TECHNICAL FIELD OF THE INVENTION

The present invention relates to the treatment of dyspnea or persistent cough caused or exacerbated by a viral respiratory infection (VRI) in a human subject suffering from asthma, chronic obstructive pulmonary disease (COPD) or post-viral lung complaints, said treatment comprising oral administration of rhamnogalacturonan I (RG-I) polysaccharides, said RG-I polysaccharides having a molecular weight in excess of 10 kDa and having a backbone consisting of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1— >4)-galacturonic-alpha(1— >2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides is within the range of 20: 1 to 1 : 1 .

BACKGROUND OF THE INVENTION

Shortness of breath (SOB), also medically known as dyspnea or dyspnoea, is an uncomfortable feeling of not being able to breathe well enough. The American Thoracic Society defines it as "a subjective experience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity", and recommends evaluating dyspnea by assessing the intensity of its distinct sensations, the degree of distress and discomfort involved, and its burden or impact on the patient's activities of daily living. Distinct sensations include effort/work to breathe, chest tightness or pain, and "air hunger" (the feeling of not enough oxygen).

Dyspnea is a normal symptom of heavy physical exertion but becomes pathological if it occurs in unexpected situations, when resting or during light exertion. In the vast majority of pathological cases, dyspnea is due to asthma, pneumonia, cardiac ischemia, interstitial lung disease, congestive heart failure, chronic obstructive pulmonary disease (COPD), post-viral lung complainsts or psychogenic causes, such as panic disorder and anxiety. The best treatment to relieve or even remove dyspnea typically depends on the underlying cause. Coughing is a common symptom of viral respiratory infections. In healthy individuals, coughing normally goes away shortly after the individual has recovered from the infection. A cough that lasts longer than three weeks after a viral respiratory infection is called a persistent or post- viral cough.

Viral respiratory infections (VRI) are infections by viruses of parts of the body involved in breathing, such as the sinuses, throat, airways or lungs. An infection of this type usually is further classified as an upper respiratory tract infection or a lower respiratory tract infection. Lower respiratory infections, such as pneumonia, tend to be far more severe than upper respiratory infections, such as the common cold.

VRI can be a serious illness for people who are already ill or weakened in some other way. In patients suffering from asthma, COPD or post-viral lung complaints, VRI can cause severe dyspnea. Whereas dyspnea that is linked to asthma typically responds well to medications such as bronchodilators and steroids, these medications are often ineffective in treating dyspnea in asthma patients that is caused or exacerbated by VRI.

Persistent cough caused or exacerbated by VRI is frequently observed in patients suffering from asthma, COPD or post-viral lung complaints. Treatment with inhaled ipratropium or cough-suppressants often provides only limited relief.

Viruses that can cause VRI include rhinoviruses, influenza viruses, adenoviruses, coxsackie virus, parainfluenza virus, respiratory syncytial viruses and human metapneumovirus.

Rhinovirus infections are the foremost trigger of exacerbations of chronic respiratory diseases like asthma and COPD.

Feddema et al. (Prevalence of Viral Respiratory Infections amongst Asthmatics: Results of a Meta-Regression Analysis, Resp Med 2020, 173, 106020, doi:10.1016/j.rmed.2020.106020) quantified the viral prevalence in asthmatics presenting with exacerbations and identified influencing factors. A VRI was detected in 52%-65% of the cases, and the detection rate was higher in children compared to adults. Rhinovirus was most often detected [51-71 %], followed by respiratory syncytial virus [8-18%], influenza virus [7-15%], human parainfluenza virus [4- 11 %] and metapneumovirus virus [3-9%]. According to the authors, prevention and detection of viral respiratory infections in asthmatics could reduce asthma related disease burden and decrease antibiotic misuse. Veerati et al. (Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD, Front Immunol 2020, 11 , 974, doi:10.3389/fimmu.2020.00974) conclude that respiratory viral infections, particularly those caused by rhinovirus, exacerbate chronic respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). The authors developed a low multiplicity of infection rhinovirus model of differentiated primary epithelial cells obtained from healthy, asthma and COPD donors. Using genome-wide gene expression following infection, they demonstrated that gene expression patterns are similar across patient groups, but that the kinetics of induction are delayed in cells obtained from asthma and COPD donors. The authors propose that propensity for viral exacerbations of asthma and COPD relate to delayed (rather than deficient) expression of epithelial cell innate anti-viral immune genes which in turns leads to a delayed and ultimately more inflammatory host immune response.

WO 2019/081523 describes a method of treating infections, said treatment comprising oral administration of carrot RG-I polysaccharides having the following combination features:

• a molecular weight in the range 10-300 kDa;

• a backbone consisting of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1^4)-galacturonic-alpha(1^2)- rhamnose residues;

• the following monosaccharide composition:

20-60 mol.% galacturonic acid residues, wherein the individual galacturonic acids can be methylated at the C-6 position and/or acetylated at the 0-2 and/or the 0-3 position; 8-50 mol.% rhamnose residues;

0-40 mol.% arabinose residues;

0-45 mol.% galactose residues; molar ratio of galacturonic acid residues to rhamnose residues in the range of 5:1 to 1 :1 ; galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 85 mol.% of the monosaccharide residues in the RG-I polysaccharides.

Lutter et al. (The Dietary Intake of Carrot-Derived Rhamnogalacturonan-I Accelerates and Augments the Innate Immune and Anti-Viral Interferon Response to Rhinovirus Infection and Reduces Duration and Severity of Symptoms in Humans in a Randomized Trial, Nutrients 2021 , 13, 4395, doi:10.3390/nu13124395) investigated the effects of dietary supplementation with carrot-derived RG-I (cRG-l, 0-0.3-1.5 g/day) in 177 healthy individuals (18-65 years) on symptoms following infection with rhinovirus strain 16 (RV16).

Primary outcomes were changes in severity and duration of symptoms, and viral load in nasal lavage. Secondary outcomes were changes in innate immune and anti-viral responses, reflected by interferon response gene expression, CXCL-10 and CXCL-8 levels and cell differentials in nasal lavage. Anti-viral responses, viral clearance and symptom scores at 1.5 g/d were in between those of 0 and 0.3 g/d, suggesting a negative feedback loop preventing excessive interferon responses. Dietary intake of cRG-l accelerated innate immune and antiviral responses, and reduced symptoms of an acute respiratory viral infection.

Nosal’ova et al. (Suppressive effect of pectic polysaccharides from Cucurbita pepo L var.

Styriaca on citric acid-induced cough reflex in guinea pig, Fitoterapia 82 (2011} 357-36) describe a study in which several water-soluble pectic polysaccharides were isolated from pumpkin fruit biomass. The pectic polysaccharides were tested for antitussive activity by studying the effects of citric add-induced cough reflex in guinea pigs and reactivily of the airway smooth muscle in vivo conditions in comparison to the narcotic drug codeine. Oral administration of all pectic polysaccharides from pumpkin inhibited the number of coughs induced by citric add in guinea pigs, but to various extents.

SUMMARY OF THE INVENTION

The inventors have unexpectedly discovered that in patients suffering from asthma, COPD or post-viral lung disease, dyspnea or persistent cough caused or exacerbated by viral respiratory infection can be treated effectively by orally administering a composition containing at least 0.1% by weight of dry matter of rhamnogalacturonan I (RG-I) polysaccharides having a molecular weight in excess of 10 kDa and having a backbone consisting of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1^4)- galacturonic-alpha(1^2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides is within the range of 20:1 to 1 :1.

The present treatment can prevent the development of dyspnea, especially servere dyspnea, and/or shorten the duration of dyspnea in patients suffering from asthma, COPD or post-viral lung disease, following a viral infection of the respiratory tract. The present treatment can also prevent the development or shorten the duration of persistent cough in patients suffering from asthma, COPD or post-viral lung disease, following a viral infection of the respiratory tract. DETAILED DESCRIPTION OF THE INVENTION

Accordingly, a first aspect of the invention relates to a composition for use in the treatment of dyspnea or persistent coughing caused or exacerbated by a viral respiratory infection in a human subject suffering from asthma, chronic obstructive pulmonary disease (COPD) or post- viral lung complaints, said use comprising oral administration of the composition to the subject, wherein the composition contains at least 0.1 % by weight of dry matter of rhamnogalacturonan I (RG-I) polysaccharides having a molecular weight in excess of 10 kDa and having a backbone consisting of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1— >4)-galacturonic-alpha(1— >2)-rhamnose residues, wherein the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides is within the range of 20:1 to 1 :1.

Unless indicated otherwise, the term “or” aso encompasses “and”. In other words, the phrase “composition comprising A or B” encompasses compositions containing A and B.

The term “treatment” as used herein encompasses both therapeutic and prophylactic treatment.

The RG-I polysaccharides of the present invention are a species of the genus pectic polysaccharides (or pectin). Pectin is a structural hetero polysaccharide that is present in the primary cell walls of terrestrial plants.

Pectic polysaccharides are a heterogeneous group of polysaccharides comprising varying amounts of the following polysaccharide domains:

(i) homogalacturonan (HG),

(ii) xylogalacturonan (XG),

(iii) apiogalacturonan (AG)

(iv) rhamnogalacturonan-l (RG-I), and

(v) rhamnogalacturonan-l I (RG-I I).

Figure 1 provides a schematic representation of the structure of pectic polysaccharides, including the aforementioned 5 polysaccharide domains. It is noted that the polysaccharide domains AG, XG and RG-I I typically represent only a very minor fraction of pectic polysaccharides. The polysaccharide domains HG, AG, XG and RG-I I each comprise a backbone that consists of a linear chain of a-(1-4)-linked D-galacturonic acid monosaccharide units (GalA).

Only RG-I comprises a backbone that consists of a linear chain of the repeating disaccharide units: 4)-a-D-galacturonic acid-(1 ,2)-a-L-rhamnose-(1. A schematic representation of the structure of RG-I is shown in Figure 2.

Pectic polysaccharide composition and fine structure vary depending on the plant source and the extraction conditions applied. The homogalacturonan domain can have a length of up to about 100 consecutive GalA residues. The RG-I domain containing the side chains is usually called the ‘ramified region’ or ‘hairy region’, while the homogalacturonan domain (connected to RG-I domains) is not typically substituted with glycosides or glycosidic side chains.

The GalA residues in RG-I are linked to the rhamnose (Rha) residues via the 1 and 4 positions, while the Rha residue is linked to the GalA residue via the anomeric and 2-OH positions. In general about 20-80% of the Rha residues is branched at the 4-OH position (depending on the plant source and the method of isolation), with neutral and acidic side chains. These side chains consist of Ara and Gal residues linked in various manners, constituting polymers known as arabinans, arabinogalactan I (AG-I) or AG-II. AG I is composed of a beta-(1 ,4)-linked D- Gal backbone with substitutions at 3-OH of alpha-L-arabinosyl groups; the Gal backbone can have interspacing alpha(1 ,5)-L-Ara units. AG-II consists of highly ramified galactan with predominantly interior beta(1 ,3)-linked D-Gal with substitutions of short (1 ,6)-linked chains exteriorly. The latter has further attachments of (1 ,3)- and/or alpha(1 ,5)-linked L-Ara. The oligosaccharide side chains may be linear or branched.

The term “branched polysaccharide” as used herein refers to a polysaccharide comprising a linear backbone chain of monosaccharide units bound together by glycosidic linkages, wherein at least one of the monosaccharide units within the backbone chain carries a sidechain of one or more glycosidically linked monosaccharide units.

The terms “backbone chain” and “backbone” are synonyms.

The term “pectic polysaccharide” as used herein refers to optionally branched polysaccharides having a molecular weight larger than 10 kDa and comprising a backbone that consists of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1— >4)-galacturonic-alpha(1— >2)-rhamnose residues. The term “stretch” as used herein refers to a sequence of two or more glycosidically linked monosaccharide units within the backbone of a polysaccharide, excluding any sidechains that are attached thereto.

The term “domain” as used herein refers to a stretch plus any sidechains that are attached to said stretch.

The term “rhamnogalacturonan-l stretch” or “RG-I stretch” refers to a stretch consisting of galacturonic acid (GalA) and rhamnose (Rha) pairs, wherein the GalA residues are linked to the Rha residues via the 1 and 4 positions, while the Rha residues are linked to the GalA residue via the anomeric and 2-OH positions, i.e. alternating alpha(1^4)-galacturonic- alpha(1^2)-rhamnose residues. The RG-I domain can comprise side chains such as, for example galactan, arabinan and arabinogalactan side chains.

The term “rhamnogalacturonan-l polysaccharide” or “RG-I polysaccharide” refers to optionally branched pectic polysaccharides that comprise a backbone that contains one or more rhamnogalacturonan-l stretches. The backbone of RG-I polysaccharide can comprise one or more side chains. These sidechains consist of residues of arabinose and/or galactose.

The term “alpha(l,4)-linked galacturonic acid stretch” refers to a stretch consisting of alpha(1 -^4)-galacturonic residues.

The HG domains, XG domains, AG and RG-I I domains that are optionally present in the RG- I polysaccharides of the present invention comprise a backbone that consists of a linear chain of two or more a-(1-4)-linked D-galacturonic acids.

HG domains do not contain any sidechains. The carboxyl groups of galacturonic acid residues within the backbone of HG domains may be esterified. Esterified galacturonic acid may occur in the form of the methyl ester or acetyl ester.

The backbone of XG domains contains one or more sidechains in the form of D-xylose.

The backbone of AG domains contains one or more sidechains that are composed of one or more D-apiose residues.

The backbone of RG-I I contains one or more side chains that are not exclusively composed of D-xylose or D-apiose. The carboxyl groups of galacturonic acid residues within the backbone of RG-II domains may be esterified. Galacturonic acid may be esterified either by methyl or acetyl groups, forming methyl or acetyl esters, respectively.

The terminology “degree of acetylation” refers to the number of acetyl residues per galacturonic acid residue, expressed as a percentage.

The terminology “degree of methylation” refers to the number of methyl residues per galacturonic acid residue, expressed as a percentage.

The concentration of different polysaccharides and their monosaccharide composition can be determined by analytical techniques known to the skilled person. After acid hydrolysis (methanolysis), the monosaccharide composition of neutral sugars, can suitably be determined by High Performance Anion Exchange Chromatography combined with Pulse Amperometric Detection (HPAEC-PAD). llronic acids (Galacturonic acid being the dominant form of uronic acids) can be determined using the colorimetric m-hydroxydiphenyl assay.

The molecular size distribution can be determined by High Performance Size-Exclusion Chromatography (HPSEC) using refractive index (Rl) detection (concentration).

The above mentioned analytical methods are described in: Analytical Biochemistry Vol. 207, Issue 1 , 1992, pg 176 (for neutral sugar analysis) and in Mol. Nutr. Food Res., Vol 61 , Issue 1 , 2017, 1600243 (for the Uronic acid analysis and the molecular size distribution).

All percentages mentioned herein, unless otherwise stated, refer to the percentage by weight.

Oral administration within the context of the present treatment encompasses selfadministration.

The present treatment is particularly effective in treating dyspnea or persistent cough in human subjects whose immune system’s capability of neutralizing the VRI is seriously impaired, as is often the case in subjects suffering from asthma, COPD or post-viral lung complaints. According to a particularly preferred embodiment, the human subject that is treated in accordance with the present invention is a non-resolver suffering from persistence of the VRI for 8 days or more, more particularly for 10 days or more even more particularly for 13 days or more. Here, persistence of the VRI means that the viral load in the respiratory tract exceeds 20%, more preferably exceeds 40%, of the maximum viral load during the preceding days, following infection.

The treatment according to the present invention preferably comprises oral administration of 0.1-3 grams of the RG-I polysaccharides per day during a period of at least 3 days, more preferably during a period of at least 6 days, even more preferably during a period of at least 8 days, even more preferably during a period of at least 10 days, yet more preferably during a period of at least 20 days and most preferably during a period of at least 30 days. According to a particularly preferred embodiment, the treatment comprises oral administration of the RG- I polysaccharides during the aforementioned periods in a daily amount of 0.15 to 1.5 grams, most prefefably of 0.2 to 0.6 grams.

The present treatment preferably compises at least daily oral administration, most preferably once daily oral administration of the composition containing the RG-I polysaccharides.

Preferably, the treatment comprises at least once daily oral administration of the composition during at least 10 days, more preferably during at least 20 days, most preferably during at least 30 days.

Preferably, the treatment of the present invention comprises oral administration of the composition to the subject after the subject has started experiencing symptoms of the viral respiratory infection. Even more preferably, the treatment comprises oral administration of the composition before and after the subject has started experiencing symptoms of the viral respiratory infection.

The present treatment is particularly effective if the VRI is an infection with a virus selected from rhinoviruses, influenza viruses, adenoviruses, corona viruses, coxsackie virus, parainfluenza virus, respiratory syncytial viruses and human metapneumovirus. According to a particularly preferred embodiment, the VRI causing or excarbating dyspnea is an infection with a rhinovirus.

The present treatment preferably is a therapeutic or prophylactic treatment of a viral infection of the upper respiratory tract.

According to a preferred embodiment, the present invention relates to treatment of dyspnea caused or exacerbated by a viral respiratory infection in a human subject suffering from asthma. According to another preferred embodiment, the present invention relates to treatment of persistent cough caused or exacerbated by a viral respiratory infection in a human subject suffering from asthma.

The composition that is used in the present treatment preferably is a nutritional formulation, a food product, a dietary supplement or a beverage.

The composition preferably contains at least 0.2% by weight of dry matter, more preferably 0.3 -10% by weight of dry matter and most preferably 0.4 -5% by weight of dry matter of the RG-I polysaccharides.

The RG-I polysaccharides that are employed in the present treatment may be obtained from different crops. In a preferred embodiment, the RG-I polysaccharides are obtained from one or more crops selected from fruit (including tomato), carrot, olive, peas, sugar beet, red beet, chicory, okra, soy, sunflower, rapeseed and maize. More preferably, the RG-I polysaccharides are obtained from one or more crops selected from apple, pear, citrus, carrot, sugar beet and chicory. Yet more preferably, the RG-I polysaccharides are obtained from one or more crops selected from apple, pear, carrot and chicory. Most preferably, the RG-I polysaccharides are obtained from carrot, apple and/or chicory.

The RG-I polysaccharides are preferably incorporated in the composition of the present invention in the form of a pectic polysaccharide isolate that is enriched in RG-I polysaccharides. Accordingly, in a particularly preferred embodiment, the RG-I polysaccharides represent at least 10 wt.%, more preferably at least 20 wt.%, even more preferably at least 30 wt.%, yet more preferably at least 60 wt.%, and most preferably at least 80 wt.% of the pectic polysaccharides present in the composition, said pectic polysaccharides being defined as optionally branched polysaccharides having a molecular weight in excess of 10 kDa and comprising a backbone that consists of galacturonic acid residues or a combination of galacturonic acid residues and rhamnose residues, said rhamnose residues being contained in alpha(1— >4)-galacturonic-alpha(1— >2)-rhamnose residues.

The RG-I polysaccharides that are employed in accordance with the present invention have a backbone that comprises rhamnogalacturonan-l stretches and optionally alpha(1 ,4)-linked homo-galacturonic acid stretches. The molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides preferably is within the range of 5:1 to 1 :1. More preferably, the molar ratio of galacturonic acid residues to rhamnose residues in the RG-I polysaccharides ranges from 4.8:1 to 1 :1 , even more preferably from 4.5:1 to 1 :1 , yet more preferably from 4.2: 1 to 1 : 1 , most preferably from 4: 1 to 1 .1 : 1.

Rhamnose residues typically represent 9-45%, more preferably 10-40% and most preferably 11-35% of all the monosaccharide residues contained in the RG-I polysaccharides, i.e. including the monosaccharide residues that are contained in sidechains.

Galacturonic acid residues typically represent 21-55%, more preferably 22-50% and most preferably 23-45% of all the monosaccharide residues contained in the RG-I polysaccharides, i.e. including the monosaccharide residues that are contained in sidechains.

Arabinose residues typically represent 4-38%, more preferably 6-36% and most preferably 8- 34% of all the monosaccharide residues contained in the RG-I polysaccharides.

Galactose residues typically represent 4-42%, more preferably 8-40% and most preferably 10- 38% of all the monosaccharide residues contained in the RG-I polysaccharides.

The combination of galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together preferably constitutes at least 88 mol.%, more preferably at least 90 mol.% and most preferably at least 92 mol.% of the monosaccharide residues in the RG-I polysaccharides.

The RG-I polysaccharides typically have a molecular weight of at least 15 kDa. More preferably, the RG-I polysaccharides have a molecular weight between 20 kDa and 300 kDa, most preferably between 40 kDa and 300 kDa.

The RG-I polysaccharides of the present invention can suitably be produced by enzymatic hydrolysis of pectic polysaccharides. Preferably, such enzymatic hydrolysis yields RG-I polysaccharides having a degree of acetylation of at least 20%, more preferably of 30-110%, even preferably of 35-90% and most preferably of 40-70%.

The RG-I polysaccharides preferably have a degree of methylation of not more than 50%, more preferably of not more than 40% and most preferably of 10-30%.

According to another preferred embodiment, the ratio of the degree of acetylation (DA) of the RG-I polysaccharides to the degree of methylation (DM) of the RG-I polysaccharides preferably is 1 or more, more preferably 2 or more, more preferably 3 or more, and most preferably 5 or more.

In accordance with a preferred embodiment, the RG-I polysaccharides of the present invention have been obtained by hydrolysis. Whereas pectic polysaccharides normally do not contain unsaturated galacturonic acid residues. Hydrolysis of pectic polysaccharides by pectin lyase and/or pectate lyase, inevitably yields polysaccharide fragments that contain a terminal unsaturated non-reducing galacturonic acid residue. Preferably, at least 10%, more preferably at least 25% and most preferably at least 50% of the terminal non-reducing galacuturonic acid residues in the RG-I polysaccharides are unsaturated galacturonic acid residues. Unsaturated galacturonic acids can easily be identified, e.g. by measuring UV absorption at 235 nm.

The arabinan side chain comprises at least one or more alpha(1 ,5)-linked arabinose residues and is substituted to the 4-OH position of a rhamnose residues in the RG-I domain. The arabinan side chain may be linear or branched. In case the side chain is linear, the side chain consists of alpha(1 ,5)-li nked arabinose residues. In case the arabinan side chain is a branched side chain, one or more alpha-arabinose residues are linked to the 0-2 and/or 0-3 of alpha(1 ,5)-linked arabinoses.

The galactan side chain comprises at least one or more beta(1 ,4)-linked galactose residues and is substituted at the 0-4 position of a rhamnose residues in the RG-I domain.

The arabinogalactan side chain is substituted at the 0-4 position of a rhamnose residue in the RG-I domain and can be a type I arabinogalactan (AGI) or a type II arabinogalactan (AGII). AGI is composed of a (1— >4)-p-D-Galp backbone on which substitutions by monomeric Galp units at the 0-6 or at the 0-3 position can occur. AGI is further substituted with a-L-Araf-p residues and/or with (1->5)-a-L-Araf short side chains. AGII is composed of a(1— >3)-p-D-Galp backbone decorated with (1— >6)-p-D-Galp secondary chains, which are arabinosylated.

Preferably, arabinose residues and rhamnose residues are present in the RG-I polysaccharides in a molar ratio of less than 4:1 , more preferably of less than 3:1 , most preferably of less than 2:1.

Galactose residues and rhamnose residues are preferably present in the RG-I polysaccharides in a molar ratio of less than 4:1 , more preferably of less than 3.2 :1 , most preferably of less than 2.5:1. The molar ratio of the combination of arabinose residues and galactose residues to rhamnose residues in the RG-I polysaccharides preferably is less than 7:1 , more preferably less than 5:1 and most preferably less than 4:1.

The combination of galacturonic acid residues and rhamnose residues preferably constitutes at least 30 mol.%, more preferably 35-90 mol.% and most preferably 40-75 mol.% of the monosaccharide residues contained in the RG-I polysaccharides.

The RG-I polysaccharides that are employed in te present treatment preferably have the following monosaccharide composition:

• 20-60 mol.% galacturonic acid residues, wherein the individual galacturonic acids can be methylated and/or acetyl-esterified;

• 8-50 mol.% rhamnose residues;

• 0-40 mol.% arabinose residues;

• 0-40 mol.% galactose residues;

• molar ratio of galacturonic acid residues to rhamnose residues in the range of 5: 1 to 1 : 1 ;

• galacturonic acid residues, rhamnose residues, arabinose residues and galactose residues together constitute at least 85 mol.% of the monosaccharide residues in the RG- I polysaccharides.

According to a particularly preferred embodiment, the RG-I polysaccharides have the following monosaccharide composition:

• 21-55 mol.% galacturonic acid residues, wherein the individual galacturonic acids can be methylated and/or acetyl-esterified;

• 9-35 mol.% rhamnose residues;

• 5-35 mol.% arabinose residues;

• 5-40 mol.% galactose residues.

The RG-I polysaccharides in the product of the present invention are preferably obtained by partial enzymatic hydrolysis of pectin. According to a particularly preferred embodiment, the RG-I polysaccharides are obtained by enzymatic hydrolysis of pectin using one or more pectinases selected from pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), endopolygalacturonase (EC 3.2.1.15), exopolygalacturonase (EC 3.2.1.67 and EC 3.2.1.82). Most preferably, the RG-I polysaccharides are obtained by enzymatic hydrolysis of pectin using one or more pectinases selected from pectin lyase (EC 4.2.2.10) and endo-polygalacturonase (EC 3.2.1.15). The product of the present invention preferably contains traces of one or more of the aforementioned pectinases. These pectinases may be present in the product in active and/or inactive form.

According to one preferred embodiment, the RG-I polysaccharides are obtained by enzymatic hydrolysis of pectin using endopolygalacturonase and/or exopolygalacturonase in combination with pectinesterase (EC 3.1.1.11).

According to another preferred embodiment, the RG-I polysaccharides are obtained by enzymatic hydrolysis of pectin using pectin lyase and/or pectate lyase.

In one advantageous embodiment of the treatment according to the present invention, the composition is a solid dosage unit having a weight of 200-3,000 mg and containing 0.1-1.5 g of the RG-I polysaccharides.

According to a particularly preferred embodiment, the pectic polysaccharides in the present composition (including the RG-I polysaccharides) have the following monosaccharide composition:

20-60 mol.% galacturonic acid residues, wherein the individual galacturonic acids can be methylated and/or acetyl-esterified;

8-50 mol.% rhamnose residues;

0-40 mol.% arabinose residues;

0-40 mol.% galactose residues; and wherein:

2x[Rha] + [Ara] + [Gal] > 50 mol.%;

[GalA] - [Rha] < 50 mol.%;

[Rha], [Ara], [Gal] and [GalA] representing the molar concentration in mol.% of rhamnose, arabinose, galactose and galacturonic acid, respectively. More preferably, the monosaccharide composition meets the following conditions:

2x[Rha] + [Ara] + [Gal] > 60 mol.%;

[GalA] - [Rha] < 30 mol.%.

In another advantageous embodiment of the present treatment, the composition is a packaged aqueous liquid having a packaged volume of 10-250 mL and containing 0.1-1.5 g of the RG-I polysaccharides. The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

Study design cRG-l, a natural extract from carrot (Daucus carota subsp. sativus), was supplied by Nutrileads (Wageningen, The Netherlands). cRG-l is a water soluble non-digestible fermentable fiber, enriched in the RG-I domain of pectin. The carbohydrate content of the extract is around 73 wt.%. The extraction method and extract characteristics (composition and structure) have been described McKay et al. (Development of an Affordable, Sustainable and Efficacious Plant-Based Immunomodulatory Food Ingredient Based on Bell Pepper or Carrot RG-I Pectic Polysaccharides, Nutrients 2021 , 13, 963).

The monosaccharide composition of cRG-l was (% mol/mol): 14.3 rhamnose; 34.8 arabinose; 19.6 galactose; 0.8 fucose; 4.3 glucose; 0.9 mannose; 0.7 xylose; 25.0 galacturonic acid.

Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x14.3 + 34.8+ 19.6 = 83%

• % HG = [GalA] - [Rha] = 25.0-14.3 = 10.7%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x14.3 I (14.3+25.0) = 72.8%

• % HG = 100% - % RG-1 = 27.2%

Test articles were prepared by mixing with maltodextrin and caramel color to obtain identical powders as follows: 0, 0.3 and 1.5 g cRG-l extract, 3, 2.7 and 1.5 g maltodextrin (MALDEX 170, Tereos, Belgium) and, for each dose, 0.5 g caramel color type 1 (Natural spices, Mijdrecht, the Netherlands) to obtain sachets with 3.5 g powdered supplement of identical volume and appearance for the no, low- and high-dose groups, respectively. A single center, randomized, double-blind, placebo-controlled dose response study was conducted that had three arms with 0, 0.3 g/day and 1.5 g/d cRG-l in a parallel design. Details on design, adverse events, safety monitoring, disposition of subjects and primary outcome have been described by Lutter et al. (The Dietary Intake of Carrot-Derived Rhamnogalacturonan-I Accelerates and Augments the Innate Immune and Anti-Viral Interferon Response to Rhinovirus Infection and Reduces Duration and Severity of Symptoms in Humans in a Randomized Trial, Nutrients 2021 , 13, 4395, doi:10.3390/nu13124395).

The study period comprised four periods: enrolment (screening, eligibility), dietary supplementation for 8 weeks, response to Rhinovirus16 (RV16) infection (exposure on day 0, dO) with continued supplementation for 2 weeks, and 3-week follow-up without supplementation as shown in Figure 3

An online Jackson questionnaire from d-7 till d-1 was used to determine subject eligibility for infection with RV16, and a throat swab on d-1 was used to detect natural viral infections by polymerase chain reaction (PCR). Only subjects not suffering from a common cold (assessed by the Jackson questionnaire) or fever within 7 days prior to the viral challenge were eligible for RV16 inoculation. The presence of a cold was defined as two of the following three criteria being present (i) a cumulative Jackson symptom score of at least 14 over a 6-day period, (ii) the subjective impression of a cold by the volunteer and (iii) rhinorrhea on at least 3 days.

Eligible subjects who were symptom-free and PCR-negative, completed the Wisconsin Upper Respiratory Symptom Score-21 (WURSS-21) questionnaire, online, the day before infection (d-1) and then every day until d13, in the morning. The WURSS-21 questionnaire provides a comprehensive set of validated questions that is divided into 3 sections: (A) the total score, and “how do you feel today?” (item 1), (B) upper respiratory tract infection (URTI) symptoms (items 2-11) to rate the severity of cold symptoms over the last 24h, and (C) quality-of-life (items 12-20) to assess the impact of a common cold on daily activities. In addition, item 21 “compared to yesterday, I feel that my cold is:” provides a relative indication of symptom severity.

Sampling was performed as depicted in Figure 3. In a nested subset (16 participants selected randomly per group), nasal brushes were performed to collect nasal epithelial samples for transcriptome analyses at the same timepoints as for nasal lavage .

The study physician assessed participants’ health based on medical history and use of medication, and participants were between 18 and 65 years of age with a BMI between 18.5 and 30.0 kg/m ² . Volunteers with an RV16 antibody titer >1 :6 at screening, with a medical history of hay fever, rhinosinusitis, asthma, COPD, other underlying pulmonary, cardiovascular, or auto-immune disease, or food allergy were excluded. A complete overview of inclusion and exclusion criteria, the minimal dietary restrictions and other relevant criteria have been reported in the earlier cied paper by Lutter et al.

Participants were instructed to take the dietary supplement, supplied as a powder in a sachet, once a day with food and drink items of choice during the first meal (preferably breakfast).

Participants were challenged by instillation of 100 tissue-culture infectious doses (TCID50) RV16 (GMP-prepared RV16: UBiopred Ell/IMI) in the nasal cavity (see Ravi et al., Rhinovirus- 16 Induced Temporal Interferon Responses in Nasal Epithelium Links with Viral Clearance and Symptoms, Clin Exp Allergy 2019, 49, 1587-1597, doi: 10.1111/cea.13481).

Analyses

Cells were separated from the nasal lavage fluid by centrifugation (10 mins at 465g at room temperature (RT)) then processed for cell differentiation using a cytospin. The supernatant was used to determine RV16 viral load by PCR and soluble mediators using multiplex by Luminex. Sequential samples were analysed batch-wise to limit variability and internal controls were used to verify consistency.

Results

One hundred and seventy-seven healthy adults (18-65 years) met the inclusion and exclusion criteria and were randomly assigned to receive 0, 0.3 or 1 .5 g/d cRG-l as powdered dietary supplement. Subjects consumed one sachet of the dietary supplement daily with their breakfast for 8 weeks prior to infection and for a further 2 weeks during the response phase of the trial.

Thirty-one people (14, 9 and 8 in the 0, 0.3 and 1.5 g/d groups respectively) dropped out of the study around the time of the scheduled infection with RV16 due to either common cold symptoms (natural infections), scheduling issues and personal reasons or health issues (not related to the dietary supplement).

A total of 146 subjects (ITT population) was intranasally exposed to an experimental infection with 100 tissue-culture infectious doses RV16. Of the infected participants, 48 were randomly selected to participate in the nested sub-study which involved additional measurements (subset). Disposition of subjects, demographic characteristics of the different (sub)populations, compliance, adverse events and safety as well as initial outcomes are detailed in the earlier cited paper by Lutter et al.

Average viral load was measured by PCR in nasal lavage samples before and after a challenge with rhinovirus. The RV16 viral load data in nasal lavage obtained from the study revealed that the participants could be clustered in three main groups:

• Early resolvers (viral load reduced to 10 ² (limit of detection) in 6 days or less)

• Late resolvers (viral load reduced to 10 ² between days 6 and 13)

• Non-resolvers (viral load still above 10 ² after 13 days)

The development of the viral load in nasal lavage from day 1 till day 13 for each group is shown in Figure 4.

The distribution of the particpants across the three groups is shown in Table 1.

Table 1

Figure 5a shows, for each of the three aforementioned groups, the effect of cRG-l treatment on viral load by RT-PCR. From this data it can be concluded that the effect of treatment on viral load was not statistically signifant.

Figure 5B shows, for each of the three groups, the effect of cRG-l treatment on WURSS-21 symptom score. It is clear from this data that the treatment had a pronounced favourable impact on the WURSS-21 symptom scores for the non-resolver group.

Figure 50 shows, for each of the three groups, the effect of cRG-l treatment on WURSS-21 Quality of Life score. This data shows that the treatment also had a pronounced favourable impact on the WURSS-21 Quality of Life score for the non-resolver group.

Figure 6A-6C show the effect of cRG- treatment on upper respiratory symptoms (Figure 6A), lower respiratory symptoms (Figure 6B) and coughing (Figure 6C) for the non-resolver group. Upper respiratory symptoms include: runny nose, plugged nose, sore throat, scratchy throat and head congestion. Lower respiratory symptoms: cough, hoarseness, chest congestion and feeling tired. The data of Figures 6A-6C show the beneficial effect of the treatment on upper and lower respiratory symptoms and coughing, for the non-resolver group.

Some subjects had a WURSS-21 symptom score greater than 0 on the day prior to infection, even though only symptom-free (Jackson score), PCR-negative subjects were exposed to RV16. To investigate this further, a post hoc analysis of subjects with a WURSS-21 symptom score of 0 on the day before infection was performed, a pharmacokinetic model was used to determine the symptom elimination half-life as described in the earlier cited paper by Lutter et al. In this subgroup of 87 subjects the beneficial effect of cRG-l is even more pronounced than in the full ITT population. Symptom severity (peak symptom score) was reduced by 33% and symptom duration was reduced by 43% in the 0.3 g/d group compared to the 0 g/d group.

Rhinovirus infections are the foremost trigger of exacerbations of chronic respiratory disease like asthma and COPD. Veerati et al. (Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD, Front Immunol 2020, 11 , 974, doi:10.3389/fimmu.2020.00974) have shown that the tendency for viral exacerbations of asthma and COPD relate to delayed (rather than deficient) expression of epithelial cell innate anti-viral immune genes. Thus, patients suffering from asthma or COPD are likely to be “non resolvers”.

In the absence of approved drugs to prevent RV infections in patients with chronic respiratory diseases, the prophylactic dietary intake of cRG-l can offer considerable benefits, notably by suppressing the debilitating symptoms, such as dyspnea.

Example 2

A RG-I polysaccharide fraction that can suitably be employed in the present treatment was isolated from apple pomace.

2 kg of dried apple pomace (protein 6.5-8%, carbohydrates 71% (sugars 11 %, dietary fiber 60%), ash 1.0-1.5%, fat 3.0-4.0%.) was dissolved in 18 kg of water in a 25 L stainless steel container and placed in a water bath of 45°C. The mixture was stirred continuously in order to keep insoluble components in dispersion. 20 g of Pectinex™ Ultra Mash was added after the apple pomace dispersion had reached 45°C. Incubation was continued for 2 hours. After these 2 hours the container was placed in an ice bath. Next, the dispersion was filled in centrifuge buckets and centrifuged for 5 min at 6000 g. The supernatants were collected in a 25 L stainless steel container after filtration over a 10 pm filter. The container was placed in a water bath of 98°C. After the dispersion had reached a temperature of 90°C, the mixture was heated for another 10 min in order to inactivate the enzyme. Next, the mixture was cooled down to 50°C by placing the container in an ice bath.

The pH of the mixture was set to pH 5.0 by adding a 33 % (m/m) NaOH solution. Next, the mixture was concentrated by a factor 5 and washed with 200% of water over an ultra-filtration membrane using a LAB20 set-up equipped with 10 kDa PolyEtherSulfone membrane (Microdyn Nadir; LIP010) at 50°C. After ultra-filtration/diafiltration the isolate was freeze-dried using a lab-scale freeze dryer.

The monosaccharide composition (% mol/mol) of the isolate is shown in Table 2.

Table 2

Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x8 + 48 + 9 = 73%

• % HG = [GalA] - [Rha] = 18-8 = 10%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x81 (8+18) = 61.5%

• % HG = 100% - % RG-1 = 38.5%

Example 3

A RG-I polysaccharide fraction that can suitably be employed in the present treatment was isolated from dry bell pepper powder (Paprika Mild 80-100 Atsa Steamtr- Felix Reverte S.A.) at pilot plant scale using the procedure described below.

The bell pepper material (100 kg) was washed three times under gentle steering with 80% aqueous ethanol, i.e. twice at 80°C for 2 hours and then overnight at room temperature; each time using 12.5% (w/v), to remove ethanol soluble material. The ethanol insoluble residue was recovered every time by centrifugation (1000 G for 10 min). The ethanol insoluble residue obtained after the 3 wash cycles was dried and 90 kg was extracted twice with 1000 L hot water having a temperature of 95°C for 90 minutes. Each time, the supernatant was retained after centrifugation at 1000 G for 10 minutes. The collected supernatant was subsequently filtered through cloth, and ultrafiltered using 2 KDa molecular weight cut off membranes to remove small molecular weight material. A dry RG-I enriched extract was obtained by drying the retentate, yielding approximately 5 kg of dry RG-I enriched polysaccharide extract.

Characterisation of RG-I polysaccharide enriched extract

The monosaccharide composition of the extract was determined after acid hydrolysis (methanolysis). Neutral monosaccharides were analysed using High Performance Anion Exchange Chromatography combined with Pulse Amperometric Detection (HPAEC-PAD). llronic acids (Galacturonic acid being the dominant of uronic acids) were determined using the colorimetric m-hydroxydiphenyl assay.

The degree of acetylation and methylation was determined as follows:

Polysaccharide samples (2-5mg) were treated with sodium hydroxide (0.1 M, overnight, 20°C). Released methanol was measured by using head-space GC equipped with a DB-WAX ETR column, Cryo Focus-4 cold trap and FID detection (adapted from Huisman et al., Food Hydrocolloids, 18, 4, 2004, 665-668). The samples were neutralized (1 M HCI) and then the released acetyl was quantified by using HPLC equipped with an Aminex HPX 87H column with guard column and Rl detection (adapted from Voragen et al. Food Hydrocolloids, 1 , 1 , 1986, 65-70). Sugar beet pectin with known degree of methylation and acetylation was used as standard. Degree of esterification is expressed as molar amount of methanol and acetic acid released as percentage of the amount of uronic acid.

The molecular characteristics of the extract are shown in Table 3.

Table 3 Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x9.0 + 9.0 + 9.0 = 36.0%

• % HG = [GalA] - [Rha] = 70.0-9.0 = 61.0%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x9.01 (9.0+70.0) = 22.8%

• % HG = 100% - % RG-1 = 77.2%

Example 4

A RG-I polysaccharide fraction that can suitably be employed in the present treatment was isolated from milled pea hulls powder. Dried and milled pea hulls powder (ex Cosucra, Warcoing, Belgium) was dispersed in demineralised water (100 g/L) and subjected to enzymatic pre-hydrolysis with a thermostable alpha-amylase (Megazyme) at 90°C for 30 min and further hydrolysis using pectinase (2 hr 45 °C, 0,2 v/v% Pectinex® Ultra Mash, Novozymes). Enzymolysis was terminated by heating at 100°C for 10 min, followed by centrifugation (18.000 g, 10 min) and extensive dialysis of the supernatant using a membrane with a 12-14 kDa (Visking, London, UK) cut off. The material was then lyophilized.

The monosaccharide composition of the isolate was determined. The results are shown in Table 4.

Table 4

Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x8 + 14 + 6 = 36%

• % HG = [GalA] - [Rha] = 57-8 = 49%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x81 (8+57) = 24.6%

• % HG = 100% - % RG-I = 75.4% Example 5

A RG-I polysaccharide fraction that can suitably be employed in the present treatment was isolated from milled sugar beet pulp powder. Milled sugar beet pulp powder (ex Suiker llnie, Dinteloord, NL) was processed in the same way as the pea powder of Example 4, except that this the a-amylase pre-incubation step was omitted.

The monosaccharide composition of the isolate was determined. The results are shown in Table 5.

Table 5

Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x7 + 45 + 7 = 66%

• % HG = [GalA] - [Rha] = 40-7 = 33%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x71 (7+40) = 29.8%

• % HG = 100% - % RG-I = 70.2%

Example 6

A RG-I polysaccharide fraction that can suitably be employed in the present treatment was isolated from milled chicory pulp powder. Milled chicory pulp (ex Cosucra, Warcoing, Belgium), was processed in the same way as the pea powder of Example 4, except that this the a- amylase pre-incubation step was omitted.

The monosaccharide composition of the isolate was determined. The results are shown in Table 6.

Table 6 Based on these numbers, the percentages (mol/mol) of monosaccharides contained in respectively RG-I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] + [Ara] + [Gal] = 2x8 + 48 + 8 = 72%

• % HG = [GalA] - [Rha] = 35-8 = 27%

The precentage of monosaccharides (mol/mol) contained in the respective backbones of RG- I and HG can be estimated as follows:

• % RG-I = 2 x [Rha] I ([Rha]+[GalA]) = 100%x2x8 I (8+35) = 37.2%

• % HG = 100% - % RG-I = 62.8%