WO2018220416A1

WO2018220416A1 - Lactobacillus paracasei strain capable of improving the immune response to a viral-bacterial coinfection

Info

Publication number: WO2018220416A1
Application number: PCT/IB2017/000812
Authority: WO
Inventors: Raphaëlle BOURDET-SICARD; Nouria BELKACEM; Muhamed-Kheir Taha
Original assignee: Compagnie Gervais Danone; Bioaster; Institut Pasteur
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2018-12-06

Abstract

The invention relates to the field of probiotics and provides a Lactobacillus paracasei strain capable of improving the immune response to a viral-bacterial coinfection.

Description

LACTOBACILLUS PARACASEI STRAIN CAPABLE OF IMPROVING THE IMMUNE RESPONSE TO A VIRAL- BACTERIAL COINFECTION

Respiratory tract infections are still causing severe morbidity and mortality and are among the leading causes of death in children and adults worldwide. Influenza virus is a major source of severe viral respiratory infections in adults, causing annual epidemics that result in important morbidity and mortality. Major pandemics in the 20^th century killed more than 100 million people. A major part of the flu morbidity is also linked to secondary bacterial infections by bacteria that are known to share the respiratory tract as natural habitat and portal of entry such as Streptococcus pneumoniae, Neisseria meningitidis and other staphylococci (Hubert et al., 1992, Meningococcal disease and influenza-like syndrome: a new approach to an old question. The Journal of infectious diseases 166:542-545; McCullers et al., 2006, Insights into the interaction between influenza virus and pneumococcus. Clinical microbiology reviews 19:571-582).

Epidemiological studies clearly showed a spatiotemporal association between influenza A virus (IAV) and N. meningitidis invasive infections. Both infections show seasonal pattern with most of the cases in winter. Moreover, the winter peaks of invasive meningococcal disease (IMO) usually follow the peak of clinical influenza-like illnesses (Figure 1, adapted from Rameix-Welti et al., 2009, Influenza A virus neuraminidase enhances meningococcal adhesion to epithelial cells through interaction with sialic acid-containing meningococcal capsules. Infection and immunity 77:3588-3595). This sequential pattern of flu infection and invasive meningococcal infection was reproduced in a murine animal model (Alonso et al., 2003. A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice. FEMS Microbiol Lett 222:99-106).

The mechanisms of enhancing bacterial superinfection has been addressed and suggested to be multifactor. Physical and immunological barriers and changes in the microenvironment have hereby been shown to contribute to the development of secondary bacterial infections. Another hypothesis is that, the neuraminidases (NA) of IAV enhance the adhesion process of N. meningitidis to the respiratory epithelium by direct interaction to the sialic acid containing capsules that constitute a substrate for the neuraminidase (NA) of IAV. Moreover, cleavage of capsular sialic acid at the bacterial surface may unravel sub-capsular meningococcal adhesins and enhance meningococcal adhesion to epithelial cells.

Several clinical trials suggested positive effect of preventive feeding with probiotics on respiratory infections in children and in elderly (Ozen et al., 2015, Probiotics for the prevention of pediatric upper respiratory tract infections: a systematic review. Expert Opln Biol Ther 15:9-20; Guillemard et σ/., 2010, Consumption of a fermented dairy product containing the probiotic Lactobacillus easel DN- 114 001 reduces the duration of respiratory infections in the elderly in a randomised controlled trial. Br J Nutr 103:58-68). In particular, it has been shown that compositions comprising L easel can improve resistance to some infectious diseases (WO 2010/018461).

However, there remains a significant need for enhancing protection against the more and more prevalent association between viral and bacterial infections.

The purpose of this invention is to fulfill this need by providing a Lactobacillus paracasei strain that can be used to enhance the immune response to a viral and bacterial coinfection. Thus, the invention relates to a Lactobacillus paracasei strain deposited with the CNCM under the number 1-1518 for use in the treatment, the prevention or the alleviation of a coinfection consisting of a primary viral infection and a secondary bacterial Infection in a subject.

The Lactobacillus paracasei CNCM 1-1518 was deposited, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, Paris, France) on December 30, 1994 with the reference CNCM 1-1518.

In the invention, the coinfection (also called superinfection) refers to the pathological state wherein a subject successively develops a primary infection and a secondary infection, the secondary infection occurring before the recovery from the primary infection.

A subject is considered as suffering from a coinfection when he is contaminated by the pathogen responsible of the secondary infection, while being still infected by the pathogen responsible of the primary infection and up to the recovery of both infections.

Therefore, it is deduced that a subject is necessarily infected by two distinct pathogens at the beginning of the coinfection, but he can remain infected by only one of these two pathogens during later phases of the coinfection. In the case of a viral-bacterial coinfection, the subject is necessarily infected by a virus and a bacterium at the beginning of the coinfection, but he can remain infected by said virus only, or by said bacterium only, until the end of the coinfection.

L. paracasei strain CNCM 1-1518 can be used for the treatment, the prevention or the alleviation of the viral-bacterial coinfection (the subject is infected by the virus and the bacterium), the residual secondary bacterial infection (the primary viral infection has been cured and the subject remains infected by the bacterium only) and/or the residual primary viral infection (the secondary bacterial infection has been cured and the patient remains infected by the virus only). in the invention, it is considered that a subject is infected (or suffering from an infection) if the clinical symptoms usually associated to the infection are detected and/or if the presence of the pathogen responsible of the infection is detected in the subject's body.

At the opposite, it is considered that a subject has recovered from an infection (or is no longer infected; or is no longer suffering from an infection) if the clinical symptoms associated to the infection are no longer detected and/or if the presence of the pathogen responsible of the infection is no longer detected in the subject's body.

The present invention is based on the unexpected observation made by the Inventors that L. paracasei strain CNCM 1-1518 is capable of improving the protection to a bacterial infection occurring during a viral infection, even if this bacterial infection occurred 7 days after the viral infection, timing when the immune system is dampened compared to control group.

Indeed, 7 days post-influenza infection, the Inventors observed that the cytokine responses in the lung were lower in the L paracasei CNCM 1-1518 group compared to the control group. Proinflammatory cytokines such as MIP-lcc, ΜΙΡ-Ιβ, MCP-1, IFN-y, but also anti-inflammatory cytokine such as !L-10 were dampened in the L. paracasei CNCM 1-1518 group compared to the control group (Figure 4). When looking at immune cells in the lungs 7 days post-influenza infection, the same number of innate and adaptive immune cells were found in both groups (Figure 6B), contrary to what was observed at DO, that is, the increase of immune cells in the lungs of animals receiving L paracasei CNCM 1-1518 compared to control group (Figure 6A).

In a particular embodiment, the coinfection occurs between 5 and 15 days, in particular between 7 and 10 days after the contamination by the pathogen responsible of the primary infection.

In other words, the subject can be contaminated by the pathogen responsible of the secondary infection between 5 and 15 days, in particular between 7 and 10 days after being contaminated by the pathogen responsible of the primary infection.

More particularly, the subject is contaminated by the pathogen responsible of a secondary infection during the week, or the two weeks, following the contamination by the pathogen responsible of the primary infection.

Even more particularly, the subject is contaminated by the pathogen responsible of the secondary infection 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days after the contamination by the pathogen responsible of the primary infection. In humans, the flu infection comprises an early stage with symptoms of fever, aches, diffuse pain, followed by a late stage of damage of the epithelium respiratory tract (cough, rhino pharyngitis and even bronchitis or bronchopneumonia).

If the flu infection is worsened by a secondary infection, the patient can develop a bacterial bronchopneumonia and/or an invasive bacterial infection (entry of the bacteria in the blood and dissemination).

In a particular embodiment of the invention, the coinfection occurs during the early stage of a flu primary infection.

In a particular embodiment of the invention, the coinfection occurs during the late stage of a flu primary infection.

In a particular embodiment, the subject is an animal, preferably a mammal, more preferably a human.

In an embodiment, the invention relates to the Lactobacillus poracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by a virus selected from the group consisting of Influenza A virus (IAV), influenza B virus (IBV), parainfluenza virus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus (RSV), coxsackie virus, in particular IAV.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primal viral infection is responsible of the flu.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for Its use as defined above, wherein the secondary bacterial infection is selected from the group consisting of: a meningococcal infection, a pneumococcal infection, a staphylococcal infection, an haemophilus infection, a pseudomonas infection, a mycoplasma infection, a chlamydia infection, a moraxella infection, in particular a meningococcal infection. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the secondary bacterial infection is caused by a bacterium selected from the group consisting of: Neisseria meningitidis, Haemophilus influenza, Streptococcus pneumoniae, Staphyloccocus aureus, Pseudomonas aeruginosa, Mycoplasma pneumoniae, Chlamydia pneumonia, Moraxella catarrhalis, in particular Neisseria meningitidis. In an embodiment, the Invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the secondary bacterial infection is responsible of the meningitis.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the virus and the bacterium colonize the same area of the body of the subject.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the virus and the bacterium colonize the respiratory tract.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the virus and the bacterium colonize the upper respiratory tract.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the virus and the bacterium colonize the lower respiratory tract.

In details, the "upper respiratory tract" comprises the nasal cavity and associated sinuses, the nasopharynx, the oropharynx, the larynx and the trachea, while the 'lower respiratory tract" comprises the bronchi, the bronchioles and the terminal bronchioles and the lungs.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the virus and the bacterium colonize the lungs.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by an influenza virus, preferably IAV, and the secondary bacterial infection is caused by Neisseria meningitidis.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by an influenza virus, preferably IAV, and the secondary bacterial infection is caused by Streptococcus pneumoniae.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by an influenza virus, preferably IAV, and the secondary bacterial infection is caused by Staphylococcus aureus.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by an influenza virus, preferably IAV, and the secondary bacterial infection is caused by Pseudomonas aeruginosa. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by the respiratory syncytial virus and the secondary bacterial infection is caused by Streptococcus pneumoniae.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary viral infection is caused by the respiratory syncytial virus and the secondary bacterial infection is caused by Haemophilus influenzae.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primary virai infection is caused by the respiratory syncytial virus and the secondary bacterial infection is caused by Staphyloccus aureus.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the primal viral infection is responsible of the flu and the secondary bacterial infection is responsible of the meningitis. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the level of at least one inflammatory cytokine in the respiratory tract and/or in the lungs of the subject that has consumed L. paracasei CNCM 1-1518 is increased compared to the level of said at least one inflammatory cytokine in the same body area of a control subject that has not consumed L. paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection.

In particular, the level of at least one cytokine is increased by at least 5%, 10%, 1596, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in the subject that has consumed L. paracasei CNCM 1-1518 compared to the control subject that has not consumed L paracasei CNCM 1-1518.

In the invention, the pro-inflammatory cytokines include, but are not limited to, IL6, MCP1, KC and IL12p70. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the level of IL6, MCP1, KC and IL12p70 in the respiratory tract and/or in the iungs of the subject that has consumed L. paracasei CNCM 1-1518 is increased compared to the level of IL6, MCP1, KC and IL12p70 in the same body area of a control subject that has not consumed L. paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection. The level of cytokines can be determined by various methods well known to one skilled in the art. In particular, it can be determined using an EUSA kit and compared to total protein content using a BSA protein assay as described in the examples.

The fact that no one bacterial species is responsible for all human cases of post influenza bacterial pneumonia indicates that there is a general impairment of pulmonary antibacterial defenses brought about by the viral infection.

In our experimental model, after 48h of secondary meningococcal post-infection, total immune cells in the lungs and in particular innate cells such as dendritic cells, monocytes and neutrophils showed elevated level in L. paracasei group compared to the control group. Adaptive immune cells such as B cells were comparable in number in the groups treated or untreated with L paracasei (see examples). In addition, pro-inflammatory cytokines such as MCP-1 and IL-6 were also elevated in L paracasei CNCM 1-1518 group compared to the control group.

The protective effect induced by L paracasei CNCM 1-1518 on influenza//V. meningitidis superinfection is linked to Innate immune response enhancement and can therefore apply to other types of superinfection.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the recruitment of immune cells in the respiratory tract and/or in the lungs of the subject that has consumed L paracasei CNCM 1-1518 is increased compared to the recruitment of immune cells In the same body area of a control subject that has not consumed L paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the recruitment of at least one population of immune cells in the respiratory tract and/or in the lungs of the subject that has consumed L. paracasei CNCM 1-1518 Is increased compared to the recruitment of said population of immune cells in the same body area of a control subject that has not consumed L paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection, said population of immune cells being selected from the group comprising dendritic cells (DCs), macrophages, monocytes, neutrophils, eosinophils and B cells, preferably from the group comprising dendritic cells, monocytes and neutrophils. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the recruitment of dendritic cells (DCs), macrophages, monocytes, neutrophils, eosinophils and B cells in the respiratory tract and/or In the lungs of the subject that has consumed L. paracasei CNCM 1-1518 is Increased compared to the recruitment of dendritic cells (DCs), macrophages, monocytes, neutrophils, eosinophils and B cells in the same body area of a control subject that has not consumed L paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection.

In particular, the recruitment of at least one population of immune cells Is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in the subject that has consumed L paracasei CNCM 1-1518 compared to the control subject that has not consumed L paracasei CNCM 1-1518.

The recruitment of immune cells can be determined by various methods well known to one skilled in the art. In particular, it can be determined by flow cytometry as described in the examples.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the bacterial load of the bacterium responsible of the secondary infection in the respiratory tract and/or in the lungs of the subject that has consumed L paracasei CNCM I- 1518 is reduced compared to the bacterial load of said bacterium in the same body area of a control subject that has not consumed L paracasei CNCM 1-1518, in particular 24 hours, more particularly 48 hours after the contamination by said bacterium.

In the Invention, the bacterial load corresponds to the measurable quantity of a bacterium in the body of a subject.

In particular, the bacterial load of the bacterium responsible of the secondary infection is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in the subject that has consumed L. paracasei CNCM 1-1518 compared to the control subject that has not consumed L paracasei CNCM 1-1518.

The bacterial load of the bacterium can be determined by various methods well known to one skilled in the art. For example, the bacterial load of the bacterium can be determined by bioluminescence or by using culture from a biological sample and counting on petri dishes. In an embodiment the invention relates to the Lactobacillus paracosei strain CNCM 1-1518 for its use as defined above, wherein said subject is selected from the group comprising a vulnerable subject, an immune depressed subject, an hospitalized patient, a child and an elderly individual aged 65 or over. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein said subject does not suffer from a chronic and/or latent infection, such as an infection by HSV (Herpes Simplex Virus), CMV (Cytomegalovirus) and HIV (Human Immunodeficiency virus).

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein said subject suffers from a chronic and/or latent infection, such as an infection by HSV, CMV and HIV.

In the invention, a chronic infection refers to a long term infection, in general for more than 3 months.

In the invention, a latent infection refers to an asymptomatic infection capable of manifesting symptoms under particular circumstances or If activated.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein said strain is used in addition of a vaccination. In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein said strain is used in addition of a treatment by antibiotics.

In an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein the subject consumes at least one daily dose of L paracasei CNCM 1-1518 during the period prior to, and/or during, the coinfection.

In an advantageous embodiment, the invention relates to the Lactobacillus paracasei strain CNCM I- 1518 for its use as defined above, wherein the subject has started to consume at least one dally dose of L. paracasei CNCM 1-1518 before the secondary infection, more preferably before the primary infection.

In order to obtain an optimal effect, at least one daily dose of L. paracasei strain CNCM 1-1518 is consumed for at least one week, preferably for at least two weeks, more preferably for at least three weeks, even more preferably for four weeks, before the contamination by the bacterium responsible for the secondary infection, preferably before the contamination by the virus responsible for the primary infection.

The administration of L paracasei strain CNCM 1-1518 may subsequently be continued for as long as desired so as to maintain the effect of the L paracasei strain against the coinfection.

The amount of L paracasei strain CNCM 1-1518 consumed dairy will preferably be at least 10¹⁰, advantageously at least 2x1ο¹⁰ CFU. This amount can be administered in one or more daily intakes. Preferably, a daily dose contains at least 10^s, preferably at least 10^s, and generally between lxlO⁸ and 1.5x10^s L paracasei cells per ml.

In an embodiment, the invention relates to the L. paracasei strain CNCM 1-1518 for its use as defined above, wherein said L. paracasei strain is contained in an oraHy administrate composition. in an embodiment, the invention relates to the Lactobacillus paracasei strain CNCM 1-1518 for its use as defined above, wherein said composition is a food product or a food supplement, in particular a fermented dairy product, more particularly a yoghurt. In the invention, the L paracasei strain CNCM 1-1518 may be used alone, or in combination with other tactic acid bacteria of the L. paracasei species or of other species. Advantageously, it may be used in combination with yogurt ferments, namely Lactobacillus bulgaricus and Streptococcus thermophilus. When Lparacasei strain CNCM 1-1518 is used in combination with yogurt ferments, the composition advantageously comprises at least 10⁷, preferably between 2x10^s and lxlO⁹ S. thermophilus cells per ml, and at least 5x10^s, preferably between 4xl0⁶ and 2xl0⁷ L. bulgaricus cells per ml.

The present invention also relates to a method of treatment, prevention or alleviation of a coinfection consisting of a primary viral infection and a secondary bacterial infection in a subject comprising administering to said subject an effective amount of Lactobacillus paracasei strain CNCM 1-1518. The present invention also relates to the use of Lactobacillus paracasel strain CNCM 1-1518 for the manufacture of a medicament for the treatment, the prevention or the alleviation of a coinfection consisting of a primary viral infection and a secondary bacterial infection in a subject. The following Figures and Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

FIGURE LEGENDS

Figure 1. Invasive meningococcal Infections and influenza-like Illnesses recorded in France by the Reference Center for Meningococci and the Sentinelles Network from January 2000 to May 2007. Reporting of invasive meningococcal infections is mandatory. All invasive meningococcal isolates in France are sent to the National Reference Center for Meningococci for full characterization and typing. The general practitioners of the Sentinelles Network report on influenza-like illnesses on a weekly basis by sending patient deidentified data via the Internet to a G IS database. The monthly incidence of MD (right axis) and the weekly incidence of influenza-like illnesses (left axis) during the period of January 2000 to May 2007 are represented on the same graph, (adapted from Rameix- Welti et ai, 2009).

Figure 2. Effects of L paracasei consumption on health status of influenza/Nm-infected mice. (A) Schematic representation of the experimental design. (B) Body weight loss, (C) score appearance and (D) mortality of mice after influenza infection. Results are expressed as mean ± SEM for each group (n=50). (*p<0.05, **P<0.01, ***P<0.001).

Figure 3. Dissemination of N. meningitidis in BALB/c-flu infected mice. Sequential IAV (250 PFU per mouse) and meningococcal infection (10⁷ CFU per mouse) were performed by intranasal route. Bacterial infection was analyzed for bioluminescence at the indicated times. Images depict photographs overlaid with colour representations of luminescence intensity, measured in total photons/sec and indicated on the scales, where red is most intense and blue is least intense. (A) Ventral views of 9 mice (PBS-fed) and 10 mice (L paracasei-ied mice). A non-infected mouse was added as a control. (B). The luminescence was quantified and expressed as means ± SEM from each category at the indicated times by defining specific representative region of interest encompassing the entire animal.

Figure 4. Cytokine profiles in the lungs of mice treated with L. paracasei CNCM 1-1518 or with PBS (control) at day 0 or at day 7 after the primary influenza infection. Results are expressed as mean ± SEM for each group (n=8 per group and per time point. Cytokines for which the differences between the two groups were significant are indicated by a star.

Figure 5. Effects of consumption of L. paracasei on cytokine profiles in the lungs, compared with the control (PBS) group after 48h of secondary meningococcal infection (day 9). Results are expressed as mean ± SEM for each group (n=20). Cytokines for which the differences between the two groups were significant are indicated by a star.

Figure 6. Effect of consumption of L. paracasei CNCM 1-1518 (N=30) on the recruitment of immune ceils in the lungs at different days at day 0 (A) and at day 7 (B) after influenza infection, compared with the control group (N=30). aM: Alveolar Macrophage, i^'M: Interstitial Macrophage, DC: Dendritic cells, iM: Inflammatory Monocyte, pM: Patrolling monocytes, Eo: Eosinophils, N: Neutrophils, B: B cells, T: T cells. Results are expressed as mean ± SEM for each group. (*p<0.05, **P<0.01). Figure 7. Effect of consumption of L paracasei on the recruitment of immune cells in the lungs compared with the control (PBS) group after 48h of secondary meningococcal infection (day 9). Results are expressed as mean ± SEM for each group (n=16). Cells for which the differences between the two groups were significant are indicated by a star.

EXAMPLES

MATERIALS AND METHODS Bacterial and viral strains

L paracasei strain CNCM 1-1518 was cultured at 37 "C overnight in aerobiose in MRS (Man, Rogosa, Sharpe) broth (DIFCO). The cultured bacteria were harvested by centrifugation (4500 g) and washed with sterile PBS (Gibco). After washing, the optical density was measured at 600 nm (ODB^) and adjusted with additional PBS to obtain a final ODuonm of 1. The bacterial suspension was stored at - 80"C in aliquots until use.

Neisseria meningitidis (Nm) bioluminescent derivative of strain 24198 (serogroup C) was cultured on GCB agar medium (Difco).

Influenza A virus (IAV) (A/Scotland/20/74 (H3N2)) adapted to mice was made from lung homogenates in 3096 glycerol and stored at -80"C. The virus was prepared, stored in aliquots at -80*C and thawed prior to infection as previously described (Alonso et a/., 2003).

Mice

Six-week old female BALB/c mice were purchased (Janvier, Genest-Saint-lsle, France) and kept one week prior to the experiment in biosafety containment facility. All mice were housed under specific pathogen free condition at the Institut Pasteur, provided with food and water ad libitum. Experimental animals were performed in accordance with guidelines of the Animal Care Use Committee at the Institut Pasteur and were approved by the French Research Ministry.

Probiotic treatment, viral and secondary bacterial Infection.

Mice were daily orally gavaged (200 μΙ) with L paracasei (2x10* colony forming unit, CFU) or PBS (control) for 7 days (Figure 2A). At day 0 (DO), mice were lAV-infected. At day 7 (D7), 7 days after IAV infection, mice were surinfected with N. meningitidis and followed up to 48 hours after secondary infection. Oral gavage with probiotic was performed during the whole period of viral Infection and secondary infection.

Viral infection was performed intranasally on mice that were anesthetized with sodium pentobarbital (Sanofi, Sante Animale, Libourne France) before an intranasal infection with 50 μΙ of virus H3N2 (260 plaque forming unit, PFU). The secondary meningococcal infection was performed 7 days post- influenza virus infection. The secondary bacterial infection was followed by bioluminescence using bioluminescent N. meningitidis LNP24198lux, a serogroup C isolate belonging to the hyper invasive clonal complex ST-11. Intranasal infection was performed similarly to the flu infection by administration of 50 μΙ of or bacterial suspension at 2x10^s CFU/ml (10⁷ CFU per mouse) as previously described (Alonso et al., 2003).

Health status, appearance score and survival of mice was followed after IAV infection at day 0 and followed during 16 days. Weight and fur appearance of each mouse were daily measured. Results were expressed as body weight loss compared to values at DO. Fur appearance was also scored after infection at DO as follows: 3-Coat is smooth; 2-Patches of hair piloerected; 3-Majority of back is piloerected, and O-Piloerection may or may not be present, mouse appears "puffy" ( Shrum et al., A robust scoring system to evaluate sepsis severity in an animal model, BMC research notes 7:233, 2014). Survival of mice was scored and represented as percent survival.

Mice were sacrificed at the end of experiment. After perfusion, lungs were extracted, homogenized and divided into two lots and used to perform the cytokine assay and flow cytometry analysis.

Quantification of <V. meningitidis (Bioluminescence imaging

Mice infected intranasal^ with bioluminescent N. meningitidis were anesthetized using a constant flow of 2.5% isoflurane mixed with oxygen, using an XGI-8 anesthesia induction chamber (Xenogen Corp.). Bacterial infection images were acquired using an MS 100 system (Xenogen Corp., Alameda, CA) according to instructions from the manufacturer. Analysis and acquisition were performed using Living Image 3.1 software (Xenogen Corp.). Images were acquired using l min of integration time with a binning of 16. All other parameters were held constant. Quantifying was performed using the photons per second emitted by each mouse by defining regions of interest (lung). An uninfected mouse in the same conditions of acquisition was used for subtracting the background.

Determination of cytokines levels

Various cytokines (MCP-1, IL10, KC, IL12p70 and IL6) were quantified in the cleared lung homogenate from mice scarified on day 9 {48h after N. meningitidis), using EUSA kit (R&D Systems, Abeam) according to the manufacturer's instructions. The cytokine levels in the lungs were expressed as the amount of cytokine per unit weight of total protein of lung. The quantification of total protein was determined using BSA protein Assay Kit (Thermos scientific, Meridian Road, USA) according to the manufacturer's instructions.

Antibodies and flow cytometry

Single-cell suspensions were prepared from the lungs that were mechanically disrupted using collagenase IV (Sigma-Aldrich, St. Quentin Fallavier, France) as previously described (18). Red blood cells from lung were lysed using ACK buffer (Gibco). Flow cytometry data were collected with the LSR Fortessa (BD Biosciences) and the results were analyzed with FlowJo software (Tree Star). Antibodies specific to mouse were purchased from BD Biosciences, Biolegend and eBioscience. Statistical Analysis

Data were expressed as meanstSEM. Student's t tests were used to analyze group differences. Values of p<0.05 were considered statically significant.

EXAMPLE 1. Effects of L oaracasel on secondary bacterial-Infected mke hearth status

The impact of oral administration of L. parocasei CNCM 1-1518 has been assessed on secondary meningococcal infection. Mice were infected intranasally with H3N2 influenza virus at day 0 and surinfected intranasally with N. meningitidis 7 days after H3N2 influenza virus infection (Figure 2A). After infection with influenza virus, infected control mice rapidly lost body weight. This loss was significantly less important in L paracasei -fed mice than in PBS-fed mice at days 1, 4, 6 and 7 post- infection (Figure 2B). Mice also seemed ill according to a clinical score based on the fur appearance, this score was significantly better in L paracasei -fed mice than in PBS-fed mice at days 3, 4, S, 6 and 7 post-infection (Figure 2C). At day 7, after A/, meningitidis infection, the body weight lost continued in both groups of mice until day 9. However, we observed less weight lost in L paracasei group, with a better clinical score (Figures 2B and 2C). Moreover, L paracasei group of mice showed better survival (90%) at day 9 compared to the 75% survival rate in PBS-fed-mice (Figure 2D). These data suggest that consumption of L paracasei before N. meningitidis infection was associated with improvement in health status in mice.

EXAMPLE 2. Effects of consumption of L parocasei on N. meningitidis respiratory Infection

N. meningitidis infection was followed by dynamic bioluminescence imaging. The quantification of bacterial load was performed in LAV infected mice in both the two groups: treated with L paracasei or with PBS, using the total photons per second emitted by each mouse after 0.5h, 3h, 6h, 24h and 48h of infection (Figure 3).

The dynamic bioluminescence imaging showed that 0.5 h after the intranasal bacterial inoculation, bacteria were present in the upper respiratory tract of infected mice but started to reach the lower respiratory pathways and lungs. The infection signal increased after 3h and 6h after N. meningitidis infection in mice of both groups. The infection seemed to be established in the lower respiratory tract and the lungs (Figure 3A) with no significant difference between the two groups of infected mice [L. paracasei-fed mice and PBS fed mice). Bioluminescent signals decreased after 24h and 48h of meningococcal infection. However, the signals were significantly lower in L. paracasei fed-mice compared to PBS-fed mice after 24h of meningococcal infection and was also lower after 48h of (Figure 3B). These data indicate that L paracasei gavage was associated with a faster clearance of meningococcal secondary infection. Example 3. Effects of consumption of L oaracasei on cytokine profiles in the lungs

Various cytokines were quantified in the lungs from mice fed with L paracasei or PBS at day 0 (just before primary infection by IAV, Figure 4A), at day 7 (just before secondary infection by N. meningitidis, Figure 4B) or at day 9 (48h after the N. meningitidis secondary infection, Figure 5). The cytokine levels in the lungs were determined and expressed as the amount of cytokine per mg of total protein of tested lungs.

We observed higher levels of pro-inflammatory cytokines, reaching statistical significance for IL6 and MCP1, but not for KC and IL12p70 in L paracasei-group compared to PBS-group (Figure 5). Concerning the anti-inflammatory cytokine IL10, although there was higher in L. paracasei-group, the difference compared to PBS-group was not significant (Figure 5).

Example 4. Effect of consumption of L paracasei on the recruitment of immune cells In lungs

The total cells counts in the lung cell suspensions were quantified using trypan blue, at day 0 (just before primary infection by IAV, Figure 6A), at day 7 (just before secondary infection by N. meningitidis. Figure 6B) or at day 9 (48h after the secondary infection with N. meningitidis, Figure 7). This quantification showed higher significant counts of total cells at D9 in the lungs of group of mice orally gavaged with L. paracasei than in control group (Figure 7A).

The flow cytometry analysis (Figure 7B) revealed a significant increase in the number of dendritic ceils, neutrophils and inflammatory monocyte in L paracasei-ted mice sacrified at day 9 (48h after N. meningitidis infection) compared to control group (PBS-fed mice). An increase was also observed for interstitial macrophage, patrolling monocytes and eosinophils.

Claims

1. A Lactobacillus paracasei strain deposited with the CNCM under the number 1-1518 for use in the treatment, the prevention or the alleviation of a coinfection consisting of a primary viral infection and a secondary bacterial infection in a subject.

2. The Lactobacillus paracasei strain CNCM 1-1518 for use according to claim 1, wherein the primary viral infection is caused by a virus selected from the group consisting of Influenza A virus, influenza B virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, coxsackie virus, in particular Influenza A Virus.

3. The Lactobacillus paracasei strain CNCM 1-1518 for use according to claims 1 or 2, wherein the secondary bacterial infection is caused by a bacterium selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Staphyloccocus aureus, Haemophilus influenza, Pseudomonas aeruginosa. Mycoplasma pneumoniae. Chlamydia pneumonia, Moraxella catarrhalis, in particular Neisseria meningitidis.

4. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

3, wherein the virus and the bacterium colonize the same area of the body of the subject, in particular the respiratory tract and/or the lungs.

5. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

4, wherein the primal viral infection is responsible of the flu and the secondary bacterial infection is responsible of the meningitis.

6. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

5, wherein the primary viral infection is caused by an influenza virus, preferably IAV, and the secondary bacterial infection is caused by Neisseria meningitidis.

7. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

6, wherein the level of at least one inflammatory cytokine in the respiratory tract and/or in the lungs of the subject that has consumed L. paracasei CNCM 1-1518 is increased compared to the level of said at least one inflammatory cytokine in the same body area of a control subject that has not consumed L. paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection.

8. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

7, wherein the recruitment of immune cells in the respiratory tract and/or in the lungs of the subject that has consumed L. paracasei CNCM 1-1518 is increased compared to the recruitment of immune cells in the same body area of a control subject that has not consumed L. paracasei CNCM 1-1518, in particular 48 hours after the contamination by the bacterium responsible of the secondary infection.

9. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

8, wherein the bacterial load of the bacterium responsible of the secondary infection in the respiratory tract and/or in the lungs of the subject that has consumed L. paracasei CNCM 1-1518 is reduced compared to the bacterial load of said bacterium in the same body area of a control subject that has not consumed L. paracasei CNCM 1-1518, in particular 24 hours, more particularly 48 hours after the contamination by said bacterium.

10. The Lactobacillus paracasei strain CNCM 1-1518 for use according to any one of claims 1 to

9, wherein said subject is selected from the group comprising a vulnerable subject, an immune depressed subject, an hospitalized patient, a child and an elderly individual aged 65 or over.

11. The Lactobacillus paracasei strain CNCM 1-1518 for its use according to any one of claims 1 to 10, wherein said L paracasei strain is contained in an orally administrable composition.

12. The Lactobacillus paracasei strain CNCM 1-1518 for its use according to any one of claims 1 to 11, wherein said composition Is a food product or a food supplement, in particular a fermented dairy product.