Field of the Invention

This invention relates to perinatal administration of a combination of at least two probiotic bacteria to the expecting / pregnant women capable of modulating the allergic response of their infants.

Background to the Invention

The prevalence of allergic diseases increased rapidly within the last decades. As by now over a third of the worldwide population is afflicted, allergy has been considered as the new epidemic challenge of the industrialized countries. The reasons for the steady increase in allergic diseases are not yet fully understood. Genetic background of the host is a prominent factor, and recently discovered genes have been shown to be associated with respiratory allergies/asthma and skin symptoms [Holloway, J.W. et al. (2010); Genetics of Allergic Disease, J.A.C.I., 125: S81 -94]. Environmental factors such as lifestyle, pollution, decreasing family size, and reduction of microbial stimulation of the immune system in early life stage as a consequence of an improved hygienic situation seem to play an important role also.

Allergic sensitization in childhood, especially in early childhood and especially to food allergens, is critical and of highest interest as development of an "allergic phenotype" or "atopy" has been shown to facilitate subsequent adverse reactions to other allergens. Hence allergies in childhood can be the first step of an allergic cascade leading to multiple allergies later in life, a situation commonly referred to as "The Atopic March". For example, it has been demonstrated in human cohorts that children with persistent food hypersensitivity early in life have a dramatically increased risk to develop allergic rhinitis (hay fever) or asthma later in childhood (Ostblom, E. et a/. (2008); Phenotypes of food hypersensitivity and development of allergic diseases during the first 8 years of life, Clinical and Experimental Allergy, 38 (8): 1325-1332). Therefore, preventing onset or attenuating the severity of food hypersensitivity may be crucial for slowing down the "Atopic March". In this context the management of allergic episodes and moreover prevention of allergies are, in childhood and infancy, of the highest importance.

Prevention of allergies can be achieved on different levels "Primary prevention" is the effect of preventing or reducing the risk of sensitization of patients to allergens, characterized by absence or reduced levels of allergen-specific IgE antibodies. Preventing or reducing sensitization will result in absence or reduction of allergic symptoms upon re-exposure to the same allergen. The current invention is concerned with primary prevention of allergies.

"Secondary prevention" is the effect of modulating the symptoms of allergies, i.e. the occurrence or intensity of the allergic reaction in patient already sensitized to one or several allergens when the patient is re-exposed to said allergen(s). By modulating the occurrence or intensity of the allergic symptoms (secondary prevention), the negative impact on the quality of life of the allergy sufferer, that is associated with allergies, is minimized.

Given these distinct concepts of allergy prevention it may be hypothesized that by virtue of their inherent mechanisms of action, some compounds might act solely at one or at both of these specific levels of prevention. Some may, for example, solely reduce the risk of the sensitization to a specific allergen (primary prevention), while other compounds may solely have an effect on the secondary prevention and reduce the severity of allergic reactions. Other compounds may be able to influence both sensitization and symptoms and thus are effective in promoting primary and secondary prevention. Food allergens are among the first allergens that infants encounter in their early life: typically, cow's milk proteins may be encountered by infants not receiving exclusive breast-feeding. Milk-proteins are indeed among the most frequently observed causes for food allergy in infancy, followed by eggs and wheat proteins. In general, food allergies can manifest by cutaneous (rash, eczema, others) and gastrointestinal symptoms (abdominal cramps; pain, especially in the abdomen; vomiting) in infants and young children. Further sensitization and episodes of allergies can also appear when the infant/young child is exposed to a novel food such as cereals, vegetables, fruits, nuts or fish and also to air borne allergens such as pollen.

Air borne allergens such as pollen, house dust mites and animal dander are also a major cause of allergy in children and adults. It is known that birch pollen-allergic people frequently present allergic symptoms after ingestion of several kinds of plant- derived foods [Vieths, S., Scheurer, S. and Ballmer-Weber, B. (2002); Current Understanding of Cross-Reactivity of Food Allergens and Pollen, Annals of the New York Academy of Sciences, 964; 47-68.]. The majority of these reactions is caused by four distinct cross-reactive protein structures that are present in birch pollen. Proteins that share common epitopes with Bet v 1 , the major birch pollen allergen, occur in pollens of several tree species and in fruits and vegetables, such as: apples, stone fruits, celery, carrot, nuts, and soybeans. It was shown in the study of Vieths et a/, that approximately 70% of patients studied who are allergic to birch pollen may experience symptoms after consumption of foods from these groups. Thus, development of allergies to air borne allergens like birch pollen is also directly linked to food allergy manifestations, known as cross reactivity in oral syndrome.

Gastrointestinal Microbiota and the Immune System

Commensal gastrointestinal microbes constitute the earliest and most substantial stimulus for the development of the gut associated lymphoid tissue and associated immune system. There is solid evidence from epidemiological studies that Western-type living conditions, e.g. reduced consumption of fermented food, substantial use of antibiotics and other drugs, and increased hygiene, are associated with the rise in allergic diseases. The so-called "hygiene hypothesis" thus suggests that a lack of exposure to microbial stimulus early in childhood is a major factor involved in this trend [Von Mutius, E. (2007); Allergies, Infections and the hygiene hypothesis - The epidemiological evidence, Immunobiology, 212(6); 433-9].

Indeed, epidemiological studies have demonstrated an association between the development of allergic diseases and disturbance of the gastrointestinal microbiota. For example, atopic children in Western societies have been reported to be less frequently colonized with lactobacilli or bifidobacteria than healthy children [Suzuki, S., Shimojo, N., Tajiri, Y., Kumemura, M., Kohno, Y. (2007); Differences in the composition of intestinal Bifidobacterium species and the development of allergic diseases in infants in rural Japan, Clin Exp Allergy; 37; 506-51 1 ; Ouwehand, A.C., Isolauri, E., He, F., Hashimoto, H., Benno, Y., Salminen, S. (2001 ); Differences in Bifidobacterium flora composition in allergic and healthy infants, J. Allergy Clin. Immunol. 108; 144-145 ; Kalliomaki, M., Salminen, S., Arivilommi, H., Kero, P., Koskinen, P., and Isolauri, E. (2001 ); Probiotics in primary prevention of atopic disease: a randomised placebo- controlled trial, Lancet 357: 1076-9].

Interestingly, clearcut differences exist with respect to the composition of the gut microbiota in response to the infant's feeding. The faecal microbiota of breast-fed infants includes appreciable populations of bifidobacteria with some Lactobacillus species, whereas formula-fed infants have more complex microbiota, with Bifidobacterium species and Bacteroides species, Clostridia and streptococci being usually present. After weaning, a pattern of gut microbiota resembling that of an adult pattern becomes established over time [Penders, J., et al. (2006); Gut microbiota composition and development of atopic manifestations in infancy: the KOALA birth cohort study. Gut 56: 661 -667]. These findings provide a rational for therapies to prevent allergic disease by supplementation of human diet with probiotic bacteria. Furthermore, as it is known that breast fed children have a lower frequency and/or severity of allergies than do formula fed children, thus supporting probiotic interventions that promote a gut microbiota in non-breast fed children that resembles as much as possible that of breastfed children.

There is also evidence that immunoprogramming by environmental factors may act within a narrow window of opportunity, either during pregnancy or early childhood when immune responses are still developing [Aaltonen, J., Ojala, T., Laitinen, K., Poussa, T., Ozanne, S., Isolauri, E. (201 1 ); Impact of maternal diet during pregnancy and breastfeeding on infant metabolic programming: a prospective randomized controlled study. E. Eur. J. Clin. Nutr. 65; 10-19]. Thus, the immunological status of the newborn is important. This status includes the protection present at the birth of the infant and the acquisition of such immunological protection during the first hours, days or weeks of the infant life. The ability to acquire and maintain such protection is a crucial factor in the health of the infant faced with his new environment. Thus, pre-, peri-, and/or postnatal interventions correspond to a promising approach to modulate immune responses promoting and/or sustaining a non-atopic status. Moreover, interventions during pregnancy/lactation may have considerable advantages in terms of convenience and compliance compared to child- directed interventions. However, recent reports confirm that peri- and/or post-natal probiotic treatment does not yield predictable results. Indeed many studies have reported contradictory results. This is especially true as for the intervention based on probiotics, and more specially for nutritional intervention combining 2 or more probiotics. It may appear that probiotics may be able to antagonise or synergize in vivo as for their effect (especially long term effect and their effect on children). Hence it is a of crucial importance to provide clinical data analysing potential nutritional interventions, especially during pregnancy and their effect on the progeny For example, in WO 01/97822 it is disclosed that, Lactobacillus rhamnosus GG, given to mothers starting 2-4 weeks before delivery and during the breastfeeding period or directly to infants in the first 6 months of life reduced the incidence of eczema at 2 years by around 50% in children who were at high risk. However, contradictory results for the same strain were reported in a paper from Kopp et al. in 2008 [Kopp et al., (2008); Randomized, Double Blind, Placebo Controlled Trial of probiotics for Primary Prevention : No clinical Effects of Lactobacillus GG supplementation Pediatrics, vol. 121 no. 4]. Lactobacillus rhamnosus GG administration to mothers 4 to 6 weeks before expected delivery and then to the offspring during a period of 6 months neither reduced the incidence of atopic dermatitis nor altered the severity of atopic dermatitis in affected children. The paper concluded that Lactobacillus rhamnosus GG cannot be generally recommended for primary prevention. These experiments were carried out on a specific sub-group of the general population, i.e. progeny at high risk of allergy.

In WO2009/1 18243 it was disclosed that specific probiotic microorganisms administered to female mice during the gestation period or the lactation period or directly administered to progeny after birth boosted the immunity of the progeny. Thus, administration of Lactobacillus rhamnosus CGMCC 1 .3724 (LPR) increased peripheral IgG responses to measles vaccine in the mice pups. However, the effect was specific to the strain used.

The article "Probiotics for the prevention of childhood eczema" (B. W. Ritz, Natural medicine Journal, dated 5/3/201 1 ) reports a list of many clinical studies involving probiotics and their effect on allergies. It highlights the various , and often conflicting, results obtained as for the allergy. It reports in particular the study of Soh et al 2009 (Probiotic supplementation in the first 6 months of life in at risk infants - effect on eczema and atopic sensititzation at the age of 1 year, Clin. Exp. Allergy, 2009, 39:571 -578) that used a combination of Bifidobacterium BL999 and lactobacillus LPR in infants but that showed no effect on eczema on the infants. Similarly, the article "Guidance for substantiating evidence for beneficial effect of probiotics : prevention and management of allergic diseases by probiotics" (The Journal of Nutrition, supplement, J Nut. 140:713S-721 S,; 2010) provides an overview of related studies.

The potential neutralizing (or antagonist) effect of probiotics when administered concomitantly is of crucial importance for the present invention. In vivo the concomitant administration can create opposite effects. Indeed such effect renders any prediction of the combined administration of the probiotics very hazardous even if each single probiotics, administered alone, has been shown to deliver some effect. Only clinical data using the tested combination can be used to highlight the effect of the combinations of probiotics.

Thus, in the light of these cited prior art documents, there is a need for a further progress in area of probiotic administration as a way to improve the health of newborn babies and young infants.

There is therefore a need to positively impact the health of the progeny by a targeted nutritional diet of the expecting mothers.

There is, in particular, a need to help guaranteeing the best immune system in the progeny, in order to best prepare them to the early life antigenic challenges as well as to enhance the future maturation of their immune system to better promote protection during later infancy.

There is a need to impact the building of the immune system of progeny at the earliest possible stage during gestation as well as during the early phases of their newborn life when the immune system is rapidly maturing. Summary of the invention

The present invention provides two combinations of probiotics :

Lactobacillus Rhamnosus LPR (LGG) and Bifidobacterium Longum BL999 (BB536),

Lactobacillus paracasei ST1 1 CMCN-l-21 16 and Bifidobacterium Longum

(BB536) for use by administration to expectant females and/or lactating mothers, and optionally to their progeny for the reduction or prevention of the development of allergy in progeny. The reduction in allergic immune response is with respect to progeny of mothers not treated with such combination of probiotics.

The expecting mothers and their progeny may be those at risk of atopic diseases. Expecting mothers as risk of atopic diseases are defined as pregnant women with atopic sensitization and either a history of allergic disease or active allergic disease. Progeny at risk of atopic diseases is intended as the progeny of such mothers. The assessment of meternal allergic disease is typically based on reported clinical history of atopic eczema, allergic rhinoconjunctivitis, food allergy or asthma. Atopic sensitization is typically verified by skin prick testing. Skin prick testing is a common sensitization testing that is well known to the person skilled in the art of allergy treatment and prevention. Such skin prick test is carried out by applying a small quantity of the purified allergen to the skin and pricking the skin surface that is in contact with the purified allergen. Intradermal injections are done by injecting a small amount of allergen just beneath the skin surface. Reaction or absence of reaction to the allergen on the subject skin is then assessed visually to identify sensitization. The test pricks are complemented by a positive (usually histamine) and a negative control to check the reliability of the test results. Skin prick test positive mothers are intended as mothers presenting a reaction to at least one allergen in the performance of the skin prick test described above. The administration according to the invention is generally made according to the clinical data results presented below. To a certain it can be extended as for various periods of administrations and various dosage of probiotics.

According to the invention, the probiotics are administered to the expectant mother. Optionally, it is also administered to the same mothers after delivery, while lactating. Optionally it is further administered to the progeny born of said mothers. Preferably the probiotics are administered to expecting mothers during at least the last 2 months of pregnancy, more preferably for at least 4 months before delivery.

According to the invention, the probiotics are administered to expectant mothers for at least two weeks, preferably for at least two months, more preferably for at least 4 months before delivery and, after delivery, for at least 1 or 2 months, preferably up to six months. In one embodiment the probiotics are administered during the full duration of the pregnancy.

According to a preferred embodiment, the probiotic is administered to the expectant mother (preferably for at least two months) and to the same mother while lactating (preferably for at least two months).

Thus, the mother receives the probiotics as a dose during at least part or all of her pregnancy and, after delivery, during at least part or all of the lactation period if she is lactating. The probiotics may be administered orally to the expectant or lactating mothers, preferably in foods, drinks, dietary supplements or pharmaceutical compositions. The dose of probiotics can be administered daily, every second day or weekly.

In one embodiment a dose of probiotics is administered to the newborn infant for at least two months, preferably up to six months, after delivery either directly or via the mother's milk. It may be administered in its pure form or diluted in water, breast milk or in an infant formula.

The daily dose of each probiotic that is administered to the expectant or lactating mother is from 1 x10 ⁶ to 1 x10 ¹¹ cfu, preferably 1 x10 ⁸ to 1 x10 ¹⁰ cfu (cfu = colony forming unit), whereas the daily doses administered the newborn infant are from 1 x10 ² to1 x10 ¹⁰ , preferably 1 x10 ² to1 x10 ⁴ cfu (cfu = colony forming unit).

In other words, the invention relates to the use of the probiotic combination in the manufacture of a composition to be administered to pregnant women for at least 1 months during pregnancy and optionally for at least 1 month during pregnancy and optionally for at least 1 month after delivery for reducing the allergic response or for the prevention of allergy in their progeny. In such a case, the manufactured composition is for pregnant woman in the last month of pregnancy or earlier in the pregnancy period, preferably in the last two months of pregnancy or earlier in the pregnancy period and optionally for lactating women in the first month of lactation, or later in the lactation process, preferably women in the first two months of lactation or later in the lactation process.

Brief Description of the Drawings Figure 1 : is the flow diagram of the study reported. Detailed description Definitions:

In this specification, the following terms have the following meanings:

"Weaning period" is the period during which infants are adapting from pure liquid nutrition to solid or semi-solid foods, and adapting from quasi unique food type (generally mother milk or infant formula) to a variety of foods. "Sensitization" means induction/development of allergen-specific IgE antibodies.

"Probiotic" means microbial cell preparations or components of microbial cells with a beneficial effect on the health or well-being of the host. (Salminen S, Ouwehand A. Benno Y. et al "Probiotics: how should they be defined" Trends Food Sci. Technol. (1999):10 107-10). The definition of probiotic is generally admitted and in line with the WHO definition. The probiotic can comprise a unique strain of micro-organism, a mix of various strains and/or a mix of various bacterial species and genera. In case of mixtures, the singular term "probiotic" can still be used to designate the probiotic mixture or preparation. For the purpose of the present invention, micro-organisms of the genus Lactobacillus are considered as probiotics.

"Prebiotic" generally means a non digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of micro-organisms present in the gut of the host, and thus attempt to improve host health.

Lactobacillus paracasei (L. paracasei) strain is the L. paracasei with the alternative name of ST1 1 , used throughout the text and the international identification reference CNCM 1-21 16 (Collection Nationale de Cultures de Microorganismes at Institute Pasteur, Paris, France). The CNCM identification refers to the Collection Nationale de Cultures de Microorganismes at Institut Pasteur, 22 rue du docteur Roux, 75724 Paris, France. The probiotics is identical to the one referenced under NCC 2461 (Nestle Culture collection, Vevey Switzerland)

Bifidobacterium Longum BL999 (deposited under ATCC BAA-999 ; BB536) is commercially available.

Lactobacillus Rhamnosus LPR (deposited under CGMCC 1 .3724; LGG) is also available as NCC4007 (Nestle Culture Collection, Vevey Switzerland). Alternative is ATCC-53103.

Combined use: combined use of 2 probiotics is a use where the probiotics are administered to the same subject(s) at the same time or in a relatively concomitant timing such as to allow the synergy of effect between the 2 probiotics. Preferably the 2 probiotics are administered within a 24 hours period, more preferably within a 6 hours period, most preferably at the same time.

Abbreviations

BL999 Bifidobacterium longum BL999 CI confidence interval

LPR Lactobacillus rhamnosus LPR

ST1 1 Lactobacillus paracasei ST1 1

OR Odds' ratio

Doses of probiotic:

The preferably daily doses of each of the probiotics administered to the expectant or breast feeding mother is from 1 x10 ⁶ to 1 x10 ¹¹ cfu, preferably 1x10 ⁸ to 1x10 ¹⁰ cfu (cfu = colony forming unit). The daily dose suitable for newborn babies can range from 1 x10 ² to 1 x10 ¹⁰ , preferably, 1 x10 ² to 1x10 ⁴ cfu.

Thus probiotics may be present in the composition in a wide range of percentages provided that it delivers the allergy protective effect described. However, preferably, the probiotics is present in a composition in an amount equivalent to between 1 x10 ² and 1 x10 ¹¹ cfu/g of dry composition. This includes the possibilities that the bacteria are live, inactivated or dead or even present as fragments such as DNA or cell wall materials. In other words, the quantity of bacteria which the formula contains is expressed in terms of the colony forming ability of that quantity of bacteria as if all the bacteria were live irrespective of whether they are, in fact, live, inactivated or dead, fragmented or a mixture of any or all of these states. Preferably, for administration to the expectant or lactating mother, the probiotic is present in an amount equivalent to between 1 x10 ⁴ to 1 x10 ⁹ cfu/g of dry composition, even more preferably in an amount equivalent to between 1 x10 ⁶ to 1x10 ⁸ cfu/ g of dry composition.

The amount of probiotic present per gram of dry composition for administration to the progeny may be lower and the daily doses described above should be respected. Method of administration:

(i) Administration to expectant mothers:

The composition can be administered to the expectant mothers by various ways as long as it induces a contact between the composition and the gastro-intestinal tract of the females. Preferably, the composition is orally administered as part of the food, drinks or dietary supplements of the expectant mothers. The composition can also be administered in a pharmaceutical composition. Preferably the administration is oral. However, in pathological conditions or when enteral feeding is otherwise used, the administration of the composition can be added to the enteral feeding. (ii) Administration to newborn progeny:

In some embodiments the probiotics can be administered via the milk of the lactating mother or can also be administered orally directly to the progeny alone (pure or diluted in water or mother's milk for example) as a food supplement or as an ingredient in an infant milk formula. Such a formula may be an infant "preterm formula" if the progeny is born before term or has a low birth weight, a "starter formula" or a "follow-on formula". The formula may also be an hypoallergenic (HA) formulas in which the cow milk proteins are hydrolysed.

Administration with other compounds:

The probiotics can be administered alone (pure or diluted in water or milk, including breast milk for example) or in a mixture with other compounds (such as dietary supplements, nutritional supplements, medicines, carriers, flavours, digestible or non- digestible ingredients). Vitamins and minerals are examples of typical dietary supplements. In a preferred embodiment, the composition is administered together with other compounds that enhance the described effect on the immunity of the progeny. Such synergistic compounds may be carriers or a matrix that facilitates the probiotics delivery to the intestinal tract of the expectant mother or they may otherwise enhance the effect of the composition on the immune system of the progeny. Such compounds can be other active compounds that synergistically or separately influence the immune response of the infant and/or potentiates the effect of the probiotic. An example of such synergistic compounds is maltodextrin. One of the effect of maltodextrin is to provide a carrier for the probiotic, enhancing its effect, and to prevent aggregation.

Other examples include known prebiotic compounds such as carbohydrate compounds selected from the group consisting of inulin, fructooligosaccharide (FOS), short-chain fructooligosaccharide (short chain FOS), galacto-oligosaccharide (GOS), xylooligosaccharide (XOS), glangliosides, partially hydrolysed guar gum (PHGG) acacia gum, soybean-gum, apple extract, lactowolfberry, wolfberry extracts or mixture thereof. Other carbohydrates may be present such as a second carbohydrate acting in synergy with the first carbohydrate and that is selected from the group consisting of xylooligosaccharide (XOS), gum, acacia gum, starch, partially hydrolysed guar gum or mixture thereof. The carbohydrate or carbohydrates may be present at about 1 g to 20g or 1 % to 80% or 20% to 60% in the daily doses of the composition. Alternatively, the carbohydrates are present at 10% to 80% of the dry composition.

The daily doses of carbohydrates, and all other compounds administered with the probiotics should always comply with the published safety guidelines and regulatory requirements. This is particularly important with respect to the administration to newborn babies.

In one embodiment, a nutritional composition preferably comprises a source of protein. Dietary protein is preferred as a source of protein. The dietary protein may be any suitable dietary protein, for example animal proteins (such as milk proteins, or meat proteins), vegetable proteins (such as soy proteins, wheat proteins, rice proteins or pea proteins), a mixture of free amino acids, or a combination thereof. Milk proteins such as casein and whey proteins are particularly preferred.

The composition may also comprise a source of carbohydrates and/or a source of fat.

If the composition of the invention is a nutritional composition and includes a fat source, the fat source preferably provides about 5% to about 55% of the energy of the nutritional composition; for example about 20% to about 50% of the energy.

Lipid making up the fat source may be any suitable fat or fat mixture. Vegetable fat is particularly suitable, for example soy oil, palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canola oil, lecithin and the like. Animal fat such as milk fat may also be added if desired.

An additional source of carbohydrate may be added to the nutritional composition. It preferably provides about 40% to about 80% of the energy of the nutritional composition. Any suitable carbohydrate may be used, for example sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, or a mixture thereof. Additional dietary fibre may also be added if desired. If added, it preferably comprises up to about 5% of the energy of the nutritional composition. The dietary fibre may be from any suitable origin, including for example soy, pea, oat, pectin, guar gum, acacia gum, fructooligosaccharide or a mixture thereof. Suitable vitamins and minerals may be included in the nutritional composition in an amount to meet the appropriate guidelines.

One or more essential long chain fatty acids (LC-PUFAs) may be included in the composition. Examples of LC-PUFAs that may be added are docosahexaenoic acid (DHA) and arachidonic acid (AA). The LC-PUFAs may be added at concentrations so that they constitute greater than 0.01 % of the fatty acids present in the composition.

One or more food grade emulsifiers may be included in the nutritional composition if desired; for example diacetyl tartaric acid esters of mono- and di- glycerides, lecithin and mono- or di-glycerides or a mixture thereof. Similarly suitable salts and/or stabilisers may be included. Flavours can be added to the composition.

Administration period:

The expectant mother may start to take the probiotics as soon as she is aware of her pregnancy. However, the administration period may also start before pregnancy starts, for example if the female is trying to become pregnant. Administration may start at any time after the pregnancy starts. It may start relatively late in the pregnancy, preferably at month 3, 4, 5, 6, 7 or 8 of the pregnancy, in the case of human pregnancy, or in corresponding periods for other mammals, or up to two weeks before the expected delivery date.

The period of administration can be continuous (for example, up to and including lactation up to weaning), or discontinuous. Continuous administration is preferred for a more sustained effect. However, it is speculated that a discontinuous pattern (for example, daily administration during one week per month, or during alternate weeks) can induce positive effects on the progeny. The duration of the administration may vary. While positive effects are expected with relatively short duration of administration (for example, daily administration during one week for newborns and one or two months for adults), longer durations are believed to provide enhanced effect (for example, a duration of three, five or eight months in humans, and corresponding periods in other mammals).

The administration should cover at least part of the gestation period and at least part of the lactation period, or the equivalent period should the newborn not be breastfed. Preferably, the administration period to the expectant mother covers substantially the full length of the gestation period, although this may be less. Similarly, the administration period for the lactating mother preferably covers substantially the full length of the lactation period, although, again, this period may be less. Preferably, the administration to the mother is by daily intake (to be taken once or twice a day), or weekly intake (to be taken one or twice a week).

The probiotics may also be administered to the infant directly. This is the case particularly if the mother does not breastfeed or after she discontinues breastfeeding. However, an infant who is being breastfed may also receive the probiotics by direct administration. The administration to the infant, either via breastfeeding, or by direct administration, or both methods, may be continued up until the age of six months or even one year. Thus, the probiotics may be administered during lactation if lactation takes place, or after partial or full weaning.

Preferably, the administration to the infant is by daily intake (to be taken once or twice a day), or weekly intake (to be taken one or twice a week).

Effect of the probiotic administration: Probiotics administered to expectant mothers protects the progeny from the development of allergy. This is show in the reported study.

Example 1 :

An example of a composition comprising the probiotics of the invention is given below. This composition is given by way of illustration only. The protein source is a conventional mix of whey protein and casein. The composition is administered to the mothers and/or to their infants.

Nutrient per l OOkcal per litre

Energy (kcal) 100 670

Protein (g) 1 .83 12.3

Fat (g) 5.3 35.7

Linoleic acid (g) 0.79 5.3

a-Linolenic acid (mg) 101 675

Lactose (g) 1 1.2 74.7

Prebiotic (100% GOS) (g) 0.64 4.3

Minerals (g) 0.37 2.5

Na (mg) 23 150

K (mg) 89 590

CI (mg) 64 430

Ca (mg) 62 410

P (mg) 31 210

Mg (mg) 7 50

Mn ( g) 8 50

Se ( g) 2 13

Vitamin A RE) 105 700

Vitamin D ( g) 1 .5 10

Vitamin E (mg TE) 0.8 5.4 Vitamin K1 (μς) 8 54

Vitamin C (mg) 10 67

Vitamin B1 (mg) 0.07 0.47

Vitamin B2 (mg) 0.15 1 .0

Niacin (mg) 1 6.7

Vitamin B6 (mg) 0.075 0.50

Folic acid ^g) 9 60

Pantothenic acid (mg) 0.45 3

Vitamin B12 ( g) 0.3 2

Biotin ^g) 2.2 15

Choline (mg) 10 67

Fe (mg) 1 .2 8

l (M9) 15 100

Cu (mg) 0.06 0.4

Zn (mg) 0.75 5

DHA Any usual amount according to

regulations

Lactobacillus Rhamnosus LPR 2x10 ⁷ cfu/g of powder of each

(LGG) and Bifidobacterium probitiocs

Longum BL999 (BB536) or alternatively

and/ or Sufficient amount to deliver 1x

Lactobacillus paracasei CNCM 10 ⁹ cfu / day

1-21 16 (ST1 1 ) and

Bifidobacterium Longum

BL999 (BB536)

In an alternative embodiment the composition administered to the mothers comprise the above probiotics in a neutral matrix (such as maltodextrine).

Example 2 / Reported Study Methods

A double-blind, randomized, placebo-controlled trial was devised to assess the effects of maternal probiotic administration on the risk of eczema in the infant. Pregnant women with atopic sensitization and either a history of or active allergic disease and the intention to breastfeed for a minimum of 2 months were considered eligible for the study. The assessment of maternal allergic disease was based on reported clinical history of atopic eczema, allergic rhinoconjunctivitis, food allergy or asthma. Sensitization was verified by skin prick testing. Women with immune- mediated disease other than atopic or allergic disease were excluded from the study. Infants born of multiple pregnancies were excluded from the analyses in order to ensure independence of the study subjects.

The study was conducted in a single tertiary center in Turku, Finland. Recruitment took place between August 2005 and April 2009. Information regarding the study was distributed to pregnant women at prenatal care centers in the Turku region in Southwest Finland. All families who were interested in participating and contacted the research nurse during the recruitment period were assessed for eligibility. Altogether 241 pregnant women were randomized in a parallel design to receive a dietary food supplement containing minerals including calcium, vitamins including vitamins B12, A and D, folic acid and other micronutrients including iron, zinc and iodine with composition and dosage in compliance with recommended daily allowances supplemented with either the combination LPR and BL999 consisting of Lactobacillus rhamnosus LPR (CGMCC 1 .3724) and Bifidobacterium longum BL999 (ATCC: BAA- 999) or the combination ST1 1 and BL999 consisting of L. paracasei ST1 1 (CNCM 1 - 21 16) and B. longum BL999. Daily dose for each probiotic was 1 x 10 ⁹ cfu provided in one sachet of 7 g per day (powder form) which was diluted in a glass of water. The same dietary supplement without probiotics served as placebo. The study preparations were provided by Nestle S.A. and were similar in appearance. Viable probiotics or placebo were prepared within the powder by the manufacturer. Control of preparation quality was conducted by the supplier and fresh new sachets were provided at frequent intervals to ensure viability. Maternal probiotic supplementation was commenced 2 months before the expected day of delivery and continued during breastfeeding until the child was 2 months of age. Information regarding products containing probiotics available in the market during the study period was given when the intervention was commenced and subsequently at five scheduled study clinic visits 1 , 3, 6, 12 and 24 months after delivery. Use of such products was discouraged. Compliance with the intervention was controlled by interview during the scheduled visits.

The number of subjects enrolled in the study is based on an assumed 50% prevalence of the primary outcome measure eczema up to the age of 2 years and a clinically relevant reduction of 25% by intervention. Assuming a type I error of 2.5% and a power of 80%, the required number of subjects to be analyzed per group should be 64 and in total 192 (Fisher exact test, one-sided). The random allocation was computer-generated independently from the investigators by the manufacturer of the study products. All investigations were performed double-blind and the code was opened after all the infants had completed the follow-up, outcomes had been assessed and the data had been finalized. The study was deemed ethically acceptable by the by the Ethics committee of the Intermunicipal Hospital District of Southwest Finland and registered (NCT00167700). Oral and written informed consent was obtained from the mothers.

Outcome measures

The infants were followed up until the age of 24 months. Clinical examination of the infants was performed at scheduled visits at the ages of 1 , 3, 6, 12 and 24 months. The primary outcome measure of the trial was cumulative incidence of eczema in the infant up to the age of 2 years. Eczema was diagnosed according to the criteria introduced by Hanifin ¹³ based on the following features: pruritus, typical morphology and distribution and a chronic relapsing course. The last criterion was fulfilled if the infant had 2 episodes of eczema with duration of at least one month each during the first two years of life. If the skin condition persisted without periods of remission the eczema was considered chronically persistent. All adverse effects possibly related to the study products were systematically monitored and recorded.

To objectively assess atopic sensitization in the infants, skin prick tests were performed at the ages of 6, 12 and 24 months as described previously. ¹⁴ Skin prick testing was used because of its high sensitivity and equal or superior accuracy as compared to serum allergen-specific IgE antibody concentrations in predicting allergic reactions, albeit the specificity of both methods is poor. ^14"16 The antigens tested included cow's milk, egg white, wheat and rice flour both diluted 1/10 (w/v) with 0.9% (w/v) sodium chloride, gliadin diluted 1 mg/ml with an ethanol/glyceroleum/ALK- diluent (Allergologisk Laboratorium A S, Horsholm, Denmark) mixture, cod, soy bean, birch, six grasses, cat, dog, Dermatophagoides pteronyssimus allergen Der p1 (Allergologisk Laboratorium A/S, Horsholm, Denmark), latex (Starallergens S.A., Anthony Cedex, France) and potato, carrot and banana by prick-prick technique. The chosen panel of allergens represents the most common sources of allergic reactions in the target population. The testing was performed on the volar side of the forearm with a 1 -mm, one-peak lancet (Allergologisk Laboratorium A/S, Horsholm, Denmark) with a shoulder to prevent deeper penetration. 10 mg/mL Histamine dihydrochloride (Allergologisk Laboratorium A/S) was used as the positive control and the negative control solution was provided by the same manufacturer. Reactions were read at 10- 15 minutes. Reactions with a mean diameter of the wheal of at least 3 mm were considered positive on the condition that the mean diameter of the wheal to the positive control was at least 3 mm and the negative control reaction was 0 mm. Statistical analyses

Continuous background data are expressed as means with range and categorical data as percentages. Differences between the groups were assessed using ANOVA for continuous variables and chi-squared test for categorical variables. To assess the effects of the interventions on outcome, comparisons between groups were conducted using logistic regression analysis and expressed as odds' ratios (OR) with 95 % confidence interval (CI). A p value <0.05 was considered statistically significant. All analyses were conducted intention to treat.

Results Altogether 205/241 (85%) mother/infant pairs completed the follow-up (Figure 1 ). The rate of discontinuing the study or lost to follow-up was similar in the three study groups. The background characteristics of the infants in the study groups are presented in Table 1. Altogether 10 mothers in the study (2 mothers receiving placebo, 3 mothers receiving LPR and BL999 and 5 mothers receiving ST1 1 and BL999) ceased to breastfeed before the infant was 2 months of age. The infants of these mothers were included in the analyses according to the intention to treat principle. Eczema was detected during the first 24 months of life in 85/205 (41 %) infants who completed the follow-up and chronically persistent eczema was diagnosed in 27/205 (13%) infants. Maternal consumption of either the combination of LPR and BL999 or the combination of ST1 1 and BL999 were both associated with a statistically significant reduction in the risk of developing eczema and chronically persistent eczema during the first 24 months of life when compared to infants whose mothers received placebo (Table 2). Atopic sensitization was detected in 53/214 (25%) of the infants by skin prick testing during the follow-up. The rate of skin prick test positivity was comparable between infants born to mothers receiving either the combination of LPR and BL999 or the combination of ST1 1 and BL999 or placebo (Table 2). Atopic sensitization was significantly associated with the risk of developing eczema or chronically persistent eczema, as 54/155 (35%) of skin prick test negative infants who completed the follow-up developed eczema as compared to 31/50 (62%) skin prick test positive infants (p=0.001 ). The respective rates for chronically persistent eczema were 12/155 (8%) and 15/50 (30%) (p<0.001 ). However, no statistically significant interactions between skin prick test status and the probiotic interventions with regard to the risk of eczema (p=0.328) or chronically persistent eczema (p=0.283) were detected using logistic regression analysis.

Table 1. Background characteristics of mother-infant pairs receiving placebo, the combination of Lactobacillus rhamnosus PR and Bifidobacterium longum BL999 (LPR+BL999) or the combination of L. paracasei ST1 1 and B. longum BL999 (ST1 1 +BL999). The data are presented as means with range or ratios with percentages, the groups were compared using ANOVA for continuous variables and chi-squared test for categorical variables

Placebo LPR+BL999 ST1 1 +BL999

Maternal age 30 (22-42) 31 (22-43) 30 (22-40) p=0.97 (years)

Gestational age 39.4 (34.5- 39.8 (34.6-41.0) 39.8 (33.5-41.5) p=0.25 (weeks 41.0)

Birthweight 3561 (2380- 3558 (2580- 3582 (1910- p=0.94 (grams) 4670) 4800) 4660)

Cesarian section 14/73 (19%) 4/80 (5%) 9/78 (12%) p=0.024

Boys 40/74 (54%) 39/80 (49%) 37/79 (47%) p=0.69

Older siblings 27/75 (36%) 31/81 (38%) 34/81 (47%) p=0.74

Pets 25/75 (33%) 27/81 (33%) 30/81 (37%) p=0.85

Exclusive 3.3 (0.0-6.0) 3.2 (0.0-7.0) 3.1 (0.0-1 1.0) p=0.93 breastfeeding

(months)

Total 9.5 (1.5-24.0) 9.1 (1.0-24.0) 10.0 (0.0-24.0) p=0.59 breastfeeding

(months Table 2. Occurrence of eczema, chronically persistent eczema and skin prick test positivity in infants whose mothers received placebo, the combination of Lactobacillus rhamnosus LPR and Bifidobacterium longum BL999 (LPR+BL999) or the combination of L. paracasei ST1 1 and B. longum BL999 (ST1 1 +BL999). The data are presented as odds' ratios with 95% confidence intervals (CI) compared to placebo group. The p values correspond to logistic regression analyses.

Since the rate of cesarian section delivery was different between the study groups (Table 1 ), further analyses were conducted. The difference in cesarian section rate between mother receiving LPR and BL999 (5%) and placebo (19%) was statistically significant (p=0.01 1 ) but that between mothers receiving ST1 1 +BL999 (12%) and placebo was not (p=0.20). Of note, mode of delivery did not interact with the impact of the interventions on the occurrence of eczema (p=0.94) or chronically persistent eczema (p=0.46) as assessed using logistic regression analysis.

No adverse effects related to probiotic supplementation were detected in the study subjects. Gastrointestinal symptoms were reported in 4/70 (6%) mothers receiving placebo, in 6/80 (8%) mothers receiving LPR and BL999, and in 3/74 (4%) mothers receiving ST1 1 and BL999, p=0.66. One mother receiving placebo and one mother receiving LPR and BL999 experienced aggravation of eczema during supplementation. Gastrointestinal symptoms were observed in 87/223 (39%) of the infants during the first 2 months of life when the study products were administered to the breastfeeding mothers. The rates of gastrointestinal symptoms among infants whose mothers received placebo, the combination of LPR and BL999 or ST1 1 and BL999 were 24/70 (34%), 35/79 (44%) and 28/74 (38%), respectively, p=0.44. Discussion

Maternal probiotic supplementation during the last two months of pregnancy and the first two months of breastfeeding significantly reduced the risk of developing eczema in high-risk infants in this study. It is of note that the probiotics were administered exclusively to the mothers whereas the infants did not receive probiotics. The results of this randomized, controlled trial corroborate our initial observation from a subgroup of 62 breastfed infants ¹² from a clinical study in which 159 high-risk mother-infant pairs received the probiotic Lactobacillus rhamnosus GG or placebo. ¹ Recently, similar data have been published from trials conducted in Norway ⁶ and Korea ⁷ using different combinations of probiotic lactobacilli and bifidobacteria but these studies suffered from high rates of subjects lost to follow-up. We interpret our data to suggest a feasible and effective, relatively short-term exclusively maternal intervention to reduce the risk of eczema in the infant. Importantly, we describe objective criteria for a clearly defined target population, which may be identified by maternal allergic disease and skin prick test positivity.

The use of viable bacteria to reduce the risk of disease in early infancy has raised obvious safety concerns related to risk of bacterial translocation and developing septicemia. Instances of probiotic-induced sepsis have been reported in infants but no serious adverse effects have to our knowledge been observed in clinical trials assessing the effects of probiotics in neonates or infants. It is vital to acknowledge, however, that clinical trials may not have sufficient numbers of subjects to detect rare but significant adverse effects since statistical power is typically calculated based on the primary outcome measures of the study. Recent reports from our unit ¹⁹ and Italy ²⁰ indicate that routine use of the probiotic lactobacilli in a neonatal intensive care setting has been safe and well-tolerated over a period of several years. In the present study, we observed no adverse effects associated with probiotics in the pregnant or breastfeeding mothers or their infants. The fact that the infants received no direct intervention and that the probiotics were solely administered to the mothers may be interpreted to further improve the safety of the intervention. Thus, according to our results, probiotics administered to the pregnant and breastfeeding mother appear to be safe and effective in reducing the risk of eczema in infants with high hereditary risk. Notwithstanding the accumulating evidence of health benefits of specific probiotics in neonates and infants, relatively little is known about the mechanisms mediating these effects. Conventionally, it has been assumed that probiotics exert their effects by modulating intestinal microbiota composition or by directly stimulating the intestinal immune system. Interestingly, however, careful review of the published clinical trials aiming to reduce the occurrence of eczema in infants suggests that the intervention is effective only if the probiotics are administered already prenatally to the mother. Our present data are also consistent with the notion of prenatal probiotic effects. Prenatal probiotic supplementation may modulate the composition of maternal vaginal and intestinal microbiota, which provide an important colonizing inoculum to the newborn infant and thus have an effect on neonatal gut colonization. However, the mechanisms of prenatal probiotic effects may also be more indirect. Epidemiological data suggest that maternal contact with farm animals and thus presumably increased microbial exposure during pregnancy reduces the risk of eczema in infancy, which in turn is associated with alterations in innate immune gene expression. In line with these observations, maternal mucosal contact with the microbe Acinetobacter Iwoffii during pregnancy reportedly modulates placental innate immune gene expression and protects the offspring from asthma in a murine model. ²³ Given that maternal immune cells have recently been demonstrated to cross the placenta and induce tolerogenic immune responses in the human fetus, it is conceivable that maternal probiotic supplementation modulates immune physiology in the feto-placental unit. To support this notion, we have recently observed that maternal prenatal probiotic intervention significantly affects immune gene expression in the placenta and in the fetal gut in humans. Even though maternal probiotic supplementation during pregnancy appears necessary for reducing eczema risk in the infant, prenatal intervention alone is not be sufficient to achieve the desired clinical effects according to a recent report. ¹¹ Based on our present data and those from previously published trials, we conclude that in addition to prenatal intervention, maternal probiotic supplementation during breastfeeding may modulate disease risk in the infant. While nonspecific microbial transfer from the mother through nursing and skin-to-skin contact undoubtedly occurs, we suggest that maternal probiotic intervention exerts its effect also via specific modulation of the immunologic properties of breast milk. We have previously shown that maternal consumption of Lactobacillus rhamnosus GG during pregnancy and breastfeeding increases the concentration of the immunomodulatory cytokine transforming growth factor (TGF)-p2 in breast milk and is associated with reduced eczema risk in the infant. ¹² TGF-p2 has been demonstrated to be necessary for breast milk-induced tolerance in an animal model ²⁶ and we have provided experimental evidence suggesting that TGF-p2 at a concentration comparable to that in breast milk modulates immune responses in the immature human gut ²⁷ and promotes immune maturation. ²⁸ In addition to its obvious nutritional function and immunomodulatory properties, breast milk has a profound impact on the composition of neonatal gut microbiota development. ²⁹ Human milk oligosaccharides promote the growth of intestinal bifidobacteria and breast milk has also been discovered to be a source of potentially colonizing bacteria to the newborn infant. Both cells in human breast milk and maternal peripheral blood mononuclear cells have recently been shown to contain bacteria and bacterial DNA. Specific live Lactobacillus have been recovered in breast milk after maternal supplementation with the probiotic in an intervention study failing to reduce eczema risk but showing decreased risk of IgE-mediated eczema after subgroup analysis. ^2,32 Experimental animal models demonstrate increased intestinal bacterial translocation during pregnancy and lactation which results in the presence of bacteria within dendritic cells in the mammary gland. Taken together, these observations suggest a unique mechanism by which breast milk serves as a vehicle to introduce maternal gut microbiota to the infant in a tolerogenic immune milieu. ³³ We hypothesize that probiotic intervention during lactation modulates this process and supports healthy immunologic maturation and intestinal microbiota development. ³³ Collectively, the data reviewed above suggest that maternal probiotic supplementation during pregnancy and breastfeeding exerts its effect via several unconventional, indirect mechanisms including modulation of placental and fetal immunophysiology and by promoting the protective potential of breast milk.

In conclusion, our data suggest that maternal supplementation with either the combination of the probiotics LPR and BL999 or the combination of ST1 1 and BL999 during the last two months of pregnancy and the first two months of breastfeeding may reduce the risk of infant eczema in a clinically and statistically significant manner in infants with skin prick test positive mothers with allergic disease. Maternal probiotic intervention appears to be safe, inexpensive and relatively easy to implement even during exclusive breastfeeding without need to administer probiotics to the infant.