WO2014038929A1

WO2014038929A1 - Probiotics for producing antiviral factors

Info

Publication number: WO2014038929A1
Application number: PCT/NL2012/050625
Authority: WO
Inventors: Belinda Van 't Land Potappel; Johan Garssen; Rodion-Guillaume Johannes Lambertus GAAJETAAN; Franciscus Rainier Maria STASSEN
Original assignee: N.V. Nutricia
Priority date: 2012-09-07
Filing date: 2012-09-07
Publication date: 2014-03-13

Abstract

The invention pertains to the use of a livestrain of the Lactobacillus casei groupfor stimulating dendritic cellsin an individual so as to increase the level of plasmacytoid dendritic cells.The plasmacytoid dendritic cells are capable of protecting the individual against herpes simplex virus type 1 infection. The live Lactobacillus caseistrain is preferably the L. casei strain with deposit number CNCM I-1518or the L. rhamnosus strain with deposit number LMG P-22799.The strain can be incorporated in a food product.

Description

Probiotics for producing antiviral factors

The invention is in the field of controlling viral infection. The invention is particularly concerned with reducing or preventing viral infection by administration of specific probiotics.

Background

Immunomodulating effects of probiotics are known in the art. Such immunomodulating effects include antiviral effects. Watanabe et al, Microbiol. Immunobiol. 1986, 30(2): 111-22, Kansenhogaku Zasshi 1989, 63(3): 182-8, describe resistance to herpes simplex virus infection by heat-killed Lactobacillus casei strain LC-9018.

WO2007/040445 claims the use of certain Lactobacillus strains, including L. plantarum and L. (parajcasei strains, for the treatment of prevention of virus infections. The claim is based on activation of cytotoxic T cells and KT cells upon intake of the probiotic strains. WO2008/129418 (FR2912657) describes the use of strain CNCM 1-1518 of L. (parajcasei for enhancing protection against influenza by subsequent vaccination. WO 2001/89541 describes enhancing a systemic immune response against influenza by this strain. WO2010/043696 describes synergetic combinations of elderberry extracts and certain probiotics, in particular L. (parajcasei CNCM 1-1518, for stimulating immune defences and for the treatment and prevention of conditions caused by influenza viruses. The effects were based on enhanced production of IFN-γ, IL-6 and IL-10.

Introduction

Type I interferons (IFNs) are the first antiviral cytokines produced by various cells during viral infection to limit replication and dissemination of the virus. These type I IFNs (IFNa/β) stimulate the interferon receptor (IFNAR), present on almost all cells, which results in the expression of many so-called interferon-stimulated genes (ISGs) ². After ISG activation, cells acquire an antiviral state and thereby hinder viral replication and dissemination. In addition, dendritic cells (DCs) are activated and stimulate B and T cells to eradicate the virus or virally-infected cells, respectively^{7, 3'5}. Therefore, type I IFNs are the main orchestrators of the immune response towards viral infections. After recognition of viral components using toll-like receptors (TLRs), DCs and plasmacytoid DCs (pDCs) in particular are able to produce large quantities of these type I IFNs. While conventional DCs (cDCs) mainly sense viruses by TLR3 and cytoplasmic receptors, pDCs use TLR7 and 9 to recognize viral RNA or DNA, respectively ^6'10. As pDCs are relatively unique in their TLR-repertoire and their massive production of type I IFNs, these DCs are indispensible for the immediate antiviral response. Previously it was demonstrated that specific TLR-ligands display profound antiviral effects both in vitro and in vivo ^77-76 Also the direct application of type I IFNs has a protective effect against viral infections ^77-79 In addition to the TLR ligands and IFNs, immunomodulating effects have also been attributed to specific probiotic strains ²⁰' ²¹.

Probiotics have been defined by FAO/WHO as specific live micro-organisms which, when administered in adequate amount, confer a health benefit on the host. These probiotic properties could occur through modulation of both mucosal and systemic immune responses. This beneficial effect is usually achieved through transient colonization of the gastrointestinal tract, thereby preventing infection of the gut by potentially pathogenic bacteria ²⁰. More recently, also antiviral effects have been attributed to certain probiotic strains. For example, recent studies have shown a positive effect of specific Lactobacillus spp. in the treatment of rotavirus infections in children ²² ²³. Grabryszewski and colleagues demonstrated that priming of the respiratory mucosa with specific Lactobacillus species markedly protected mice from the lethal sequelae of a severe respiratory virus infection, which was probably due to markedly diminished inflammatory responses upon a viral challenge ²⁴. Alternatively, bacterial strains have the capacity to stimulate dendritic cells resulting in the release of different cytokines (including ΠΤΝΓβ) depending on the species or strains used ²⁵. However, it remains to be determined whether stimulation of DC subsets with different bacterial strains is sufficient to limit viral replication. This prompted the inventors to investigate the antiviral activity of specific probiotic strains in an in vitro bioassay model as described previously ¹². Furthermore, it was analysed which DC subsets are most important and whether interferons are involved in the antiviral effect detected. Description of the invention

It was found that live cells of specific Lactobacillus strains are capable of directing immune maturation in an individual in such a way that plasmacytoid dendritic cells (pDC) are selectively increased in the differentiation of bone marrow cells into pDC and conventional (myeloid) dendritic cells (cDC) in mammals. In this way, the strains are capable of increasing INF levels. The stimulation of pDC and/or the increase of ΠΤΝΓβ levels was found to be useful for inducing an antiviral effect against herpes simplex virus type 1 (HSV-1) as further explained below. An increase of pDC can be determined using standard bioassays for human IFNa of ΠΤΝΓβ such as commercially available ELISA kits.

The specific strains to be used according to the invention are members of the facultatively heterofermentative Lactobacillus casei group, which encompasses L. casei, L. paracasei (which may be the same species, depending on nomenclature changes), L. rhamnosus, and L. zeae. Although the precise taxonomy of this group of Lactobacilli is still under discussion, there is broad agreement about the separate status of this Lactobacillus group (see e.g. Felis and Dellaglio, Curr. Issues Intestinal Microbiol. 8: 44-61 (2007)). The strain is particularly a J. para(casei) strain. Most preferred strain is L. casei strain with deposit number CNCM 1-1518. Another useful strain according to the invention is L. rhamnosus strain with deposit number LMG P-22799 (deposited at BCMM). The live strains can be produced by conventional cultivation methods.

The live strains can be administered by oral or other route. Oral administration forms include tablets, capsules, sachets, elixirs, tube feeds and the like, containing the live strains in the presence of suitable carriers. For an effective stimulation of pDC's capable of preventing or treating HSV-1 infections, the live strains can preferably be administered at a daily dosage level of 10⁵ - 2x10⁹ cells, in a single dose or in multiple doses. Preferred daily administration levels are between 10⁶ - 10⁹ cells, more preferred 2xl0⁶ - 2xl0⁸ cells.

Preferred oral administration forms include solid, semi-solid or especially liquid compositions containing, in addition to the live strains, food components, such as carbohydrates, proteins, lipids, nutritional fibres, vitamins, mineral and combinations thereof, and can be provided as nutritional supplements, complete foods, beverages, snacks, etc. Liquid nutritional compositions can contain the live strain at a level of between 10⁵ - 10⁹ live cells per 100 ml, preferably 10⁶ - 10⁸ cells of the live strain per 100 ml. Alternatively or additionally, for compositions containing digestible carbohydrates, proteins and/or lipids, preferably at least carbohydrates, more preferably at least proteins and carbohydrates, nutritional compositions can contain the live strain at a level of between 10⁵ - 10⁹ live cells per 100 kcal, preferably 10⁶ - 10⁸ cells of the live strain per 100 kcal.

In a preferred embodiment, the live strain is contained in a food product containing proteins, in particular milk proteins. The food product preferably contains 0.01-10 g, more preferably 0.02-5 g, most preferably 0.1-1 g of milk protein per 10⁶ cells of the live strain. Whey proteins are especially interesting, and are preferably present in the food product at a level of 0.05-5 g, more preferably 0.01-1 g, most preferably 0.025-0.5 g per 10⁶ cells of the live strain. Among the whey proteins, lactoferrin is advantageously present in combination with the live cells, at a ratio of 2-1000 mg lactoferrin per 10⁶ cells of the live strain, more advantageously 5-500 mg, most advantageously 10-200 mg lactoferrin per 10⁶ cells of the live strain.

The live cells of the L. casei group, in particular of the strain CNCM 1-1518 strain, can be combined with other probiotics, such as strains of other Lactobacillus strains, of Streptococcus thermophilus or of Bifidobacterium strains. In such combinations, the cell count ratio between the live L. casei group strain (in particular CNCM 1-1518) and the other probiotics is preferably between 1 :9 and 9: 1, more preferably between 1 : 1 and 4: 1. The live cells of the L. casei group, in particular of the strain CNCM 1-1518 or LMG P-22799, can be combined with prebiotics such as galacto-, fructo-, xylo-, manno-, arabino-, fuco-, rhamno-, indigestible gluco- and hetero- (e.g. glucomanno-, galacto- manno-, arabinogalacto-, arabinoxylo-, galactofructo-, etc.), as well as uronic oligosaccharides, and combinations of two or more thereof. Preferred prebiotics comprise galacto-, fructo-, galactomanno- and galactofructo-oligosaccharides and combinations thereof. In such combinations, the ratio between the L. casei group strain (e.g. CNCM I- 1518) and the prebiotics is preferably between 0.01 and 5 g prebiotics per 10⁶ cells of the live strain, more preferably between 0.05 and 1 g prebiotics per 10⁶ cells. The live strains and the compositions containing them can be used for treating or preventing herpes simplex virus, in particular HSV-1 infection in individuals in need thereof. Individuals in need of such treatment or prevention obviously include patients suffering from such HSV infections, as presented by the known characteristics such as facial, labial, buccal, or mucosal sores, blisters or other lesions. Individuals in need of such treatment or prevention also include patients in which the visible or otherwise sensible manifestations of the infection have been cured or have disappeared, but where the virus has become latent and a risk of reactivation exists. Other individuals include those for whom environmental, historical, hereditary or other factors increase the risk of HSV-1 infection, or individuals for whom, e.g. as a result of their immunological status, a HSV infection has the risk of factors resulting in more serious health damage than for average healthy people. A further target group include individuals, where the immunological system has not been fully developed, in particular infants. The administration form can be adapted to the particular target group. For example, a food supplement or beverage, containing the live strains in a food component such as a milk product, or a snack can be used for preventive purposes. For treatment of manifest infections, pharmaceutical forms such as tablets, elixirs and the like, as described above, are suitable, in addition to food supplements. For persons having specific risks, such as immune-compromised persons, or elderly and/or malnourished persons, the compositions containing the live strains may be complete food compositions containing carbohydrates, lipids, proteins, and further food constituents. For infants, the composition may be a milk-based infant formula, to which the live strains are added. The levels of the live strains, and further assisting components such as prebiotics, whey proteins, and the like etc. for the pharmaceutical products, food supplements, and complete foods, can advantageously be as described above.

Detailed description of the invention

It was found that the specific bacterial strains as defined above can stimulate antiviral immunity. The pronounced antiviral effects were only observed when a mixed population of pDCs and cDCs was exposed to the specific lactobacilli strains. On the other hand, stimulation with a specific Bifidobacterium breve did not show an effect in the concentration tested. Moreover, it was shown that this antiviral effect was associated with high IFNp mRNA levels. In general, probiotic bacteria are primarily thought to be effective in the gastrointestinal tract by preventing colonization or infection by pathogenic microorganisms. Additionally, the immunomodulatory properties of probiotics are now being more and more recognized. Recent data indicate that certain probiotics trigger the expression of viral defence and may as such have a role in the prevention or treatment of viral infections ²⁷' ²⁸. Yet, additional studies are required to compare the antiviral potency of specific strains and how this is mediated. Bacteria interact with TLRs and other pattern recognition receptors (PRRs) on DCs,

21 29 31

which results in the activation of the immune system ' ^" . Since DCs are the central players in the antiviral response, we initially tested whether exposure of these cells to three different bacterial strains resulted in any antiviral effect. Interestingly, both lactobacilli induce strong antiviral effects in contrast to the Bifidobacterium strain, an effect that may be mediated by high IFNP levels. A variability in immunomodulating properties of different probiotics has been reported recently by Weiss and colleagues ²⁵. The present inventors have found a similar phenomenon, in that stimulation of BM-DC differentiated in the presence of GM-CSF (GM BM-DC) with L. casei (CNCM 1-1518) revealed an antiviral effect while this effect was virtually absent when cells were stimulated with L. rhamnosus (LMG P-22799). Moreover, in our study as well as in the study by Weiss, less IFNP was found when GM BM-DCs were stimulated with any of the bifidobacteria.

In the study by Weiss mentioned above, only GM BM-DCs were used. It was found according to the invention that the antiviral effect of lactobacilli is strongly enhanced when pDCs were present in the cell culture. pDCs are the principal producers of large amounts of type I IFN. Thus, pDCs probably recognize specific components of lactobacilli, which results in the production of IFNP . In a previous study, a pronounced antiviral effect was observed when BM-DC differentiated in the presence of Flt-3L (FL BM-DCs) were stimulated with non-methylated CpG oligodeoxynucleotides (CpG ODNs) ¹² , a well-recognized TLR9 ligand. This was only observed in the FL BM-DC subset, but not in the GM BM-DC subset and the antiviral effect was also IFNP- dependent. A similar difference was observed when the two DC subsets were stimulated with the probiotics. This suggests that the antiviral effects are most likely TLR9 mediated, which is supported by the fact that non-methylated CpG motifs are also widely present in bacteria 6 ' 10 ' 20 ' 32. Thus, the intracellular TLR9 might be the receptor engaged in pDCs after phagocytosis of the lactobacilli. This is supported by recent data from Plantinga et al, who demonstrated that the same lactobacilli strains as used the present study, indeed stimulated primary immune cells through TLR9 ³³. Remarkably, although TLR9 is also present in cDCs ^34~36, this does not seem to result in protection against viral infection ¹² and it has been speculated that in these cells TLR9 might be primarily involved in immune responses towards fungal pathogens ^34~36. Alternatively, other studies have shown the involvement of different TLRs or intracellular receptors in the immunomodulating effects of probiotics and further research is required to unravel the molecular pathways which are implicated in the observed antiviral effects.

Description of the Figures

Figure 1: Viral copy numbers after BM-DC stimulation with probiotics.

Antiviral properties of supernatants, derived from bacterial stimulation of FL BM-DCs (A) or GM BM-DCs (B) stimulated with different strains, on L929 cells subsequently infected with HSV-1. Symbols indicate BM-DC supernatant from individual mice (n=5). Viral copies were determined by qPCR. ** = P <0.01 and * = P <0.05 versus control.

Figure 2. Viral copy number after direct stimulation ofL929 cells with bacteria.

All bacteria were administered directly to L929 cells for 18h. Symbols indicate independent experiments (n=4). Viral copies were determined by qPCR. Figure 3. IFNfi expression in BM-DC subsets stimulated with different probiotics.

IFNp expression in FL BM-DCs (A), GM BM-DCs (B) and L929 cells (C). IFN is displayed as relative expression compared to GAPDH values. Symbols indicate BM- DCs from individual mice (n=5) or independent experiments. ** = P <0.01 versus control. # = P <0.01 of Lactobacillus versus Bifidobacterium. EXAMPLES

Materials & Methods

Mice

Bone marrow was derived from male BALB/c mice (Charles River, 8-14 weeks of age). Mice were euthanized by intraperitoneal injection of Nembutal^® (150 mg/kg, Sanofi Sante B.V. Maassluis, the Netherlands). The study was approved by the ethical committee for animal experiments of Maastricht University.

Isolation and differentiation of bone marrow cells

Bone marrow (BM) cells were isolated as described previously ¹². BM cells were cultured in 24-well tissue culture plates (Becton Dickinson, NJ, USA) at 10⁶ cells/ml in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA) with 10% FCS (Lonza, Verviers, Belgium). The medium was supplemented with either 200 ng/ml human Flt- 3L or 20ng/ml GM-CSF (both from Miltenyi Biotec. Leiden, the Netherlands) for differentiation into a mixed culture of pDCs and cDCs or a monoculture of cDCs only, respectively. When GM-CSF was used, the medium was refreshed 3 and 6 days after seeding the cells in the plates. Cells were allowed to differentiate for 8 days at 37°C and 5% C0₂ before stimulation with specific bacterial strains was started. Bacterial fermentation and enumeration

Two different Lactobacillus strains (LMG P-22799 and CNCM 1-1518) and a Bifidobacterium strain were grown at 37°C in a 400 ml reactor containing MRS supplemented with 0.5 g/1 L-cysteine for bifidobacteria. The pH was maintained at 6.5 by addition of NaOH. To ensure anaerobic conditions the headspace was flushed with N₂ or a gas mixture consisting of 5% H₂, 5% C0₂ and 90% N₂ for bifidobacteria. Bacteria were harvested in the early stationary phase, washed in PBS and stored with glycerol 20% (w/v), in aliquots at -80°C. Cell counts were determined by plating serial dilutions (CFU) and fluorescent microscopy by staining with DAPI. Stimulation of DCs

At day 8, the medium of the DCs was removed and replaced with medium containing 150 μg/ml gentamycin (Eurovet, Bladel, the Netherlands) and one of the bacterial strains (10⁷ bacteria/ml). Differentiated BM cells were stimulated with Lactobacillus rhamnosus (L. rhamnosus), Bifidobacterium breve (B. breve) or Lactobacillus casei (L. casei) for 24h at 37°C and 5% C0₂. Afterwards, cells were snap-frozen in liquid nitrogen and stored at -80°C for future gene expression analysis. Supernatants of the stimulated DCs were stored at -80°C until further use. Cells and virus

L929 cells (CCL-1, ATCC) (Rockville, MD, USA) were cultured in Earle's Minimal essential medium (EMEM) (Invitrogen) supplemented with non-essential amino acids (MP Biomedicals, Solon, Ohio, USA), L-glutamine (2mmol/L), sodium pyruvate (lmmol/L) and 10% FCS (Lonza). Cells were allowed to grow in T75 flasks at 37°C and 5% C0₂.

HSV-1 was obtained from ATCC (VR-539) and was propagated in Vero cells (ATCC CCL-81) in EMEM (Invitrogen) with 2% FCS (Lonza), non-essential amino acids (MP Biomedicals), L-glutamine (2mmol/L) and sodium pyruvate (lmmol/L). When 100% cytopathogenic effect (CPE) was achieved, cell debris was removed by centrifugation and viral titres in the supernatant were determined by plaque assay.

Stimulation and infection protocol

The antiviral potency of the conditioned media were tested in a bioassay as described previously ¹². Briefly, L929 cells were exposed to the conditioned media for 18h. After removal, cells were infected with HSV-1 (MOI 0.1), harvested 30h post infection (p.i.), snap-frozen in liquid nitrogen and stored at -80°C for DNA isolation. The supernatant was stored at -80°C until being used for cytopathogenic effect (CPE) test.

Quantitative PCR (qPCR)

DNA was extracted from frozen cell pellets according to the Wizard^® Genomic DNA Purification Kit (Promega Benelux B.V. Leiden, the Netherlands) according to the manufacturer's instructions. DNA purity and quantity were measured with the Nanondrop® ND-1000. The DNA isolates were amplified in a volume of 25μ1 containing 5μ1 HOT FIREPol^® EvaGreen^® qPCR mix plus (Solis BioDyne, Tartu, Estonia), HSV-1 forward and reverse primer, and DNA sample. HSV-1 was detected by using a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Thermal cycling was started with UNG activation for 2min at 50°C, followed by HotStarTaq activation during 15min at 95°C. Subsequently, 40 cycles of amplification were run consisting of 15s at 95°C (denaturation) and lmin at 60°C (annealing and attaching).

To determine the actual number of HSV-1 DNA copies, a DNA standard curve was used. Dilutions were made from a plasmid, which contains the HSV-1 PCR-target sequence. Used concentrations ranged from 10⁷ to 10° copies, with a dilution factor of 10. Copy numbers were quantified by the standard curve using the iQ™5 version 2.0 Optical System Software.

RT-qPCR

RNA was isolated from frozen cell pellets with the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Remaining DNA was removed by DNAse treatment (Turbo DNA-free™ kit, Ambion, Austin, TX, USA). Subsequently, RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). qPCR was performed as described above. To control for DNA contamination, in every PCR run a sample was included which was not reverse transcribed. Relative expressions were determined by using the 2^"ACt, normalized to GAPDH values. All samples were measured in duplicate.

CPE test

To determine the presence of infectious HSV-1 particles, collected supernatant from infected L929 cells (2x, 4x and 8x diluted) was added to Vero cells (ATCC CCL-81) grown until being confluent in 24-well plates. After 48h incubation at 37°C and 5% C0₂, CPE was visualized by staining and fixation with 0.13% crystal violet in 5% formaldehyde.

Statistical analysis

The Student's t-test was used to analyse differences between control and stimulated samples. Differences between multiple groups were determined by one-way ANOVA with a Bonferroni post-hoc test. Values of p < 0.05 were considered statistically significant. Data are expressed as mean ± SEM, unless stated otherwise. Results

Supernatant of lactobacilli-stimulated DCs limits viral infection of L929 cells

HSV-1 infection of L929 cells could be inhibited significantly (p<0.05) when cells were pre-treated for 18h with conditioned supernatants collected from Flt-3L (FL) BM-DC cultures, which had been stimulated before with both lactobacilli strains for 24h (figure

IA) . In contrast, when FL BM-DC were treated with the Bifidobacterium strain, no antiviral effects were observed in the bioassay. Remarkably, when GM-CSF (GM) BM- DCs were stimulated with the bacterial strains, only the L. casei was able to reduce viral copies, although the antiviral effect was less pronounced (26.7% viral copies left) compared to the effects after FL BM-DC stimulation (1.9% viral copies left) (figure

IB) .

To control for possible antiviral effects because of bacteria still present in the supernatant of stimulated DCs, all strains were administered directly to L929 cells for 18h. However, direct application of all strains had no antiviral effect at all, demonstrating that the observed antiviral effect was clearly DC dependent (figure 2).

In addition to the DNA levels, it was also investigated whether the bacterial strains were able to reduce the formation of infectious virus particles. Therefore, Vero cells were exposed to supernatants collected from infected L929 cells, which had been treated prior with different conditioned media from DC cultures. In agreement with the reduced viral DNA copies, the observed CPE in the Vero cells was also significantly diminished when cells were treated with supernatants from L929 cells pre-treated with conditioned media from lactobacilli-treated FL BM-DCs. In contrast, stimulation of FL BM-DCs with B. breve was not sufficient to prevent the formation of infectious particles in the bioassay. Moreover, and also in concert with the viral DNA levels found in L929 cells, the antiviral effect was limited after GM BM-DC stimulation. Although L. casei exposure resulted in a significant reduction in viral DNA copies, the degree of CPE was variable between trials. Also, stimulation of GM BM-DCs with either L. rhamnosus or B. breve was not sufficient to limit CPE. Direct stimulation of L929 cells did not confer any reduction of CPE.

Overall, these data imply that stimulation of FL BM-DCs, but not GM BM-DCs, with the two Lactobacillus strains (CNCM 1-1518 and LMG P-22799) resulted in a pronounced antiviral effect, while B. £reve-dependent effects were practically absent. The data were representative of 4-5 independent experiments.

IFNfi is essential for the antiviral effects following Lactobacilli-stimulation of FL BM- DCs

It was investigated whether IFNP is required for the induction of the antiviral effects. Normally, both high levels of IFNP as well as IFNa are produced after viral infection which exerts potent antiviral effects. Since stimulation of BM-DCs with the two Lactobacillus strains resulted in clear antiviral effects, the mRNA levels of IFNP in the stimulated BM-DCs were determined. As expected, the IFNP-levels were significantly increased when FL BM-DCs were exposed to either the Lactobacillus strains (figure 3A). B. breve stimulation of FL BM-DCs also resulted in increased IFNP mRNA expression, but levels were significantly lower compared to Lactobacillus- duced IFNP mRNA expression and, as demonstrated above, insufficient to reduce viral copy numbers or CPE. No increased IFNP mRNA was detected in GM BM-DCs or L929 cells following stimulation with either of the bacterial strains tested (figure 3B and 3C).

Next to IFNP, also the IFNa4 subtype belongs to the primary IFN responders in mice ²⁶. However, IFNa4 mRNA levels, which are barely detectable under basal conditions, remained extremely low after FL BM-DCs bacterial exposure with all three strains (data not shown).

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Claims

A live strain of the Lactobacillus casei group for use in stimulating

plasmacytoid dendritic cells.

A strain for use according to claim 1, which comprises L. casei CNCM 1-1518.

A strain for use according to claim 1, which comprises L. rhamnosus LMG P-22799.

A strain for use according to any one of the preceding claims, wherein plasmacytoid dendritic cells induce prevention or treatment of an infection by herpes simplex virus type 1 (HSV-1).

A strain for use according to any one of the preceding claims, wherein the strain is contained in a food product comprising 10⁶ - 10⁸ cells of said live strain per 100 ml and/or per 100 kcal.

A strain for use according to any one of the preceding claims, wherein the strain is contained in a food product comprising 0.02-5 g, preferably 0.1-1 g, of milk protein per 10⁶ cells of said live strain.

A strain for use according to any one of the preceding claims, wherein the strain is contained in a food product comprising 0.01-1 g, preferably 0.025-0.5 g, of whey protein per 10⁶ cells of said live strain.

A method for stimulating plasmacytoid dendritic cells, comprising

administering to a subject in need thereof an effective amount of a live strain of the Lactobacillus casei group.