Photo (colorized) of scanning electron microscopy of a bacterium lyzed by the phages (© L. Debarbieux, Institut Pasteur; M. and C. Rohde, Helmholtz Centre for Infection Research).

Antibiotic resistance represents a major public health challenge, associated with a high mortality rate. While bacteriophages – viruses that kill bacteria – could be a solution for fighting antibiotic-resistant pathogens, various obstacles stand in the way of their clinical development. To overcome them, researchers from Inserm, Université Sorbonne Paris Nord and Université Paris-Cité at the IAME Laboratory, in close collaboration with their counterparts at Institut Pasteur and the Paris Public Hospitals Group (AP-HP), have developed a model to better predict the efficacy of phage therapy and possibly develop more robust clinical trials. Their findings have been published in Cell Reports.

The discovery of antibiotics had revolutionized the history of medicine in the 20th century, allowing us to effectively fight bacteria for the first time. However, antibiotic resistance – a phenomenon during which bacteria become resistant following mass, repeated use – has become a major public health issue in recent decades. Each year, these resistant bacteria are estimated to be responsible for 700,000 deaths worldwide. Yet the discovery of new antibacterial agents has been stagnating for several years.

In this context, phage therapy has recently generated renewed interest. This therapeutic approach involves the use of bacteriophages that target and destroy pathogenic bacteria whilst being unable to infect humans. While the concept has been in existence for a long time, its clinical development has been hampered by various limitations. Unlike “conventional” medicines, bacteriophages are complex biologics, whose action in the body, optimal dose, and most effective route of administration are difficult to study and anticipate.

In order to remove some of these obstacles, Jérémie Guedj’s research team at Inserm, in collaboration with Laurent Debarbieux’s team at Institut Pasteur, has developed a new mathematical model to better define the interactions between bacteriophages and pathogenic Escherichia coli bacteria in animals and to identify the key parameters that influence the efficacy of phage therapy.

Supporting clinical development

Various data from in vitro and in vivo experiments were used to construct this model. In particular, the researchers used the bacteriophages’ infection parameters determined in the laboratory (for example, the duration of the infectious cycle of the bacteria, the number of viruses released when a bacterium is destroyed…) and information collected during experiments using a mouse model of lung infection.

Some of the animals were infected with a bioluminescent strain of E. Coli (in order to best monitor it within the body). Among them, some were treated with bacteriophages at different doses and using different routes of administration. The quantities of bacteria and bacteriophages thus measured over time helped to feed the mathematical model and test which were the most important parameters for effective phage therapy. 

Using their model, the scientists show that the route of administration is an important parameter to consider when it comes to improving the animals’ survival: the more rapidly it brings the bacteriophages into contact with the bacteria, the more it is effective. In the animal model, the phage therapy administered intravenously was therefore less effective in comparison with the intratracheal route because fewer bacteriophages were reaching the lungs. On the other hand, when administered by intratracheal route, the model suggests that the dose of the medication given has little effect on the efficacy of the therapy.

Another important point is that this model incorporates data on the animals’ immune response in the context of phage therapy. The model confirms and extends the principle that bacteriophages act in synergy with the immune system of infected animals, enabling more effective elimination of pathogenic bacteria.

“In this study, we propose a new approach to streamline the clinical development of phage therapy, which otherwise continues to have its limitations. Our model could be reused to predict the efficacy of any bacteriophage against the bacteria it targets, once a limited number of in vitro and in vivo data are available on its action. Beyond phage therapy, the model could also be used to test anti-infective therapies based on the association between bacteriophages and antibiotics,” concludes Guedj.

Human respiratory epithelial cells (in red) infected with an influenza virus (in green). © A. Cezard, D. Diakite, A. Guillon, M. Si-Tahar, PST ASB-Microscopy Department.

Seasonal influenza is a major public health issue because it continues to remain associated with considerable mortality, particularly among people who are elderly, immunocompromised, or both. It also has a significant socioeconomic cost. With vaccination and current treatments still being of limited efficacy, research teams are trying to develop new therapeutic approaches. At the Research Center for Respiratory Diseases in Tours, scientists from Inserm, Université de Tours and Tours Regional University Hospital have shown that in a context of influenza infection, a metabolite[1] called succinate, which is naturally present in the body, has an antiviral and anti-inflammatory action. These findings open up new therapeutic prospects based on the use of succinate derivatives. The study has been published in EMBO Journal.

Often considered a mild disease, influenza continues to cause the deaths of 10,000 to 15,000 people each year in France. The socioeconomic cost of the disease is also significant because it is associated with high levels of absenteeism and a major burden on hospitals.

Seasonal influenza vaccination is a central pillar of the preventive strategies deployed to reduce the number of cases and fight the disease. However, its efficacy can vary from year to year, depending on the influenza viruses in circulation and the suitability of the vaccine to them. Drugs that directly target the influenza virus are available for severe cases, but the window of time for effective action with these treatments is very short. What is more, influenza viruses have become resistant to their action.

In this context, the development of innovative therapies is a priority. While current treatments work by targeting certain components of the virus, Inserm Research Director Mustapha Si-Tahar and his colleagues at the Research Center for Respiratory Diseases are trying to better understand the host’s cellular and molecular responses to viral infection, with the long-term aim of developing novel therapeutic strategies aimed at strengthening these responses.

The role of metabolites in immune response

While metabolism¹ has long been considered to be a purely energetic mechanism essential for cell function, recent research has shown that some metabolites can also regulate the immune response.

Based on these data, Si-Tahar’s team wondered whether an influenza infection could cause the reprogramming of the metabolism of the target cells of the virus and whether specific metabolites play an especially active role in the immune response.

In mice infected with influenza, the researchers observed that a metabolite called succinate accumulates in the lungs. A phenomenon which was then confirmed in humans by comparing respiratory fluids from intensive care patients with and without influenza pneumonia. The presence of succinate was found to be significantly higher in the influenza patients.

They then exposed cells from the pulmonary epithelium to succinate, thereby demonstrating that the molecule has antiviral activity by blocking multiplication of the virus. Succinate also helps to reduce the strong inflammatory response that is triggered in the lungs following infection with influenza.

The researchers also found that mice exposed to the virus receiving intranasal succinate are better protected against infection and have a higher survival rate than those that do not receive it.

In search of molecular mechanisms

In order to better understand these different phenomena, the scientists sought to decipher the molecular mechanisms behind the antiviral action of succinate.

This involved analyzing the impact of succinate on the various stages of the viral replication cycle and demonstrating that, while there is no influence on the early stages of the cycle (entry, transcription, and translation), there is an influence on a later stage. The findings show that this metabolite prevents a major structural protein of the virus, the “nucleoprotein”, from exiting the nucleus of infected cells, thereby preventing the assembly of the final viral particle and interrupting the multiplication cycle of the virus.

These data all point to the key role of succinate in controlling influenza infection, as well as its therapeutic value.

Additional studies are needed in order to test the therapeutic potential of succinate and identify other metabolites of interest.

[1] Metabolism refers to all of the chemical reactions that take place inside the body’s cells. A metabolite is an organic substance derived from metabolism.

[2] In keeping with this research, Si-Tahar has since early 2021 coordinated a French National Research Agency (ANR) program entitled “Development of succinate-based formulations and analogues against SARS-CoV-2-induced respiratory infections and influenza viruses.” His team is also the beneficiary of an “ERS-RESPIRE4 Marie Skłodowska-Curie fellowship” to develop a project entitled “Succinate-Producing Probiotics as an Innovative Therapy for Viral Respiratory Infections: a proof-of-concept study”.

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A very small percentage of people with HIV-1, known as “post-treatment controllers” (PTCs), are able to control their infection after interrupting all antiretroviral therapy. Understanding the fundamental mechanisms that govern their immune response is essential in order to develop HIV-1 vaccines, novel therapeutic strategies to achieve remission, or both. A recent study investigated the humoral immune response – also known as antibody-mediated immunity – in some PTCs in whom transient episodes of viral activity were observed. The researchers have shown their humoral immune response to be both effective and robust, which could help to control the infection in the absence of treatment. The findings of this study, carried out in collaboration with teams from Institut Pasteur, Inserm and Paris Public Hospitals Group (AP-HP) and supported by ANRS | Emerging Infectious Diseases and the National Institutes of Health (NIH), were published in Nature Communications on April 11, 2022.

A very small percentage of people with HIV-1 and who received early treatment maintained over several years have the capacity to control the virus over the long-term when their treatment is interrupted. However, the mechanisms of this control have not been fully elucidated.  

The team of researchers, led by Dr. Hugo Mouquet, director of the Laboratory of Humoral Immunology at Institut Pasteur (partner research organization of Université Paris Cité), conducted an exhaustive study in PTCs in order to characterize their humoral response (i.e. their production of B cells and specific antibodies), compared with non-controllers.

The scientists have shown that the humoral immune response profiles vary according to the activity of the virus observed in the subjects.

In PTCs who experience short episodes in which the virus resumes low-level activity after interruption of treatment, transient exposure to the viral antigens induces:

• a strong anti-HIV-1 humoral response, involving more frequent intervention of HIV-1 envelope-specific memory B cells;
• the production of antibodies with a cross-neutralizing action and which possess “effector” antiviral activities in which the innate immune cells recognize the infected cells bound to the antibodies, thereby inducing their elimination;
• the increase in the blood of atypical memory B cells and subpopulations of activated helper T cells.

This specific, multifunctional, and robust humoral response could help to control their infection in the absence of treatment.

However, other PTCs in whom the virus continuously remains undetectable after treatment interruption do not develop a strong humoral response. The control mechanisms in these patients continue to be investigated in the VISCONTI study.

The discovery of these two types of humoral immune response, which depend on the profile of the PTCs, sheds new light on the phenomenon of HIV control. For Dr. Mouquet, researcher at Institut Pasteur and principal investigator of the study, “these findings show that early antiretroviral treatment can facilitate the optimal development of humoral immune responses, in some cases countering viral rebound after treatment interruption.” The example of the immune response of the PTCs having short episodes of “awakening” of the virus could even inspire novel therapeutic or vaccine strategies.

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In response to the 2014-2016 Ebola epidemic, the clinical development of a two-dose regimen of vaccines Ad26.ZEBOV and MVA-BN-Filo was accelerated. Approved in 2020 by the European Commission for use in epidemic emergencies, this regimen continues to demonstrate its relevance. An Inserm study led by Rodolphe Thiébaut (Inserm, INRIA, Université de Bordeaux, Vaccine Research Institute) has played a role in evaluating its safety and immunogenicity in healthy adults and in those with HIV and compared different time intervals between the two doses. It has confirmed that this regimen is well tolerated, that the antibodies acquired persist for at least one year, and that they can be easily reactivated by a booster shot. The findings of this trial have been published in Plos Medicine.

Since its discovery in 1976, Ebola has been found in several countries in Equatorial Africa with increasingly frequent epidemics (more than 30 to date). Infection with the virus begins in the form of flu-like illness but is often complicated by life-threatening organ failure and hemorrhage. During the largest epidemic in West Africa between 2014 and 2016, a total of 28,616 cases were identified leading to 11,310 deaths. During this epidemic, an initial single-dose vaccine was approved and used in the field (Ervebo). At the same time, pharma company Janssen launched the accelerated development of a different vaccine regimen1. This involves the administration of two different vaccines: the first being Janssen’s Ad26.ZEBOV, composed of an adenoviral vector containing the envelope protein of the Ebola Zaire virus that triggers antibody production, and the second being Bavarian Nordic’s MVA-BN-Filo, which uses a different viral vector and contains four different antigens of the Ebola virus family, including the Ebola Zaire virus glycoprotein.

“Using the viral vector makes it possible for the Ebola virus antigen (the glycoprotein) to penetrate the immune cells in the manner of a Trojan horse, triggering the immune response. With this dual-administration strategy, we assumed that the immune response would last longer than with a single-dose vaccine,” explains Rodolphe Thiébaut, Inserm team leader and Professor of Public Health at Université de Bordeaux, which is participating in this EBOVAC accelerated development program.

This hypothesis has recently been validated by the publication of the findings of a Phase II clinical trial (EBOVAC 2) intended to assess the safety and immunogenicity of this regimen in healthy people and in those with HIV living in Africa. In the meantime, it was approved in July 2020 by the European Medicines Agency in adults and children over one year of age, based on data already available.

Nevertheless, this regimen continues to be developed in different populations (adults, children, pregnant women, caregivers), in different parts of the world (to confirm the robust immune response and tolerability profile) and with different follow-up durations, to confirm the preliminary data and evaluate its utility in prevention in populations potentially exposed to the virus – particularly rural populations in Equatorial African countries. The aim being to prevent the occurrence of a new epidemic. “While the epidemics are generated by the virus passing from bats to humans, their progression is facilitated by mobility that is increasing thanks to the advances in infrastructure,” warns Rodolphe Thiébaut, and “having vaccines tested in Africa with the collaboration of research teams from African countries to protect populations is a major development challenge for our countries with their limited means and limited hospital resources,” adds Houreratou Barry, investigator responsible for the trial at the Muraz Center in Bobo-Dioulasso, Burkina Faso.

This Phase II trial is moving in that direction. It enrolled 668 healthy adults between the ages of 18 and 70 and 142 adults between the ages of 18 and 50 with HIV (a common condition in African populations liable to influence immune response to the vaccine) whose viral load was controlled by antiviral therapy. Participants were recruited in Kenya, Burkina Faso, Côte d’Ivoire, and Uganda. They received the vaccine or a placebo solution according to the following regimen: one dose of Ad26.ZEBOV or placebo, followed 28, 56, or 84 days later by MVA-BN-Filo or placebo. In the group of healthy subjects, 90 people also received an additional booster dose of Ad26.ZEBOV one year later.

The researchers analyzed adverse event reports over the follow-up year as well as changes in the levels of antibodies to the Ebola virus glycoprotein. No vaccine-related serious adverse events were observed, only mild to moderate events common with vaccination, such as pain at the injection site, fatigue, headache, and muscle pain.

The increase in the interval between the vaccinations from 28 to 56 days improved the immune response and the antibodies persisted for at least one year in both the healthy subjects and in those with HIV. These antibodies were found in 78 to 88% of the participants. Extending the interval to 84 days provided no additional benefit (and would only extend the vaccination schedule unnecessarily), which confirmed the 56-day interval as optimal for this vaccine regimen.

In addition, the booster one year later sufficiently stimulated the production of antibodies with levels multiplied by 55, indicating that the first vaccination triggered an easily reactivable memory immune response, “which is very important in the context of the recurring epidemics observed in Africa,” concludes Thiébaut.

1 Supported by the IMI EBOLA+ program (a joint initiative of the European Commission and the European Federation of Pharmaceutical Industries and Associations [EFPIA]) within the framework of a consortium with the academic teams from Inserm, the London School of Hygiene and Tropical Medicine, the University of Oxford, and the Muraz Center in Burkina Faso.  This project was funded by the joint Innovative Medicines Initiative 2 (www.imi.europa.eu) under EBOVAC2 grant agreement no. 115861. This joint initiative is supported by the European Union and EFPIA’s Horizon 2020 research and innovation program.