile Goujard, Université Paris-Sud – INSERM U1018.


(French) : Découverte d’une nouvelle cible thérapeutique chez E. coli

Quels progrès médicaux pour les personnes vivant avec le VIH en France ?

Vesicular transport: a requirement for T immune response

T-lymphocyte (TL) activation requires recognition by the T-cell receptor (TCR) of the ligands present on the antigen-presenting cell (APC). The ligand–TCR link bound to the TL plasma membrane  introduces a signalling cascade that converts a signal received from the outside into a suitable response, for example cytokine secretion. Claire Hivroz, INSERM Research Director (INSERM Unit 932, Institut Curie, Paris) and her colleagues recently showed in an article published in Nature immunology how a specific protein known as VAMP7 is essential for transporting the signal produced by the ligand–TCR link. 

The setting up of this activation cascade has been the subject of numerous research papers and yet remains little known. In particular, the location of the cell in which the signalling occurs and the mechanisms governing the formation of the protein complexes needed for such activation remain the subject of controversy.

Claire Hivroz’s team showed in 2004 that a protein that played a key role in TCR signalling, the LAT molecule, acts as a framework on which the other signalling proteins are assembled. It is present on the plasma membrane and in each of the intra-cellular vesicles. In response to TCR stimulation, these vesicles are attracted to the area in which the stimulation takes place, namely, the contact area between the TL and the APC, known as the immunological synapse (IS).


Crédit photo : © Inserm-Institut Curie/C. Hivroz

Evanescent wave microscopy image showing recruitment into the immunological synapse of LAT-containing vesicles (magenta), VAMP7 (green) or the two proteins (white) in a T-lymphocyte interacting with a glass slide covered in activating antibodies (INSERM–Institut Curie/C. Hivroz)


Researchers wanted to discover the role played by this intracellular LAT ‘pool’ and the mechanisms that lie behind its recruitment by the IS. The study being published this month in Nature Immunology was performed in collaboration with Thierry Galli, a specialist in the proteins involved in the transport of vesicles to the neurological synapse, known as SNARE proteins, involved in membrane fusion reactions during vesicular transport.

By using genetically modified mice, RNA interference mechanisms and high-resolution microscopy, Claire Hirvoz’s team showed that VAMP7, a SNARE protein, is necessary for transporting LAT-containing vesicles to the IS. This transport controls LAT activation and the formation of the protein complex that sets up the signalling cascade. Consequently, T-lymphocytes deprived of VAMP7 will not respond normally to stimulation of their TCRs.

In conclusion, this new data will contribute to understanding the way in which information is propagated over time and space after TCR stimulation and shows for the first time how the transport of vesicles is involved in setting up a response to T-lymphocytes.

These results also show that molecules present in the neuronal synapses and involved in their functions are also involved in the functions of the immunological synapse.

Chikungunya: discovery of a human-specific factor involved in the virus replication

Scientists from the Institut Pasteur, Inserm and CNRS, have identified a human-specific factor involved in the replication of Chikungunya virus which accounts for the species specificity of this virus. Chikungunya virus is an emerging virus that in 2005 caused, for the first time, an outbreak in La Réunion island, a French overseas district where more than 30% of the population was infected, and has recently emerged in temperate regions of Europe. The identification of this new host factor enriches our understanding of the molecular bases of Chikungunya virus infection, which were characterized so far. This work also paves the way for the development of a more relevant humanized animal model to better understand the pathophysiology of infection.
This research has been published online on April 26, at the European Molecular Biology Organization reports (EMBO reports).

Chikungunya virus was first identified in Tanzania in the 1950s and emerged in the islands of the Indian Ocean in 2005 where it caused a large outbreak, most notably in La Réunion island. In 2007, this virus was imported to Europe, causing an outbreak in Italy. In late summer 2010, the two first autochthonous cases were reported in the French Riviera.

Virus Chikungunya à la surface d'une cellule

Copyright Institut Pasteur / Thérèse Couderc

The molecular mechanisms of Chikungunya virus infection in human cells have been poorly characterized. Scientists at the Institut Pasteur, Inserm and CNRS, under the leadership of Marc Lecuit, head of the Biology of Infection Unit at the Institut Pasteur in Paris, have made a significant advance: they have identified a human-specific intracellular molecular factor called NDP52 that is involved in the replication of Chikungunya virus. The scientists have demonstrated that in human cells, NDP52 is able to bind to a protein of Chikungunya virus, nsP2, and that this interaction promotes the replication of the virus inside its target cells, thereby contributing to the development of human Chikungunya virus infection. In contrast, mouse NDP52 does not interact with nsP2, and NDP52 therefore does not promote infection in murine cells.

In 2008, the team headed by Marc Lecuit had developed the first animal model to experimentally reproduce the human disease caused by Chikungunya virus. This animal model has since allowed a better understanding of the physiopathology of infection, and has enabled the identification of Chikungunya virus target cells and tissues to test the efficacy of preventive and curative approaches. The discovery of the role of the human factor NDP52 in Chikungunya virus infection opens new avenues for the development of a humanized animal model, which would be instrumental for deeper understanding of the pathophysiology of Chikungunya virus infection.

(French) Légionellose : reprogrammation inédite des cellules hôtes à l’avantage de la bactérie Legionella pneumophila