Patent Application
20180256633
The invention relates to a combination of a long chain inulin and a vaccine, wherein the long chain inulin is orally administered. The invention also relates to a long chain inulin for influencing the immune response against a pathogen wherein the long chain inulin is orally administered.
Dietary fibers are considered an essential part of healthy nutrition. The health benefits of sufficient fiber intake comprise prevention of colorectal cancer1, type 2 diabetes2, cardiovascular disease3, reduced risk of hyperlipidemia, hypercholesterolemia and hyperglycemia4 5, and regulation of bowel habit6-10.
Inulin-type fructans are prebiotic dietary fibers with many health benefits. They are oligomers and polymers of fructose subunits, and often terminate in a glucose molecule13. Besides the established effects on gut health and metabolism11 12 14, evidence for immunostimulatory effects of inulin-type fructan consumption is accumulating. Results from ex vivo and in vitro experiments in human cells, cell lines, and from animal studies, support the notion that inulin-type fructans exert immune modulating effects15-19. The underlying mechanisms for immune modulation by inulin-type fructans have been attributed to the selective stimulation of beneficial bacteria in the intestine, and their SCFA fermentation products17. In addition, previous results from our group support the notion that these fibers can also exert direct effects on immune cells, by activating or inhibiting Toll-like receptors (TLRs) on monocytic cells, and modulating, preferably inducing cytokine production in human peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation20.
There is no evidence that inulin-type fructan supplementation could behave as a compound that is able to quantitatively and qualitatively influence the immune response induced in response to a pathogen or to an antigen from a pathogen such as present in a vaccine in humans. Results obtained so far with infants to elderly from supplementation trials21-24 are not consistent for at least the following reasons. The compound used as “inulin-type fructan” is not always well defined in terms of degree of polymerization (DP), and often mixtures of fructans having different DP and/or with other carbohydrates are applied. This study demonstrates that the effects on the immune system is at least depending on the DP of the inulin-type fructan used. In addition, several studies treated infant or toddler populations or use different types of infant/toddler populations within one single study. The immune system of an infant population is expected to be Th2 and less susceptible to a skewing towards Th1 than the one of an older infant/toddler population, let alone the one of an elderly population. Absence of significant effects observed in one specific population does not per se indicate the same absence of significant results would be obtained in another population having an immune system more susceptible to be skewed towards a Th1 immune response.
There is always a need for compounds that could further optimize the efficacy of vaccines or promote immunity against infections. Ideally such compounds may also confer new attractive properties to vaccines. For example, they can influence the type and direction of the immune response induced.
We surprisingly show that long chain inulin-type fructan supplementation influences the immunity response against pathogens such as during vaccination, where an immune reaction is evoked against a pathogen without inducing a disease. Using common vaccination programs against hepatitis B surface antigen (HBsAg) to immunize subjects, we demonstrate that by supplementing young adults with inulin-type fructans from 7 days before, until 7 days after the first injection of an HBsAg vaccination program, the antibody response against the vaccine has been significantly improved compared to placebo supplemented participants. This has been shown by an earlier onset of titer development, an increased titer response on the final measuring time point, and/or the presence and number of ‘responders’, characterized as subjects with titers equal to or above 10 IU/mL. More importantly, we demonstrated that in the treated group, a shifted Th1/Th2 balance may have been induced which was skewed towards Th1 cell responses. This is the first in vivo study which demonstrates a structure-function relation of a dietary fiber on a human immune response.
In a first aspect, the invention relates to a combination comprising a long chain inulin and a vaccine, wherein the long chain inulin is orally administered. In said first aspect, the combination is preferably for influencing the immune response against a pathogen. In a second aspect, the invention also relates to a long chain inulin for influencing the immune response against a pathogen, wherein the long chain inulin is orally administered.
Unless otherwise indicated, the definition and text of the description applies to both aspects.
In the first aspect of the invention, the word “combination” preferably means that a long chain inulin and a vaccine are not comprised within one single composition. The invention provides the insight that both compounds (i.e. a long chain inulin and a vaccine) are needed or are used in order to get an optimal or maximum or significant or measurable effect on the immune response as defined later on. If a long chain inulin and a vaccine are not present in a same composition, each compound may be used sequentially or simultaneously administered.
However it is not excluded that a long chain inulin and a vaccine may be together or present together or combined together or physically in contact with the other forming one single composition.
Inulin is the generic name covering all β(2,1) fructans (see
Long chain inulin refers to the degree of polymerisation of inulin. The degree of polymerization (DP) or number of fructose units in the fructose polymer of standard/native inulin ranges from mainly 2 to 60 or from 2 to 60 with average DP of approximately 8-13 or 8-13 for example for native chicory. Long chain inulin is generally obtained by crystallization and removal of impurities. Long chain inulin therefore is a fructose polymer made of poly-β-D-(2→1)-fructofuranosyl-α-D-glucopyranose but also of derivatives thereof such as poly-β-D-(2→1) polyfructofuranose and which has preferably a DP ranged from mainly 8 to 60 or from 8 to 60. In this context, mainly preferably means that at least 60%, 70%, 80%, 90%, 95% or 99% of the molecules has its DP comprised in this range. It is also encompassed by the invention that long chain inulin has a DP ranged from mainly 10-60, 14-30, 10-42 or mainly 20-30, 21-30, or mainly 21-29 of from 10-60, 10-42, 14-30, 20-30, 21-30, 21-29. A preferred average DP of long chain inulin is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.
Short chain inulin made from standard inulin ranges from 2 to 35 with an average DP of ranged from mainly 6-10 or 7-9 or from 6-10 or 7-9. Oligofructose and fructo-oligosaccharide (FOS) may also be considered as short chain inulin. Typically oligofructose has a DP of 2-8. Oligofructose may be produced from inulin by partial enzymatic hydrolysis. Typically FOS has a DP of 2-5 and may be enzymatically produced from sucrose.
An estimation of longest inulin chains present in for example chicory inulin can be made from an inulin chain length distribution analysed for example by a chromatographic method (HPAEC-PAD).
A preferred natural inulin is chicory (Cichorium intybus) inulin. Other sources for inulin production include artichoke, agave, amongst others. Inulin from chicory is a polydisperse mixture of linear fructan oligomers and polymers coupled by means of β(2-1) bonds, and mostly with a terminal glucose unit. Native inulin and its partial enzymatic hydrolysis product oligofructose (OF) are usually purified from the chicory plant (preferably from the root of the chicory plant) and its partial enzymatic hydrolysis product oligofructose (OF), and long-chain inulin, are produced (or are usually produced) from native inulin.
A preferred long chain inulin is Frutafit®TEX! from Sensus, Roosendaal, the Netherlands. Frutafit®TEX! is a natural powdered food ingredient based on or derived from native chicory inulin, and known for its texturizing properties. In order to obtain Frutafit®TEX!, chicory roots are first pretreated and subsequently the obtained mixture is purified. The pretreatment may comprise a washing, a slicing and extraction steps. The extraction step is preferably carried out using only hot water. Hot water extraction of inulin from chicory takes place at similar conditions of extraction of sugar from sugar beets. It means that preferably no other solvent is being used. Subsequently long chain inulin is generally obtained by crystallization and removal of impurities. (Meyer & Blaauwhood, 2009, edited by G. O. Philips and P. A. Williams, pages 829-848, Woodhead, Cambridge, UK).
The DP ranges of the Frutafit®TEX! product are from mainly 8 to 60 or from 8 to 60. In this context, mainly preferably means that at least 60%, 70%, 80%, 90%, 95% or 99% of the molecules of the product has its DP comprised in this range. More preferably, the DP of the product is ranged from mainly 14-30 or 14-30, 10-42, or mainly 20-30, 21-30, or mainly 21-29 or 20-30, 21-30 or 21-29. A preferred average DP of this product is 22, 23, 24, 25, 26, 27, 28, 29. Experimental evidence are provided in this application using a combination of the invention comprising Frutafit®TEX! as long chain inulin.
Suitable inulin derivatives included within the scope of this term are derivatives of inulin in which the free hydroxyl groups or parts thereof have been for example oxidised, acetylated, methylated, etherified or esterified, for example by chemical substitution with alkyl, aryl or acyl groups, by known methods or by enzymatic substitution.
The invention also encompasses a particle that is constituted by, contains or is coated by long chain inulin, or a derivative or mimetic thereof. The long chain inulin particle may be solid or hollow and may be wholly comprised of long chain inulin molecules or may alternatively have a non-sugar core, skeleton or shell comprising, for example, carbohydrate compounds, metal compounds, proteins or lipids but which at its surface expresses long chain inulin molecules either covalently or non-covalently bonded to the components comprising the core.
In one embodiment, long chain inulin as used in the present invention is at least partly soluble and/or is not detectable as a crystalline form at a temperature ranged from room temperature and body temperature of the subject to whom it is to be administered. Preferably, the solubility and absence of crystalline form of said long chain inulin is assessed when said long chain inulin is present at a concentration of no greater than 0.5 mg/ml or 1 mg/ml or 2 mg/ml in distilled water or saline or phosphate buffered saline, for at least 10, 20, 30, 40, 50, or 60 minutes.
Therefore a preferred combination of the first aspect of the invention comprises a long chain inulin with a DP of mainly 10-60, (or a DP of 10-60), preferably an average DP of 22, 23, 24 and a vaccine, wherein the long chain inulin is orally administered.
Accordingly in the second aspect of the invention, a long chain inulin with a DP of mainly 10-60, (or a DP of 10-60), preferably an average DP of 22, 23, 24 is orally administered for influencing the immune response against a pathogen.
The dose of long chain inulin used in the invention may vary depending from the subject treated. The subject treated is an animal, more preferably a mammal, even more preferably a human. In a further preferred embodiment, a human subject is a baby (0 to 1 year), an infant or a toddler/small child (1 year to less than 5 years), a child (5 to 18 years) an adult (18-60 years) or an elderly (60 years and older). Other preferred animals include companion animal as dog, cat. Other preferred animals include pig, cows, horse.
In an embodiment, the dose of long chain inulin is ranged from 1 to 25 g per day for human adult and elderly, or 1 to 8 g per day or 1 to 5 g per day.
In another embodiment, the dose of long chain inulin is ranged from 1 to 15 g per day for children and small children, or 1 to 10 g per day or 1 to 8 g per day, or 1 to 5 g per day. The dose may be further adapted depending on the age of the children.
In another embodiment, the dose of long chain inulin is ranged from 0.01 to 15 g per day for baby and toddler/small child, or 0.1 to 12 g per day or 0.5 to 8 g per day, or 1 to 5 g per day. The dose may be further adapted depending on the age of the baby. Therefore in one embodiment, the dose of long chain inulin is ranged from:
It is therefore clear to a skilled person that the long chain inulin may be for use in baby, small children or infant, adult or elderly. Preferably, the combination is for small children to elderly.
In a combination of the invention (first aspect), a vaccine is present. A vaccine or vaccine composition is a composition that provides active acquired immunity to a particular disease. A vaccine usually comprises an agent that resembles a disease-causing micro-organism or disease-causing virus. A disease-causing microorganism (or microorganism) or disease-causing virus (or virus) is also called a pathogen. A preferred disease-causing microorganism or a preferred disease-causing virus is such that it is able to induce a systemic infection. In a preferred embodiment, a vaccine comprises an antigen from said microorganism or virus. It is clear for the skilled person that in a preferred embodiment, a vaccine does not comprise or consist of an alive and/or whole pathogen. Usually if a pathogen is used in a vaccine it is killed or attenuated.
As used herein, the term “antigen” generally refers to a substance that evokes an immune response, including humoral immunity and/or cellular immunity response, and that is capable of binding with a product, e.g., an antibody or a T cell, of the immune response. An antigen as intended herein may in an alternative be such as to induce immuno-tolerance, e.g., may be an auto-antigen (including auto- and allo-antigens) or may be allergen. Hence, in a preferred example, an antigen requires a functioning immune system of a subject to which it is administered to elicit a physiological response from such a subject. The “antigen” as intended herein also encompasses “self-antigens” which do not provoke an immune response in a healthy individual but would do so in a person suffering from auto-immune disease, i.e. the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as “self”, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease.
Accordingly, the “antigen” as intended herein also encompasses a (physiologically active) protein which would not provoke an immune response in a healthy individual but would do so in a person genetically deficient in said protein. In addition, the “antigen” as intended herein also encompasses an allergen which would not provoke an immune response in a healthy individual but would do so in a person suffering from an allergic disease.
An antigen as intended herein may be derived from any polypeptide to which an immune response in a human or animal subject would be therapeutically useful, e.g., from a pathogen, e.g., from a viral, prokaryotic (e.g., bacterial) or eukaryotic pathogen, from a non-physiological protein (e.g., a protein derived from cancer tissue), from allergen (e.g., for eliciting immune tolerance), etc. An antigen could also be a metabolite of a protein. As an example, the antigen could be a polysaccharide, a lipid or other.
An antigen which is able to induce a specific immune response from a subject when said antigen is present in said subject is said to be immunogenic or to be an immunogen. An antigen is preferably a polypeptide or a peptide. A peptide may comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids or more. An antigen may be a protein fragment or a full length protein originating from a disease-causing organism such as a microorganism or virus as identified later herein. It is also possible to use several antigens from the same organism in order to be more effective in combating the organism. An antigen may also be defined by reference to an encoding nucleic acid molecule represented by a nucleic acid sequence.
The source of an antigen may be a protein, a digest of the protein and/or a fragment thereof, which may be in a purified form or may be comprised within a crude composition, preferably of biological origin, such as a bacterial lysate, parasitic lysate, yeast lysate, viral lysate, fungal lysate, sonicate or fixate. Alternatively, an antigen may be chemically synthesized or enzymatically produced in vitro. The source of a protein, or fragment thereof as antigen, may also be a nucleic acid encoding said, or fragment thereof, from an RNA or DNA template. The RNA or DNA molecules may be ‘naked’ DNA, preferably comprised in vesicles or liposomes, or they may be comprised in a nucleic acid construct or a vector. The vector may be any (recombinant) DNA or RNA vector known in the art, and preferably is a plasmid; wherein genes encoding latency antigens are operably linked to regulatory sequences conferring expression and translation of the encoded messengers. The vector may also be any DNA or RNA virus, such as, but not limited to, Adenovirus, Adeno-Associated Virus (AAV), a retrovirus, a lentivirus, modified Vaccinia Ankara virus (MVA) or Fowl Pox virus, or any other viral vector capable of conferring expression of a polypeptide into a chosen subject. DNA vectors may be non-integrating, such as episomally replicating vectors, or may be vectors integrating in the host genome by random integration or by homologous recombination.
DNA molecules comprising genes encoding an antigen protein, or fragments thereof according to the current invention, optionally embedded in a vector such as a virus or plasmid, may be integrated in a genome of a subject. In a preferred embodiment of the invention, such a host may be a micro-organism. Preferably such a recombinant micro-organism is a Mycobacterium, for instance of the species M. tuberculosis, M. smegmatis or M. bovis and most preferably M. bovis Bacillus Calmette Guerin (BCG) or M. smegmatis, capable of delivering to a host the polypeptides or fragments thereof according to the invention (as described in Yue. Y. et al, (2007), J. Virol. Meth., 141: 41-48, Cayabiyab Y. et al, (2006), J. Virol., 80: 1645-1652). Recombinant BCG and methods for recombination are known in the art; for instance, in WO2004094469.
An antigen may originate from any organism known to be associated with a disease or a condition in a subject. An antigen may originate from a microorganism such as a bacterium, a yeast, a fungus, a parasite. Alternatively, an antigen may originate from a virus. An antigen may also be a self or auto antigen as, for example, those associated or linked to allergic diseases.
In an embodiment, a vaccine as used in a combination of the invention comprises an antigen and said antigen is from a virus. Any virus that causes a disease in humans from which antigens are known is encompassed within the scope of the present invention. Such virus include an Adenovirus, a Coxsackievirus, an Epstein Barr virus (EBV), a Hepatitis A, B or C virus, a Herpes simplex virus type I or II or VIII, a cytomegalovirus, a HIV virus, an influenza virus, a measles virus, a mumps virus, a human papilloma virus, a parainfluenza virus, a polio virus, a rabbies virus, a respiratory syncytial virus (RSV), a rubella virus, a Varicella zoster-virus and an Ebola virus.
An adenovirus may cause acute febrile pharyngitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis and/or infantile gastroenteritis.
A coxsackievirus may cause hand, foot and mouth disease, aseptic meningitis, pericarditis and/or myocarditis.
An EBV may cause infectious mononucleosis, Burkitt's lymphoma, Hodgkin's lymphoma and/or nasopharyngeal carcinoma.
A hepatitis A virus may cause an acute hepatitis.
A hepatitis B or C virus may cause acute hepatitis, chronic hepatitis, hepatic cirrhosis and/or hepatocellular carcinoma. Experimental evidence are provided in this application using a combination of the invention comprising a hepatitis B virus.
A Herpes simplex virus type I or II may cause a primary or latent infection. A herpes simplex type II may cause an aseptic meningitis.
A cytomegalovirus may cause an infectious mononucleosis and/or a Cytomegalic inclusion disease.
A HIV virus may cause AIDS.
An influenza virus may cause influenza, a measles virus may cause measles, a mumps virus may cause mumps.
A measles virus may cause measles and/or post-infectious encephalomyelitis.
A mumps virus may cause mumps.
A human papilloma virus may cause hyperplastic epithelial lesions, cervical carcinoma, and/or squamous cell carcinomas.
A parainfluenza virus may cause croup, pneumonia, bronchiolitis and/or common cold
A polio virus may cause poliomyelitis.
A rabies virus may cause rabies.
A RSV may cause bronchiolitis, pneumonia, influenza-like syndrome and/or severe bronchiolitis with pneumonia.
A rubella virus may cause German measles and/or congenital rubella.
A Varicella zoster-virus may cause chickenpox and/or herpes zoster.
An Ebola virus may cause the Ebola virus disease formerly known as Ebola haemorrhagic fever.
Therefore each of the listed virus infections and corresponding viral diseases may be treated using a combination of the invention.
In another embodiment, an antigen may originate from a yeast, a fungus, a bacterium or any other pathological cell. Any yeast or fungus or bacterium that causes a disease in humans from which antigens are known is encompassed within the scope of the present invention. Such bacteria include Mycobacterium tuberculosis, Streptococcus, Pseudomonas, Shigella, Campyobacter, Salmonella.
A Mycobacterium tuberculosis may cause tuberculosis.
A Streptococcus or Pseudomonas bacterium may cause pneumonia.
A Shigella, Campylobacter and Salmonella bacteria may cause a food-borne disease.
In another embodiment, an antigen may be an allergen.
In a preferred embodiment, the combination of the first aspect of the invention comprises a long chain inulin and a vaccine comprising an antigen from a pathogen, more preferably wherein the pathogen is a virus or a bacterium.
More preferably, the pathogen is able to induce a systemic viral or bacterial infection. Preferred bacteria inducing a systemic infection include: Mycobacterium tuberculosis, Streptococcus or Pseudomonas bacterium.
Preferred virus inducing a systemic infection include: Adenovirus, a Coxsackievirus, an Epstein Barr virus (EBV), a Hepatitis A, B or C virus, a Herpes simplex virus type I or II or VIII, a cytomegalovirus, a HIV virus, an influenza virus, a measles virus, a mumps virus, a human papilloma virus, a parainfluenza virus, a polio virus, a rabies virus, a respiratory syncytial virus (RSV), a rubella virus, a Varicella zoster-virus and an Ebola virus.
A most preferred virus is the hepatitis B virus.
In a second aspect of the invention, the oral administration of long chain inulin as defined herein is for influencing the immune response against a pathogen, preferably influencing the immune response against an antigen of a pathogen. In this aspect, a vaccine does not per se need to be administered. A subject may become infected or may become naturally infected by a pathogen. A pathogen and an antigen from a pathogen are as earlier defined herein. In this embodiment, preferred pathogens are those causing common infections like the influenza virus.
In a preferred embodiment of the first aspect, the combination comprises a long chain inulin and a vaccine, wherein the long chain inulin is orally administered to a human, the combination is for influencing the immune response of said human against a pathogen, preferably wherein the pathogen is a virus.
In a preferred embodiment of the second aspect, the long chain inulin is for influencing the immune response of a human against a pathogen, wherein the long chain inulin is orally administered and preferably wherein the pathogen is a virus.
The present invention demonstrates that oral administration of long chain inulin as defined herein (first and second aspects) influences the immune response against an antigen preferably present in a vaccine. In a more preferred embodiment, the immune response is systemic. Within the context of the invention, orally administered long chain inulin is said to influence the immune response elicited against an antigen preferably present in a vaccine when the elicited immune response has been changed, modified compared to the elicited immune response in the absence of the long chain inulin. The change in the immune response may be quantitative and/or qualitative. The change in the immune response is preferably assessed by comparison with the immune response in a control subject. To analyze the antigen-specific elicited immune response, said immune response is compared to the immune response induced in presence of an antigen preferably in the form of a vaccine without orally administered long chain inulin. An alternative or additional control may be a subject treated with an antigen, preferably in the form of the same vaccine and with a short chain inulin as defined herein. The induction is preferably assessed in a subject or in a sample from said subject or in cells from said subject. Preferred sample is blood and preferred cells are PBMCs.
In this context, the antigen-specific elicited immune response is synonymous with the induced immune response against said antigen or the increase in the induction of an immune response against said antigen or a detectable immune response against said antigen. Eliciting an antigen-specific immune response may be replaced with inducing, enhancing, or increasing an immune response against an antigen.
An immune response against said antigen may be a B and/or a T cell response. An immune response may be a B cell response, i.e. production of an antibody specifically directed against said antigen. An immune response may be a T cell response, preferably a Th1 response. The skilled person knows that depending on the disease, a B and/or T cell response may be required to be induced to control it. The production of said antibody could be assessed by ELISA, or by FACS preferably as carried out in the examples. Alternatively said immune response may be detected by measuring the production of cytokines such as, for example, IFNgamma, IL-6, TNFalpha, IL-12 or IL-10. The production of such cytokines could be assessed on PBMC cells of a treated subject by ELISA, PCR, or by a multiplex immunoassay built with luminex xmap technology from Biorad. Preferably the assay used for assessing the production of cytokines is the one used in the examples.
In short, in the context of the invention oral administration of long chain inulin as defined herein influences the immune response against an antigen preferably present in a vaccine in at least one of the following ways:
The effects observed on cytokine production are preferably that:
In the context of the invention, “higher” may be replaced by an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200% or more.
In the context of the invention, “lower” may be replaced by a decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200% or more.
The production of such cytokines may be assessed on a subject, or in a sample from said subject such as cells, preferably PBMC cells. The technique used for the assessment of the production of such cytokines is ELISA or PCR. The detection of the production of such cytokine may be carried out as in the examples. In an embodiment, the production of such cytokines is assessed after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 day of administration of the combination of the invention (i.e. first aspect, vaccine and long chain inulin) or of administration of an antigen and the long chain inulin (second aspect).
The effects observed on B cell response are preferably that:
In the context of the invention, the word “increased” may mean an increase of at least 1%, 2%, 5%, 7%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200% or more.
In the context of the invention, the number of responders is increased of at least 30%, 50%, 75%, 100%, 150%, 200%, 250% or more.
The assessment of antibody titer may be carried out on a subject, or in a sample from said subject such as in peripheral blood plasma samples. The technique used for the assessment of such titer may be an immunoassay. The assessment of such titer may be carried out as in the examples. In an embodiment, the assessment of such antibody titer is carried out after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 day of administration of the combination of the invention (i.e. first aspect, vaccine and long chain inulin) or of administration of an antigen and the long chain inulin (second aspect). The number of responders is defined as the number of subjects exhibiting an antibody titer which is above a predetermined threshold. The skilled person knows that a threshold is defined for each vaccine and corresponding antigen. In the example using a hepatitis B virus vaccine, the threshold was set to 10 IU/ml. Using this threshold, there were two responders using the combination of the invention whereas no responders were detected in the placebo control or in the subjects treated with the same vaccine and a short chain inulin.
The effect observed on T cell response is preferably that:
In the context of the invention, the word “increased” may mean an increase of at least 1%, 2%, 5%, 7%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200% or more.
The assessment of the number of Th1 cells may be carried out on a subject, or in a sample from said subject such as in peripheral blood plasma samples. The technique used for the assessment of such titer may be FACS. The assessment of the number of such cells may be carried out as in the examples using TBET as marker of Th1 cells. In an embodiment, the assessment of the number of such Th1 cells is carried out after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 day of administration of the combination of the invention (i.e. first aspect, vaccine and long chain inulin) or of administration of an antigen and the long chain inulin (second aspect).
The increase of TNFalpha, IL-12 and/or IFNgamma production and the decrease of production of IL-10 and/or IL-6 by the long chain inulin is in line with the Th1 inducing profile or shifted Th1/Th2 balance of long chain inulin.
The preferred mode of administration for the long chain inulin in the first and second aspect of the invention is oral administration.
In an embodiment of the first aspect, the long chain inulin is orally administered before or after the vaccine has been administered. The long chain inulin may have been simultaneously or sequentially orally administered with the vaccine. It does not per se mean that the vaccine and the long chain inulin are formulated together in one composition. In an embodiment, the long chain inulin is orally administered before the vaccine is being administered. In this context, “before” may mean at least 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 day before. In a more preferred embodiment, the long chain inulin is orally administered also after the vaccine has been administered. In this context, “after” means at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 day after. It is therefore encompassed to orally administer the long chain inulin for a period such as 20 days and administer the vaccine once within this period. Depending on the vaccine in question, it may have to be administered once, twice, 3 times, 4 times or more. Accordingly the period of 20 days is not limited and may be extended for at least 10%, 30%, 50% 75%, 100% or more.
In an embodiment of the second aspect, the long chain inulin is orally administered on a daily basis during a given period of time. A period of time may be at least 1, 2, 3, 4, 5, 6 month or longer.
In the context of the invention, the word “composition” is used at least for two features of the invention. It is used in connection with the long chain inulin which may be formulated in a composition (first and second aspects). It is also used in connection with the term vaccine which may also be formulated in a composition (first aspect).
In a preferred embodiment, the long chain inulin (first and second aspects of the invention) used is formulated in a composition. Such composition is preferably suitable for oral administration. This composition is preferably a food composition such as a nutraceutical, a functional food, a food additive. More preferably said food composition is a food supplement, an infant food or follow-on formula or food for elderly people. Alternatively, this composition may be a medical food or a medical composition or considered as a medicament as defined below or later herein.
As used herein, “nutraceuticals” generally encompass foods or food products that provide health and medical benefits. Nutraceuticals are edible and may be eaten directly by humans, but are preferably provided to humans in the form of additives or nutritional supplements, e.g., in the form of tablets or capsules of the kind sold in health food stores, or as ingredients in edible solids, more preferably processed food products such as cereals, breads, tofu, cookies, ice cream, cakes, potato chips, pretzels, cheese, etc., and in drinkable liquids e.g., beverages such as milk, soda, sports drinks, and fruit juices. Especially preferred processes for producing nutraceuticals involve only naturally derived solvents.
Therefore, such a long chain inulin or composition comprising such a long chain inulin (first and second aspects of the invention) is preferably formulated as a food composition such as a nutraceutical, functional food, food supplement, food additive and is more preferably for use as a medicament. This medicament is preferably for preventing, treating, regressing, curing and/or delaying a disease or a condition associated with or caused by a pathogen.
In contrast to nutraceuticals, the so-called “medical foods” are not meant to be used by the general public and are not available in stores or supermarkets. Medical foods are not those foods included within a healthy diet to decrease the risk of disease, such as reduced-fat foods or low-sodium foods, nor are they weight loss products. A physician prescribes a medical food when a patient has special nutrient needs in order to manage a disease or health condition, and the patient is under the physician's ongoing care. The label states that the product is intended to be used to manage a specific medical disorder or condition. A long chain inulin as defined herein may therefore be comprised within a medical food composition.
Functional foods may encompass those foods included within a healthy diet to decrease the risk of disease, such as reduced-fat foods or low-sodium foods, or weight loss products.
It is also encompassed by the present invention that the long chain inulin is formulated in a composition with another carbohydrate. In an embodiment, another carbohydrate is a resistant starch (Bermudez-Brito M, et al, (2015), Mol. Nutr. Food Res., doi 10.1002/mnfr 201500148).
In an embodiment, a long chain inulin or a combination comprising said long chain inulin does not comprise a fucosyllactose. In said embodiment, preferably, a long chain inulin or a combination comprising said long chain inulin does neither comprise a fucosyllactose nor a betagalactooligosaccharide. In an embodiment, a long chain inulin is the sole or the only fructan used in the context of the invention. Preferably in this embodiment of said first and second aspect, there is no oligo fructose, more preferably no Raftilose P95.
In an embodiment, a vaccine or vaccine composition (first aspect) comprises an antigen as defined herein and optionally an adjuvant dissolved in PBS or a suitable buffer. Such composition may further comprise a pharmaceutically acceptable adjuvant and/or carrier. Such a composition is preferably for use as a medicine or as a medicament.
The vaccine or vaccine composition used in a combination of the invention may comprise an adjuvant. Any known adjuvant may be used herein. The skilled person knows several suitable adjuvants. Adjuvants are most preferably selected from the following list of adjuvants: cationic (antimicrobial) peptides, saponine and Toll-like receptor (TLR) ligands such as, but not limited to, poly(I:C), CpG motifs, LPS, lipid A, lipopeptide Pam3Cys and bacterial flagellins or parts thereof, and their derivatives having chemical modifications. Other preferred adjuvants for use in the method and in compositions according to the invention are: mixtures with live or killed BCG, immunoglobulin complexes with the said latency antigens or parts thereof, IC31 (from www.intercell.com; in WO03047602), QS21/MPL (US2003095974), DDA/MPL (WO2005004911), DA/TDB (WO2005004911; Holten-Andersen et al, 2004 Infect Immun. 2004 March; 72(3):1608-17.) and soluble LAG3 (CD223) (from www.Immunotep.com; US2002192195). In addition, another preferred adjuvant includes the use of Corynebacterium paryum or Propionobacterium acnes (Aebischer T., et al, (2000) Infection and Immunity., 68: 1328-1336, Poot J et al, (2009), Vaccine, 27: 4439-4446 and Ferreira J. H. et al, (2008), Vaccine, 26: 67-685).
A composition as defined in the first and second aspects of the present invention may be in the liquid, solid or semi-liquid or semi-solid form. A vaccine composition (first aspect) is preferably liquid. A long chain inulin composition (first or second aspect) is preferably liquid but may be solid or semi-solid.
In a preferred embodiment, other compounds are used sequentially or simultaneously with a combination of the invention (first aspect) or with a long chain inulin (second aspect) in order to improve the specificity of the treatment. It is advantageous for example to use other compounds that will further enhance the immune response of the treated subject. More preferably, such compounds are not present in a single composition together with the long chain inulin (first or second aspect) or together with the vaccine (first aspect).
A vaccine (or a vaccine composition) used in a combination of the invention (first aspect) may function as a therapeutic vaccine. Typically, there is a time period between contact with an antigen, i.e. infection and apparition of the first symptom of a disease associated with said antigen. In this case, a vaccine would act as a pharmacological immune product that would prevent and/or treat the disease and/or delay its progression by eliciting in the host an immune response that counteracts the pathological effect of the disease. A therapeutic vaccine differs from a prophylactic vaccine in that a therapeutic vaccine will induce protection in a subject who already has the infection or the disease. In another embodiment, a vaccine used in a combination of the invention is a prophylactic vaccine. A prophylactic vaccine may be administered to a subject before said subject has been contacted with said antigen.
A vaccine (or a vaccine composition) is preferably a medicament. In one embodiment, the long chain inulin composition used in a combination of the invention may also be considered as a medicament. A medicament as defined herein is preferably administered parenterally, e.g. by injection or infusion by intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial or intralesional route. A preferred administration mode for the vaccine is injection. The invention is not limited to a specific mode of administration of the vaccine as defined herein. A medicament may be combined with a pharmaceutically acceptable medium or delivery vehicle by conventional techniques known in the art. For example, a medicament may be dissolved in Phosphate buffer saline (PBS). Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, Remington's Pharmaceutical Sciences, Ed. A R Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.
Alternatively, a vaccine as defined herein may be locally administered via a catheter or a pump, or a suppository. Alternatively, a vaccine as defined herein may be topically administered. The formulation of a vaccine as defined herein or of a composition comprising said vaccine depends on the intended mode of administration and (therapeutic) application. A pharmaceutical carrier can be any compatible, non toxic substance suitable to deliver said compound to a subject. E.g. sterile water, or inert solids or excipients may be used as the carrier, usually complemented with pharmaceutically acceptable adjuvants, buffering agents, dispersing agents, and the like. Compositions will either be in liquid, e.g. a stabilized suspension of said compound, or a composition comprising said compound, or in solid and/or dry forms: e.g. powder. For oral and rectal administration, said compound can be administered in solid dosage forms, such as capsules, tablets, suppositories, and powders, or in liquid dosage forms, such as elixirs, syrups, creams, ointments, enemas, and suspensions. Another form may be a semi-solid or semi-liquid form wherein said compound is present as a liquid form in, or on, a solid support such as a patch.
In a preferred embodiment of the first aspect, a combination is encompassed wherein the long chain inulin is orally administered and the vaccine is injected.
In a preferred embodiment of the second aspect, the long chain inulin is orally administered.
Accordingly a combination of the invention (first aspect) or a long chain inulin (second aspect) is preferably for use as a medicament. Such a medicament is preferably for preventing, treating, regressing, curing and/or delaying a disease or a condition associated with the antigen preferably present in the vaccine. The antigen is preferably from a pathogen as earlier defined herein.
In a preferred embodiment, such a combination (first aspect, more preferably the long chain inulin present in said combination) or such a long chain inulin (second aspect) influences the immune response against said antigen preferably present in a vaccine. The antigen is preferably from a pathogen as earlier defined herein.
More preferably, said influence is as earlier defined herein: cytokine production, B cell response, monocyte/macrophage response and/or T cell response.
In an embodiment, such influence may increase the ability of the human or animal immune system to fight such a disease or condition as defined herein.
Preferably, a therapeutically effective dose of a combination of the invention (first aspect, more preferably the long chain inulin present in said combination) or such a long chain inulin (second aspect) will prevent and/or delay the development of said disease or condition and/or it is able to elicit the proper immune response, or induce or induce an increase of the proper immune response against a specific antigen preferably present in the vaccine in a treated subject as defined herein. Even more preferably, the elicited or induced immune response is a protective immune response. Depending on the disease or the condition, the skilled person will know which parameter or symptom associated with the development of said disease to choose in order to follow the development of the disease or condition. Preferred parameters have already been defined herein to assess the effect of the long chain inulin preferably used in a combination of the invention on the immune system (i.e. B cell response, T cell response, monocyte/macrophage response and/or cytokine production).
In a more preferred embodiment, at least 5, 10, 15 or 20 micrograms of an antigen as defined herein is being used, preferably in a vaccine. Said vaccine may be administered at least once, twice, three times, four times or more. A vaccine, as defined herein, may be a prophylactic or a therapeutic vaccine. The volume in which an antigen as defined herein may be dissolved may vary from 100-500 microliters.
Accordingly, there is further provided the use of a combination as defined herein (first aspect) and/or corresponding compositions for the manufacture of a medicament for treating a disease or a condition associated with an antigen preferably as present in said vaccine as identified earlier herein.
Accordingly, there is further provided the use of a combination as identified herein (first aspect) and/or corresponding compositions for the manufacture of a medicament being a vaccine for treating a disease or a condition associated with the antigen present in said vaccine.
Accordingly, there is further provided the use of a long chain inulin or a composition comprising said long chain inulin as defined herein (second aspect) for the manufacture of a medicament for treating a disease or a condition associated with an antigen as identified earlier herein.
Each feature of these uses has already been defined herein. The antigen is preferably from a pathogen as earlier defined herein.
In a further aspect, there is provided a method of treatment of a disease or a condition associated with an antigen as present in the vaccine, wherein said treatment comprises a combination (first aspect) and/or corresponding compositions.
Accordingly, there is further provided a method of treatment of a disease or a condition associated with an antigen as present in the vaccine as identified herein, wherein said treatment comprises a combination (first aspect) and/or corresponding compositions as defined herein.
In a further aspect, there is provided a method of treatment of a disease or a condition associated with an antigen, wherein said treatment comprises a long chain inulin or a composition comprising said long chain inulin as defined herein (second aspect). Each feature of these methods has already been defined herein. The antigen is preferably from a pathogen as earlier defined herein.
Polypeptide
“Polypeptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term “polypeptide” encompasses naturally occurring or synthetic molecules. An antigen may be a polypeptide or a peptide or corresponding gene product.
Nucleic Acid Construct, Expression Vector, Operably Linked, Expression, Control Sequences
A nucleic acid construct is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject.
“Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for an antigen as defined herein such that the control sequence directs the production/expression of said antigen in a cell and/or in a subject.
Expression will be understood to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.
Control sequence is defined herein to include all components which are necessary or advantageous for the expression of an antigen. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein
An expression vector may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide in a cell and/or in a subject. As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide −1 of the transcription start site (TSS).
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of” meaning that a product or a composition or a vaccine composition or a long chain inulin or a long chain inulin composition or a combination as defined herein may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention.
In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
The term “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−10% or less, more preferably +/−5% or less, and still more preferably +/−1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.
Within the description, different aspects of the invention are defined in more detail in the form of several embodiments. Each embodiment so defined may be combined with any other embodiment or embodiments unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Inulin from chicory is a polydisperse mixture of linear fructan oligomers and polymers coupled by means of β(2-1) bonds, and mostly with a terminal glucose unit. The number of fructose units in the chain (degree of polymerization, or DP) can vary naturally between 2 and 60. Supplement A (Frutafit®TEX! Sensus, Roosendaal, the Netherlands), is a natural powdered food ingredient based on chicory inulin, and known for its texturizing properties. The DP ranges between mainly between 10 and 60 and average DP of approximately 14-30. Supplement B (Frutafit®CLR Sensus), is a powdered fructo-oligosaccharide (FOS) produced by partial hydrolysis of chicory inulin (Meyer & Blaauwhood, 2009). Frutafit®CLR is a highly soluble food ingredient with a DP ranging mainly between 2 and 35 but average DP of approximately 7-9. Supplement C consists of fructose, which is a powdered carbohydrate, and serves as placebo because it consists of the monomer building blocks of fructans and does not have β(2-1) bonds. The DP profiles of supplement A and B are depicted in
This study was approved by the ethical board of the University Medical Center Groningen, Medisch Ethische Toetsingscommissie University Medical Center Groningen, and documented in the approved application METC_097. It has been registered in the national Dutch trial register, Nederlands Trial Register (NTR41644). Written informed consent was obtained from all participants, and data was analyzed and presented anonymously. Hepatitis B vaccination and blood sampling was conducted within the University Medical Center Groningen, in the Netherlands. All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.
A randomized double-blind placebo controlled human dietary intervention trial was designed to study the effect of inulin-type fructans on vaccination efficacy and peripheral blood lymphocyte populations. To study whether the degree of polymerization (DP) of inulin-type fructans can influence whether different types of immune reactions are stimulated, three groups were included in the study. Healthy volunteers without a history of gastrointestinal symptoms and free of medication, aged 18-29 (17 males, 23 females), were supplemented for 14 days with inulin-type fructans of DP10-60, or with inulin-type fructans of DP2-25, or fructose (Sigma-Aldrich, the Netherlands) as placebo (n=13, 13, and 14 per group respectively, 8 g/d in one dose per day), and vaccinated against hepatitis B (Engerix-B, GlaxoSmithKline Biologicals s.a, Belgium) on day 7. The volunteers consumed their habitual diet and filled out a nutrition diary for the 35 days of the study. Blood samples were collected at day 0, 7, 14, 21, and 35, see
Human PBMCs of 6 healthy volunteers were isolated and cultured as previously described20. 1×106 cells were stimulated overnight with inulin-type fructans of DP10-60 or DP2-25 (100 μg/mL), and with plain culture medium as control. Cytokine profiles in the supernatant were assayed with a human Bio-PlexTM 6-plex premixed cytokine assay (group I, IL-1Ra, IL-1β, IL-6, IL-10, IL-12p70, and TNF-α) according to the manufacturer's instructions (Bio-Rad Laboratories, Veenendaal, The Netherlands), and analyzed as described previously20.
A HEK(293)-Blue reporter cell line with inserted constructs for human TLR2 and secreted embryonic alkaline phosphatase (SEAP), coupled to the NF-κB/AP-1 promotor, was purchased from Invivogen (Toulouse, France), and cultured as previously described20. 2.8×105 cells were stimulated overnight with a concentration series of DP10-60 and DP2-25 (100, 200, 400, 1000 and 2000 μg/mL), with plain culture medium as control, and with heat killed Listeria monocytogenes (1×108 cells/mL, Invivogen). Reporter assay and analysis were performed according to the manufacturer's instructions, as previously described20.
Multi-parameter flow cytometry was performed to measure percentages of different B cell, T cell, NK cell, and NKT cell populations within the total lymphocyte population and subsets within populations. Blood was drawn from the inner cubital vein and collected in 10 mL lithium heparin Vacutainer tubes (BD, Plymouth, UK). All subsequent steps were performed at 4° C. Whole blood (1.5 mL) was centrifuged at 2000 g for 15 min, and plasma supernatant was aliquotted and stored at −20° C. for anti-hepatitis B antibody titer analysis. The remaining whole blood was separated into two 50 mL tubes and erythrocytes were lyzed by incubating twice with 40 mL of ammonium chloride per tube for 10 min. Cell pellet was collected by centrifuging for 5 min at 1800 g. After washing the cell pellet twice with 15 mL FACS buffer (2% fetal bovine serum in phosphate buffered saline, FBS in PBS), cells were counted on a coulter counter (Beckton Dickinson, the Netherlands) and 1×106 cells per well were transferred to a round bottom 96 wells plate. After pelleting the cells for 5 min at 1800 g and discarding the supernatant, cell pellets were resuspended in 50 μl of blocking buffer (20% normal rat serum, Jackson laboratories, in FACS buffer) and incubated for 20 min. Cells were pelleted as described above, resuspended in 50 μl of extracellular antibody mix consisting of extracellular antibodies, 5% normal rat serum, and FACS buffer (antibodies are listed in Table 1-3), and were incubated in the dark for 30 min. After washing the cells which were stained for B cell and NK cell markers, twice with 200 μl FACS buffer per well, cells were incubated with 200 μl of FACS-lysing buffer (Beckton Dickinson BV, Breda, the Netherlands) for 30 min. Cells were then washed three times with FACS buffer, resuspended in 200 μl FACS buffer per well, and stored at 4° C. in the dark until analysis. After incubation with the extracellular antibody mix, the cells stained for T cell markers were washed three times with 200 μl of permeabilization buffer (eBioscience, Vienna, Austria) per well. Cells were resuspended in 50 μl of intracellular blocking buffer (20% normal rat serum in permeabilization buffer) and incubated for 20 min. After pelleting the cells as described above and discarding the supernatant, cells were incubated with 50 μL of intracellular antibody mix, consisting of intracellular antibodies, 5% normal rat serum, and permeabilization buffer (antibodies are listed in Table 2), and incubated for 30 min. Cells were washed three times with permeabilization buffer, resuspended in 200 μl of FACS buffer, and stored at 4° C. in the dark until analysis on an LSR II flow cytometer (Beckton Dickinson BV). The corresponding isotype control antibodies were purchased from the same company as the target antibodies, and isotype stainings were used to set positive gates, using 1% margins. UltraComp eBeads (eBioscience, Vienna, Austria) were applied to set the appropriate compensation values for each antibody panel. FlowJo VX software (FlowJo, Oregon, USA) was used to analyze lymphocyte subsets. Basal sampling at day 0 for every individual allowed repeated measures analysis on the flow cytometry data.
Flow cytometry gating strategies are described in
Anti-HBsAg titers were analyzed at day 0, 14, 21, and 35. Per sample, 500 μL of plasma was aliquotted in Architect tubes (Abbott, Ill., U.S.A.), and analyzed for Anti-HBsAg titers using an Architect Immunoassay Analyzer (Abbott Diagnostics) following the manufacturer's instructions.
GraphPad Prism 5.0 was used for statistical analysis of all data. Flow cytometry data sets were analyzed for normal distribution using a d'Agostino Pearson test. TLR2 activation data was analyzed with a Kruskal-Wallis test and Dunn's post test to determine significant differences compared to control. Data from multiplex cytokine assays was analyzed with a Wilcoxon signed rank test to identify significant differences between fructan treatments. To test for anti-HBsAg antibody titer development per supplement, statistical significance levels were determined with a Friedman test and Dunn's multiple comparison test, comparing T14, T21, and T35 to T0 (basal samples), and Mann Whitney test was used for differences between supplements. Repeated measures ANOVA and Tukey's multiple comparison test or a Friedman test and Dunn's multiple comparison test were used to analyse the flow cytometry data, comparing T14, T21, and T35 to T0 (basal samples). P-values <0.05 were considered statistically significant, and p=0.1 was considered representative for a statistical trend.
Upon 24 h of stimulation of HEK(293)-Blue hTLR2-reporter cells with DP10-60 or DP2-25 inulin-type fructans (
Supplementation with DP10-60 Fructans Increased Anti-HBsAg Antibody Production Compared to Supplementation with DP2-25 Fructans and Placebo
Effects of nutritional supplementation with DP10-60 fructans vs. DP2-25 fructans on vaccination efficacy were studied by analyzing vaccine-induced anti-HBsAg antibodies in peripheral blood plasma samples of supplemented subjects using Architect Immunoassay analysis (
Individuals developing an antibody titer against any vaccination, which is above a predetermined threshold value are indicated as so-called responders, vs. individuals who do not reach titers above this value, which are indicated as non-responders. This threshold value for the anti-HBsAg applied in the present study is generally set at 10 IU/mL29. There we no responders in the placebo groups and the DP2-25 fructan group at T35. This was different in the DP10-60 fructan group in which two responders were present at time point T35.
These results combined, demonstrate that supplementing young adults with a daily single bolus of 8 g DP10-60 fructans in the 14-day period around the first injection enhanced the efficacy of the anti-hepatitis B vaccination.
Peripheral blood lymphocyte subsets of the supplemented individuals were analyzed by multi-parameter flow cytometry to study the effects of different DP fructans. Specifically, we were interested whether changes in T cell, B cell, NK cell, and NKT cell subsets were induced. As B cells are essential in mounting antibody responses against vaccines, we will first describe the results of B cell subsets in peripheral blood of subjects in the experimental groups. The subsets and their corresponding markers of identification are summarized in Table 1, gating strategies are shown in
The percentages of B cells in the total lymphocyte population did not change as compared to basal samples (
Human NK cells (CD56+ CD3−) are part of the first line of defense against viral pathogens and their activation can modulate the outcome of the adaptive immune response30. Similar to NK cells, NKT cells (CD56+ CD3+) are also implicated in the response against hepatitis B vaccine antigens30. The subsets and their corresponding markers of identification are summarized in Table 2, gating strategies are depicted in
Gating strategies are depicted in
Previous studies from others and us have shown that inulin-type fructans can impact immunity by either serving as microbiota accessible fiber17 or by directly binding to immune cells20, but scientific evidence in humans of related immunological benefits were largely lacking. To confirm the direct signaling mechanisms of inulin-type fructans on immune cells and to form an idea of the type of reactions of the immune system which could be expected to occur in vivo, we analyzed the TLR2-activating potential of the fibers and we studied whether DP10-60 induced different cytokines in human PBMCs compared to DP2-25. Both fibers dose-dependently activated TLR2 as expected, and the activation of NF-κB/AP1 through TLR2 appeared to be slightly stronger for DP10-60 fructans. Although the activation of NF-κB/AP1 via TLR2 can give an indication of the expected immune receptor ligation, analysis of the cytokines which are produced upon contact with the fibers by immune cells may give a more detailed view of the type of reactions which can be expected. The cytokine profile induced in PBMCs differed between the two formulations, indicating a more immunostimulating, Th1-inducing profile for DP10-60 fructans as measured by significantly increased TNFα production compared to DP2-25 fructans, together with induction of IL-12. DP2-25 fructans also induced IL-12 production, but this was accompanied by increased production IL-10 as compared to control, which is generally considered an immunosuppressive cytokine, and DP2-25 fructans also increased the production of IL-6 as compared to control and DP10-60 fructans, which has been shown to exert Th1-inhibiting properties31.
Building on the in vitro results in this study, we hypothesized that DP10-60 fructans would stimulate vaccine responses in vivo due to their predominant induction of proinflammatory cytokines in PBMCs, and the relatively low IL-10/IL-12 ratio20, in combination with a strong ability to activate TLR220. We observed that supplementation with DP10-60 fructans significantly enhanced the titer response (T35 of the study) as compared to the DP2-25 fructan supplemented group, and that a strong increased trend was present as compared to the titer development in the placebo group. Another observation that supports the immune stimulating effects was the identification of two responders in the DP10-60 group vs. no responders in either the DP2-25 group or the placebo group. Inulin-type fructans have been studied in several infant vaccination trials, but in the majority the fructans were only studied in combination with GOS and/or pectic oligosaccharides. In one study though, long term supplementation with a mixture of oligofructose/inulin, i.e. short chain inulin-type fructans combined with long chain inulin-type fructans, enhanced the vaccination responses. In this study Saavedra et al.21 observed an increase in blood IgG levels after measles vaccination in a 10 week supplementation study with oligofructose (OF)/inulin (7/3, 0.2 g/kg BW/d) in 7-9 months old infants. In a study by Duggan et al.24 in which 6-12 month old infants were supplemented with OF, i.e. only short chain inulin-type fructans (0.7 g/d), no effect was observed on antibody response after vaccination with H. influenza type B vaccine. Strikingly, other studies in infants with prebiotic mixtures did not induce vaccine potentiating effects22, 32. It should be noted that the applied fructans in these mixtures are often—if not always—of a short chain nature. Dietary fibers can differ substantially in their molecular composition, and even within categories such as inulin-type fructans, different effects on the body and microbiota can be elicited15-19. To study the effect of different DP of fructans on changes in peripheral lymphocytes, we analyzed B cell-, T cell-, NK cell, and NKT cell subsets of supplemented individuals for their percentage of the total lymphocyte population, percentage of relevant subpopulations, and for differences in activation marker expression. Class-switching is one of the hallmarks of activation and maturation of B cells. If the peripheral blood is representative for the immune responses occurring after the vaccination, the increased titer response at T35 would be expected to coincide with a decrease in naïve B cells, and increases in transitional B cells or even plasma cells33. The percentage of transitional B cells which are in the process of maturation (CD38hi IgMhi) was significantly increased in the DP10-60 group and the placebo group for time points T14 and T21 compared to the basal samples but not in the DP2-25 group. Increased percentages of this population indicate that B cells are activated and stimulated to differentiate into antibody producing plasma cells, which is a functional objective of vaccination. The fact that this population was stimulated by DP10-60 fructan supplementation and not by DP2-25 fructan supplementation underscores the effective differences of these two supplements. The induction of memory B cells is important for the ability to mount an efficient secondary immune response and protection against infection upon encountering the relevant antigen33. In the non-class switched memory cells, the percentage of IgM+ cells was slightly increased for the placebo group at T21 compared to basal samples while the fructan groups did not. It is possible that the chosen time points for sampling are too early after vaccination to observe the induction of B memory cells33 and that these cells arise after the 35 day period.
NK and NKT cells did not demonstrate clear supplement dependent effects, suggesting that B cells and especially T cells may be more involved in the boosting of the vaccination response via dietary inulin supplementation, or that B cell and T cell effects may be better detectable in peripheral blood. Supplementation with DP10-60 fructans induced striking differences in T cell populations in time, the increased titer response for the long chain group on T35 was associated with an increased percentage of (TBET+) Th1 cells compared to basal samples. Contrary to Th1 cells, (CD294+) Th2 cells did not change in time for either treatment. These results suggest that in the DP10-60 group, a shifted Th1/Th2 balance may have been induced which was skewed towards Th1 cell responses.
The fact that we observed clear differences for the different DP compounds suggests that DP is an important factor which determines the reaction of immune cells in the body. This could be explained by several mechanisms. Upon consumption, inulin-type fructans of different DP may selectively stimulate different populations of the microbiota34, 35, 36, and these different populations could influence the immune system either in a stimulating or an attenuating manner37. In addition, short chain fibers could be fermented into different products than long chain fibers, thus inducing different SCFA profiles in the intestine, qualitatively and quantitatively17. However, most effects of SCFA on the immune system are attenuating38, contrary to the immune stimulation results observed in the current study. It is more likely that the effects are the result of the sum of indirect and direct effects on immune cells in the intestine17 19 20. It is striking that an oral supplement can stimulate a systemic response to an intramuscular vaccination, and this warrants further studies into the way this process of antigen uptake and presentation can be impacted by orally taken supplements. Generally, the antigens of an intramuscular vaccination such as the applied hepatitis B vaccination, are thought to be detected by circulating dendritic cells, which then recruit other immune cells and migrate towards a draining lymph node, where antigen is presented to B-, and T cells followed by a primary immune response39. Because of the natural surveillance function exerted by DCs, they circulate through the body and mount immune responses against antigens which are encountered. Ligation of innate immune receptors on DCs followed by antigen presentation toward effector cells, such as B cells, T cells, and NK cells locally, or in specialized lymphoid structures could be a mechanism which explains the induction of pro- and anti-inflammatory cytokines as observed in many prebiotic studies. Due to the fact that DCs can ‘sample’ the gut lumen40 and they are migratory cells41, they are one of the candidate cell types to mediate dietary fiber-induced immune effects occurring in the periphery. A model of the impact of inulin-type fructans of different DP is depicted in
In conclusion, this is the first in vivo study which demonstrates a structure-function relation of a dietary fiber on a systemic human immune response. We demonstrate that events in the gut which occur upon supplementing with a dietary fiber, have consequences for systemically induced changes in the immune system. The in vivo immunostimulatory potential of long-chain enriched inulin-type fructans subscribes an important nutritional health claim that these fibers can be beneficial for the immune system. Finally we feel that the use of a low efficacy vaccination model such as Hepatitis B may be more instrumental to demonstrate immunological effects of a nutritional supplement than the very efficient vaccination models21, 22, 32. These human studies can be performed with relatively low numbers of subjects and still with a high statistical power.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15185963.4 | Sep 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/072103 | 9/19/2016 | WO | 00 |
