Fucosylated oligosaccharides for prevention of coronavirus infection

Abstract

The invention provides compositions and methods for utilizing oligosaccharides, such as isolated human milk oligosaccharides, to attenuate a respiratory pathogen infection and/or to promote recovery from a respiratory pathogen infection in the respiratory system and/or the GI tract of a subject.

Claims

1. A method of attenuating or treating a corona virus infection and/or promoting recovery from said corona virus infection in a subject expressing an ACE2 receptor in need thereof, comprising administering to the subject a composition comprising a pharmaceutically effective amount of at least one isolated human milk oligosaccharide (HMO), wherein the corona virus comprises i) a severe acute respiratory syndrome virus (SARS-CoV); ii) a SARS-CoV-2 (COVID-19) virus; or iii) a hCoV-NL63, wherein the corona virus binds to the ACE2 receptor on a cell or tissue of the subject, wherein the at least one isolated HMO is identified by structural modeling to compete with the corona virus from binding to the ACE2 receptor on the cell or tissue.

2. The method of claim 1, wherein the at least one isolated human milk oligosaccharide (HMO) is present in an amount effective to reduce or inhibit the corona virus from binding to an animal cell or tissue.

3. The method of claim 1, wherein the at least one isolated human milk oligosaccharide (HMO) comprises 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lactodifucotetraose (LDFT), Lacto-N-difucohexaose I (LDFH I), or Lacto-N-difucohexaose II (LDFH II).

4. The method of claim 1, wherein the composition is in the form of a powder, a tablet, an aerosol, feed for mammalian animals, a packet of sugar, yogurt, a beverage, a weaning food, or an infant formula.

5. The method of claim 2, wherein the binding between the corona virus and the animal cell or tissue is mediated by fucosylation.

6. The method of claim 2, wherein the animal cell or tissue comprises a tissue of a lung or GI tract mucosa or a cell in a tissue of the lung or GI tract mucosa of the subject.

7. The method of claim 1, wherein the subject is a human, non-human primate, mouse, rat, dog, cat, horse, cattle, sheep, pig, chicken, or goat.

8. The method of claim 1, wherein the subject has a fucosyltransferase 2 (FUT2) secretor genotype.

9. The method of claim 1, wherein the subject has an ABO blood group type A, B, or AB.

10. The method of claim 1, wherein said pharmaceutically effective amount is equivalent to a dosage of i) about 0.2 g to 20 g per day; ii) about 1 g to 10 g per day; or ii) about 5 g to 10 g per day.

11. The method of claims 1, wherein the at least one isolated HMO is administered to the lung and/or the GI tract of the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a chart showing the ABO blood group system with the presence of one, both, or neither of the A and B antigens on the surface of erythrocytes (red blood cells or RBCs). The precursor oligosaccharide chain (“precursor chain”) containing Galactose (Gal) and N-Acetylglucosamine (GlcNAc) is further linked to a fucose (Fuc), by an α(1,2) fucosyltransferase FUT1 enzyme (encoded by H-gene), to form an H antigen (“H-substance”). The H antigen may be further modified with an N-Acetylgalactosamine (GalNAc) or a Galactose (Gal), by an α(1,3) fucosyltransferase enzyme A-transferase or B-transferase, forming an A antigen (“A-substance”) or a B antigen (“B-substance”), respectively. RBCs with only one of the H, A, and B antigens are type O, A, or B RBCs, respectively, while RBCs with both the A and the B antigens are type AB RBCs. A subject of the type O or the type AB blood group has the OO or AB genotype, respectively. A subject of the type A (or the type B) blood group may have the AA or AO (or BB or BO) genotype.

(2) FIGS. 2A and 2B are a series of charts showing exemplary MALDI-TOF mass spectra of permethylated N-glycans of human lung and bronchus. The FUT2 gene regulates expression of both H antigen (directly) and A/B blood group antigens (indirectly). Specifically, ABO N-glycan profiles on the surface of human lung (FIG. 2A) or human bronchus (FIG. 2B) were obtained from the 50% MeCN fraction from a C18 Sep-Pak column. Annotated structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M+Na]+. Structures are based on composition, tandem MS, and the biosynthetic knowledge. Due to the presence of heterogeneous multiantennary structures with extended LacNAc repeats, the annotations are simplified throughout by using biantennary structures with the extensions listed in parentheses.

(3) FIG. 3 is a bar chart comparing ages of children infected by various endemic viruses in a PREVAIL cohort study.

(4) FIGS. 4A-4D are a series of drawings showing the overall structure of the complex between angiotensin-converting enzyme 2 (ACE2) and neutral amino acid transporter B(0)AT1. FIG. 4A is a linear chart and an image of Western Blot, showing a representative size exclusion chromatography purification profile of full-length human ACE2 in complex with B(0)AT1. mAU refers to milli-absorbance units; MWM refers to molecular weight marker. FIG. 4B is an image showing a 3-D cryo-EM map of the ACE2-B(0)AT1 complex. Protomer A of ACE2 (upper right), protomer B of ACE2 (upper left), protomer A of B(0)AT1 (lower right) and protomer B of B(0)AT1 (lower left) are shown. FIG. 4C is a cartoon representation of the atomic model of the ACE2-B(0)AT1 complex. The glycosylation moieties are shown as sticks. The complex shows the PD (upper right) and CLD (middle right) subunits in one ACE2 protomer. FIG. 4D is a cartoon representation of an open conformation of the ACE2-B(0)AT1 complex. The two PDs, which contact each other in the closed conformation, are separate in the open conformation.

DETAILED DESCRIPTION

(5) Some respiratory pathogens, such as coronaviruses, infect millions of people and cause enormous mobility and mortality. The health of individuals, especially their respiratory system and the GI tract vulnerable for pathogen infection, depending on host age, disease, microorganism infection, stress, nutritional components, and pharmaceutical treatments.

(6) Described herein are compositions that contain at least one fucosylated oligosaccharide, such as at least one isolated human milk oligosaccharide (HMO), in an amount effective to prevent, attenuate/reduece, and/or inhibit a respiratory pathogen infection and/or to promote recovery from the pathogen infection in the respiratory system and/or the GI tract of a subject. Such fucosylated oligosaccharides include, e.g., 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), lactodifucotetraose (LDFT), and others described herein. The compositions described herein prevent, attenuate, and/or inhibit the binding, and thus the entry for infection, of the pathogen to a cell or tissue of the respiratory system and/or the GI tract of the subject. Not intended to be limiting, the binding and/or the infection require fucosylation on the cell or tissue, while the compositions described herein compete with endogenous receptors for the pathogen to bind the pathogen, thus preventing, attenuating or inhibiting the pathogen from binding to the cell or tissue and causing infection.

(7) As described in details below, at least one isolated human milk oligosaccharide (HMO) is administrated in an amount effective to prevent, attenuate or inhibit a respiratory pathogen (e.g., a coronavirus) infection and/or to promote recovery from a respiratory pathogen infection in the respiratory system and/or the gastrointestinal (GI) tract of a subject. For example, as described in detail below, at least one isolated and/or purified 2′-FL, 3-FL, LDFT, or other HMOs, is administered to selectively prevent, attenuate or inhibit binding of the pathogen to its receptor in the respiratory system and/or the GI tract of the subject, thus preventing, attenuating or inhibiting the pathogen infection.

(8) Respiratory Pathogens

(9) Multiple pathogens may infect the respiratory system and/or the GI tract of a subject described herein. For example, many coronaviruses, such as those in Table 5, including at least SARS-CoV, SARS-CoV-2, and hCoV-NL63, are known respiratory pathogens.

(10) Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is caused also by certain other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19. Symptoms in other species vary: in chickens, they cause an upper respiratory tract disease, while in cows and pigs they cause diarrhea.

(11) Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a positive strand RNA virus that causes severe respiratory syndrome in humans. The resulting outbreak of coronavirus disease 2019 (COVID-19) has emerged as a severe epidemic worldwide. The genome of SARS-CoV-2 shares about 80% identity with that of SARS-CoV and is about 96% identical to the bat coronavirus BatCoV RaTG13 (Zhou et al., (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270-273).

(12) According to CDC, people with COVID-19 have had a wide range of symptoms reported—ranging from mild symptoms to severe illness. Symptoms may appear 2-14 days after exposure to the virus. People having COVID-19 infection may have cough, shortness of breath or difficulty breathing, or at least two of these symptoms: fever, chills, repeated shaking with chills, muscle pain, headache, sore throat, new loss of taste or smell, etc. Children have similar symptoms to adults and generally have mild illness.

(13) In the case of SARS-CoV, the spike glycoprotein (S protein) on the virion surface mediates receptor recognition and membrane fusion. During viral infection, the trimeric S protein is cleaved into S1 and S2 subunits and S1 subunits are released in the transition to the post-fusion conformation. S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2), whereas S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE2, another cleavage site on S2 is exposed and is cleaved by host proteases, a process that is critical for viral infection. A recent publication reported the structure of the S protein of SARSCoV-2 and showed that the ectodomain of the SARS-CoV-2 S protein binds to the PD of ACE2 with a dissociation constant (Kd) of ˜15 nM [Wrapp et al., (2020) Science eabb2507].

(14) ABO Blood Groups

(15) The ABO blood group system is used to denote the presence of one, both, or neither of the A and B antigens on erythrocytes. In human blood transfusions it is the most important of the 36 different blood type (or group) classification systems currently recognized. A detailed description is shown in FIG. 1. Blood containing red cells with type A antigen on their surface has in its serum antibodies against type B red cells. If, in transfusion, type B blood is injected into persons with type A blood, the red cells in the injected blood will be destroyed by the antibodies in the recipient's blood. In the same way, type A red cells will be destroyed by anti-A antibodies in type B blood. Type O blood can be injected into persons with type A, B, or O blood unless there is incompatibility with respect to some other blood group system also present. Persons with type AB blood can receive type A, B, or O blood.

(16) The O antigen, eponymous for the O blood group type, is a glycan moiety with an α-1,2 linked fucose, also known as the H antigen when expressed in exocrine secretions. In the A blood group type, this motif is masked by an N-acetylgalactosamine, creating steric hindrance that inhibits binding by O antibodies or ligands. Similarly, in the B blood group type, the O antigen is masked by galactose. For the AB blood group type, the O antigen is masked by both, providing the strongest inhibition of binding by O ligands (Prieto et al. (1997) Expression of human H-type alpha1,2-fucosyltransferase encoding for blood group H(O) antigen in Chinese hamster ovary cells. Evidence for preferential fucosylation and truncation of polylactosamine sequences. J Biol Chem 272, 2089-2097).

(17) Human Milk Glycans or Human Milk Oligosaccharides (HMOs)

(18) Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides. For example, the purified HMO described herein, such as fucosylated oligosaccharides, e.g., 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), and lactodifucotetraose (LDFT), are typically found in human milk. Human milk oligosaccharides as a class survive transit through the intestine of infants very efficiently, being essentially indigestible (Chaturvedi, P et al. 2001 Adv Exp Med Biol, 501:315-323). Although these molecules may not be utilized directly by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy gut microbiome (Marcobal A, et al. 2010 J Agric Food Chem, 58:5334-5340), in the prevention of disease (Newburg D, et al. 2005 Annu Rev Nutr, 25:37-58), and in immune function (Newburg and Walker (2007) Protection of the neonate by the innate immune system of developing gut and of human milk. Pediatr Res 61, 2-8). Despite millions of years of exposure to human milk oligosaccharides (HMOS), pathogens have yet to develop ways to circumvent the ability of HMOS to prevent adhesion to target cells and to inhibit infection. The ability to utilize HMOS as pathogen adherence inhibitors promises to address the current crisis of burgeoning antibiotic resistance. Human milk oligosaccharides represent the lead compounds of a novel class of therapeutics against some of the most intractable scourges of society.

(19) Human milk glycans, which comprise both unbound oligosaccharides and their glycolconjugates, play a significant role in the protection and development of infant organs, such as the gastrointestinal (GI) tract. Milk oligosaccharides found in various mammals differ greatly, and composition in humans is unique (Hamosh M, 2001 Pediatr Clin North Am, 48:69-86; Newburg D, 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Approximately 200 distinct human milk oligosaccharides have been identified and combinations of simple epitopes are responsible for this diversity (Newburg D, 1999 Curr Med Chem, 6:117-127; Ninonuevo M et al., 2006 J Agric Food Chem, 54:7471-74801). Human milk oligosaccharides are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). Human milk oligosaccharides are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J, et al. 1999 Anal Biochem, 273:261-277; Martin-Sosa, et al. 2003 J Dairy Sci, 86:52-59; Parkkinen J and Finne J, 1987 Methods Enzymol, 138:289-300; Shen Z, et al. 2001 J Chromatogr A, 921:315-321). Approximately 70-80% of oligosaccharides in human milk, e.g., 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), lactodifucotetraose (LDFT), are fucosylated.

(20) Human milk glycans have structural homology to cell receptors for enteropathogens and function as receptor decoys. For example, respiratory pathogens, such as coronaviruses, bind specifically to the ACE2 receptor. Coronavirus binding and infectivity may thus be inhibited by 2′-FL and other glycans. Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by human milk oligosaccharides containing 2-linked fucose moieties. Several major strains of human caliciviruses, especially the noroviruses, also bind to 2-linked fucosylated glycans, and this binding is inhibited by human milk 2-linked fucosylated glycans. Consumption of human milk that has high levels of these 2-linked fucosyloligosaccharides was associated with lower risk of norovirus, Campylobacter, ST of E. coli-associated diarrhea, and moderate-to-severe diarrhea of all causes in a Mexican cohort of breastfeeding children (Newburg D et al., 2004 Glycobiology, 14:253-263; Newburg D et al., 1998 Lancet, 351:1160-1164).

(21) Human Milk Glycans or Human Milk Oligosaccharides (HMOs) to Attenuate a Respiratory Pathogen Infection and/or to Promote Recovery from a Respiratory Pathogen Infection

(22) Human milk contains a complex oligosaccharide mixture attenuating a respiratory pathogen (e.g., a coronavirus) infection and/or promoting recovery from a respiratory pathogen infection. As described herein, human milk oligosaccharides such as 2′-fucosyllactose (2′-FL, MW=488), 3-fucosyllactose (3-FL, MW=488), and lactodifucotetraose (LDFT, MW=635) have such properties and are administered, as described herein, to prevent, attenuate, inhibit, and/or treat a respiratory pathogen (e.g., a coronavirus) infection and/or to promote recovery from a respiratory pathogen (e.g., a coronavirus) infection. Such oligosaccharides are short-chain carbohydrates with a degree of polymerization between 2 and 60, and are indigestible by human or animal digestive systems.

(23) As described herein, human milk glycans or HMOS function as agents that prevent, attenuate, and/or inhibit the respiratory pathogen (e.g., a coronavirus) from binding (thus affecting the pathogen colonization and/or adhesiveness) to its natural receptor in the respiratory system or the GI tract of a subject. The glycans or HMOS described herein are not limited to use in human infants or adults. They may also be administered to an infant or adult mammal. The mammal can be, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a horse, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably, the mammal is a human.

(24) The invention also provides using other HMOS or glycans, or other agents, in addition to at least one HMO or glycan, for attenuating the pathogen infection. Such additional agents may include, e.g., fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS).

(25) Pharmaceutically available and/or effective administration routes may be used to deliver the fucosylated oligosaccharides (such as HMOS) described herein to a subject. With no intention to be limiting, the fucosylated oligosaccharides may be administered locally or systemically to a subject, including administering to, e.g., the respiratory system and/or the GI tract of the subject, via, e.g., inhalation, pulmonary lavage, oral ingestion, anal administration, infusion, and/or injection. Administration routes also include, but not limited to, administering to a subject intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, by injection, and by infusion.

(26) Dosages and dosing regimes may be determined by a doctor or a medical personnel accordingly to each patient's individual situation. Generally, HMOS, such as 2′-FL, can be given orally at about 1-20 g/day to reduce infection of the lung. Oral HMOS, such as 2′-FL, may be administered either by itself in tablet or powder form, or it may be conveniently dissolved in a little water or another beverage, or it may be included in foodstuffs or in food or medical supplements. The preferred adult dose is generally equivalent to about 10 g per day, but anywhere between about 0.2 g per day up to about 20 g per day may be taken. For example, potential dosages may include about 0.01 g to about 20 g per day, about 0.01 g to about 10 g per day, about 0.1 g to about 10 g per day, about 1 g to about 10 g per day, about 1 g to about 5 g per day, about 5 g to about 10 g per day, or any dosages found effective to the subject. Specifically, 2′-FL is a natural component of human milk and as such it is non-toxic, however high doses (i.e. >10 g per day) may lead to mild gut discomfort and a reversible osmotic diarrhea.

Example 1: A Correlation Between Virus Infection and Cell Surface Oligosaccharide Components of the ABO Blood Group Types

(27) Early in the COVID-19 pandemic, Zhao et al (2020) conducted a case-control study of COVID-19 hospitalization in two regions of China: Wuhan & Shenzhen, indicates that SARS-CoV-2 hospitalization rates differ by ABO blood group type (see Zhao et al. (2020) Relationship between the ABO Histo-Blood Group and the COVID-19 Susceptibility. medRxiv 2020.03.11.20031096; doi: https://doi.org/10.1101/2020.03.11.20031096). In each region, hospitalized patients' ABO blood group type was compared to blood group typing data. A summary of parts of their results is given in the Table 2 below.

(28) TABLE-US-00002 TABLE 2 SARS-CoV-2 hospitalizations by ABO blood groups in China Number (Percentage) of individuals by blood group type O Study Total All (refer- Groups no. A B AB non-O enced) Wuhan 1775 670 469 178 1317 458 COVID- (37.8%) (26.4%) (10.0%) (74.2%) (25.8%) 19 patients (from 2 hospitals) Wuhan 3694 1188 920 336 2444 1250 regional (32.2%) (24.9%) (9.1%) (66.2%) (33.8%) controls Odds ratio 1.54 1.39 1.45 1.47 1.0 (95% CI) (1.3, (1.2, (1.2, (1.3, (N/A) 1.8) 1.6) 1.8) 1.7) p-value <0.0001 <0.0001 0.0006 <0.0001 N/A Shenzhen 285 82 83 39 204 81 COVID- (28.8%) (29.1%) (13.7%) (71.6%) (28.4%) 19 patients Shenzhen 23386 6728 5880 1712 14320 9066 regional (28.8%) (25.1%) (7.3%) (61.2%) (38.8%) controls Odds ratio 1.36 1.58 2.55 1.59 1.0 (95% CI) (1.0, (1.1, (1.7, (1.2, 1.9) 2.2) 3.8) 2.1) p-value 0.048 0.003 <0.0001 <0.0001 N/A

(29) Zhao et al emphasized a small reported association with A blood group—1.2 fold increased risk. However, their data analysis and conclusions are flawed. A re-analysis of Zhao's data described here led to a different conclusion. For example, Zhao et al stated that their results showed that blood group A was associated with a higher risk for acquiring COVID-19 compared with non-A blood groups, whereas blood group O was associated with a lower risk for the infection compared with non-O blood groups.

(30) However, the error of their analytic approach is that they compared each blood group type to all others. This approach obscured the observation that there is no statistical difference across A, B, and AB blood group types; all were significantly and similarly elevated vs O blood group type. A blood group should not be singled out.

(31) Thus, the re-analysis found that only O blood group type differed from the others, and was consistently protective, and the non-O blood groups odds of COVID-19 hospitalization was increased by about 1.5-fold, which is of public health significance.

(32) SARS-CoV-2 shares ˜80% amino acid sequence homology with SARS-CoV spike protein implicated in binding. Prior studies on SARS-CoV were then re-analyzed in view of the newly found ABO-related infection risk on SARS-Cov-2. For example, a previous report Chang Y et al (JAMA, 2005) studied 45 health care workers (HCW) in Hong Kong exposed to HK SARS index case and found that SARS-CoV infection risk also differed by ABO types. ABO blood type was significantly associated with SARS, while the O blood group has decreased risk of infection and the A, B, or AB (non-O) groups have about 1.5-fold increased risk of infection (p=0.03). A summary of parts of their results is given in the below Table 3.

(33) TABLE-US-00003 TABLE 3 SARS-CoV Infection by ABO blood groups Number (Percentage) of individuals Total by blood group type All O Study Groups no. A B AB non-O (referenced) Hong Kong SARS- 34 8 12 3 23 11 infected HCW (88%) (58%) Hong Kong HCW not 11 0 3 0 3 8 infected of SARS (12%) (42%) Relative risk 1.53 1.0 (95% CI) (1.02, 2.3) P-value 0.033 N/A

(34) Therefore, for both SARS-CoV and SARS-Cov-2 infection, non-O groups have an increased risk compared to the O blood group, which provides about 1.5-fold protection.

(35) The structures of sugar chains added to lipids on cell surface, representing the differences among the ABO blood groups, are shown in FIG. 1. The increased risk of virus infection for non-O groups may be caused by the additional sugar (e.g., Gal or GalNac) found on cell surface of lung tissue of individuals of the non-O blood group. Presentation of A or B antigen requires another gene FUT2, which controls the ability to attach fucose to the precursor chain. As shown in FIGS. 2A and 2B, there are multiple fucosylated ABO glycans on the surface of human lung and bronchus. The FUT2 gene regulates expression of both H antigen (directly) and A/B blood group antigens (indirectly). For reports on the correlation between the ABO blood groups and the FUT2 function, see Walther et al. (2013) PLoS Pathog. 9(3): e1003223. doi: 10.1371/journal.ppat.1003223; and Li et al. (2011) APJCP 12(12):3201-3206.

(36) Our re-analysis concluded as follows:

(37) 1. ABO risk is similar for SARS-CoV & SARS-CoV-2. For these two types of viruses, non-O blood groups are at similarly increased risk compared to the O blood group, which enjoys about 1.5-fold protection;

(38) 2. Such risk has to do with the additional sugar (Gal or GalNAc) found on surface of lung tissue of A or B blood group individuals; and

(39) 3. Presentation of the A or B antigen requires another gene (FUT2), which controls the ability to attach fucose to the precursor.

Example 2: A PREVAIL Cohort Study of Epidemiology of Endemic hCoVs in Children

(40) In an ongoing CDC-sponsored PREVAIL cohort study (Morrow, PI), endemic hCoVs have been examined to better understand coronaviruses. Data were collected from a completed respiratory viral panel testing for >12,000 nasal swab samples submitted from Oct. 1, 2017 through Sep. 1, 2019. The Luminex panel detects coronaviruses, adenovirus, bocavirus, enteroviruses/rhinoviruses, influenza, metapneumovirus, RSV and parainfluenza viruses.

(41) Data for four exemplary globally endemic viruses (NL63, OC43, HKU1, and 229E) are shown below. Of the 91 PREVAIL children with ≥75% compliance with weekly nasal swabs, 70 (77%) were positive for a strain of coronavirus at some time. In these 70 coronavirus-positive children, a second infection occurred in nearly 60% of the children. Most repeat infections were with a different strain, but one-quarter of second infections occurred with the same strain. As shown in FIG. 3 and Table 4, there were significant strain-specific differences in carriage by age of child, while NL63 has been more common in infants. Specifically, among the 77% of children ever infected, there were, on average, 2 episodes per child (0-2 yrs). While about 10% of infections were symptomatic, one-third of symptomatic infections were associated with medical care. There were no reported uses of emergency department or hospital. Durations of viral shedding/infection were about 1-4 weeks, depending on strains (p<0.001). Longest duration shedding was with the NL63 strain.

(42) It is hypothesized that secretor status may be relevant to endemic hCoV carriage, but would vary by viral strains. Table 4 below further shows the endemic coronavirus infections in PREVAIL children by FUT2 secretor status. Carriage of strains did vary significantly by secretor status of the child. Infection by the NL63 strain was identified more often in nasal swabs from FUT2 secretors than non-secretors.

(43) TABLE-US-00004 TABLE 4 Correlation between virus infection and FUT2 secretor status FUT2 status in Children Total no. NL63 OC43 HKU1 229E Secretor 113 40 41 27 5 (100%) (35%) (36%) (24%) (4%) Non-secretor 34 4 17 12 1 (100%) (12%) (50%) (35%) (3%) P = 0.037 (Fisher's exact)

(44) The PREVAIL data regarding the hCov-NL63 binding and infection may be an important clue for SARS-CoV-2 infection. Human coronavirus NL63 is a species of group 1 coronavirus, identified in 2004 in an infant with bronchiolitis in the Netherlands. HCoV-NL63 has been found worldwide, tending to infect children and immune-compromised individuals and being the etiological agent for up to 10% of all respiratory diseases. The virus is an enveloped, positive-sense, single-stranded RNA virus which enters its host cell by the ACE2 receptor. See Abdul-Rasool et al. (2010) Understanding Human Coronavirus HCoV-NL63. Open Virol J. 4:76-84. doi: 10.2174/1874357901004010076. PMID: 20700397; PMCID: PMC2918871. Although SARS-CoV and SARS-CoV-2 are group 2B betacoronaviruses, while NL63 is a group 1 alphacoronavirus, both of them utilize the same ACE2 receptor for attaching and infection. Other coronavirus strains use other receptors. An overview of some known coronavirus entry receptors is in Table 5. See also Wilde et al. (2018) Host Factors in Coronavirus Replication. Curr. Top. Microbiol. Immunol 419:1-42.

(45) TABLE-US-00005 TABLE 5 Overview of Some Known Coronavirus Entry Receptors Binding Binding partner for partner for the viral the viral Genus Species S1-NTD S1-CTD References Alphacoronavirus Alphacoronavirus Neu5Gc and APN Tresnan et al. 1996; 1 Neu5Ac Delmas et al. 1992 PEDV Neu5Ac APN Liu et al. 2015; Li et al. 2007 PRCV APN Schultze et al. 1996 HCov-229E APN Yeager et al. 1992 HCoV-NL63 ACE2 Wu et al. 2009 Betacoronavirus Betacoronavirus 1 Neu5,9Ac2 Schultze and Herrler 1992; Krempl et al. 1995 MERS-CoV DPP4 Raj et al. 2013 MHV CEACAM1 Williams et al. 1991 HKU1 Neu5,9Ac2 Huang et al. 2015 HKU4 DPP4 Yang e tal. 2014 SARS-CoV ACE2 Li et al., 2003 Gammacoronavirus IBV Neu5Gc Schultze et al. 1993 Deltacoronavirus PDCV Unknown Unknown

Abbreviations

(46) S1-NTD: the N-terminal domain of the S1 region of the coronavirus S protein, usually binding to sialic acids, like N-glycolylneuraminic acid (Neu5Gc),

(47) N-acetylneuraminic acid (Neu5Ac), and/or 5-N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). One exception is the murine hepatitis virus (MHV) S1-NTD, which binds the N-terminal D1 domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a type-I membrane protein belonging to the immunoglobulin superfamily;
S1-CTD: the C-terminal domain of the S1 region of the coronavirus S protein, usually binding to aminopeptidase N (APN). Exceptions include HCov-NL63 and SARS-CoV, binding to angiotensin-converting enzyme 2 (ACE2), MERS-CoV and HKU4, binding to dipeptidyl peptidase 4 (DPP4);
PEDV: Porcine epidemic diarrhea virus;
PRCV: Porcine Respiratory coronavirus;
HCoV: Human coronavirus;
MHV: Murine hepatitis virus;
IBV: Infectious bronchitis virus;
PDCV: Porcine delta coronavirus;

REFERENCES

(48) Tresnan et al. (1996) Feline aminopeptidase N serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup I. J Virol 70:8669-8674; Delmas et al. (1992) Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357:417-420; Liu et al. (2015) Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol 89:6121-6125; Li et al. (2007) Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus. Virology 365:166-172; Schultze et al. (1996) Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity. J Virol 70:5634-5637; Yeager et al. (1992) Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357:420-422; Wu et al. (2009) Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc Natl Acad Sci USA 106:19970-19974; Schultze and Herrler (1992) Bovine coronavirus uses N-acetyl-9-O-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells. J Gen Virol 73(Pt 4):901-906; Krempl et al. (1995) Analysis of cellular receptors for human coronavirus OC43. Adv Exp Med Biol 380:371-374; Raj et al. (2013) Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495:251-254; Williams et al. (1991) Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci USA 88:5533-5536; Huang et al. (2015) Human coronavirus HKU1 spike protein uses O-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme. J Virol 89:7202-7213; Yang et al. (2014) Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc Natl Acad Sci USA 111:12516-12521; Li et al., (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426:450-454; Schultze et al. (1993) N-acetylneuraminic acid plays a critical role for the haemagglutinating activity of avian infectious bronchitis virus and porcine transmissible gastroenteritis virus. Adv Exp Med Biol 342:305-310.

Example 3: A Structural Study of the Binding Between SARS-CoV-2 and ACE2

(49) The crystal structure for interaction between SARS-CoV-2 and full-length human ACE2 was recently reported [Yan et al. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367:1444-1448].

(50) The spike glycoprotein (S protein) on the virion surface of SARS-CoV-2 mediates receptor recognition and membrane fusion. During viral infection, the trimeric S protein is cleaved into S1 and S2 subunits and S1 subunits are released in the transition to the postfusion conformation. S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2), whereas S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE2, another cleavage site on S2 is exposed and is cleaved by host proteases, a process that is critical for viral infection. The S protein of SARS-CoV-2 may also exploit ACE2 for host infection. A prior publication, Wrapp et al. [Science eabb2507 (2020)], reported the structure of the S protein of SARSCoV-2 and showed that the ectodomain of the SARS-CoV-2 S protein binds to the PD of ACE2 with a dissociation constant (Kd) of ˜15 nM.

(51) Since the single transmembrane (TM) helix of ACE2 makes it challenging to determine the structure of the full-length protein, Yan et al. used cryo-electron microscopy (cryo-EM) to study the structure of the full-length human ACE2, stabilized by binding to the amino acid transporter B(0)AT1, also known as SLC6A1 (see FIGS. 4A to 4D). Further, a complex between the receptor binding domain (RBD) of SARS-CoV-2 and the ACE2-B(0)AT1 complex was formed and its structure was resolved with an overall resolution of 2.9 Å and with 3.5-Å local resolution at the ACE2-RBD interface.

(52) The overall RBD-ACE2-B(0)AT1 interface is similar to that between SARS-CoV and ACE2, mediated mainly through polar interactions. An extended loop region of the RBD spans the arch-shaped al helix of the ACE2-PD like a bridge. The α2 helix and a loop that connects the β3 and β4 antiparallel strands, referred to as loop 3-4, of the PD also make limited contributions to the coordination of the RBD. The contact can be divided into three clusters. The two ends of the bridge interact with the N and C termini of the α1 helix as well as small areas on the α2 helix and loop 3-4. The middle segment of α1 reinforces the interaction by engaging two polar residues.

(53) Structural alignment of the S protein of SARS-CoV-2 with the RBD-ACE2-B0AT1 ternary complex indicates that two S protein trimers can simultaneously bind to an ACE2 homodimer. In addition, the host binding site is highly glycosylated, with seven and five glycosylation sites on each ACE2 and B(0)AT1 monomer, respectively.

Example 4: Reduction of Infection with SARS-CoV-2 (COVID-19), SARS-CoV and Other Major Respiratory Pathogens by Administration of Fucosylated Oligosaccharides

(54) SARS-CoV-2 (COVID-19), SARS-CoV, and hCoV-NL63 (an endemic coronavirus strain) bind to the ACE2 receptor, which has many glycosylation sites. It's shown in previous Examples that risk of infections with each of these strains of coronaviruses is increased in individuals with histo-blood group types (e.g., the ABO blood group types) that involve fucosylation. The binding between the viruses and the host ACE2 receptor was enabled or strengthened by A, B, and H antigens in the lung, all of which require an active FUT2 allele for expression. The role of fucosylation in binding or immune response to these coronaviruses is critical.

(55) Preliminary data from the PREVAIL cohort (Morrow, PI) indicate that infection with human coronavirus NL63, one of the major endemic coronavirus strains, differs in frequency between infants depending on their “secretor” histo-blood group type, which is determined by their fucosyltransferase 2 (FUT2) genotype. Secretor histo-blood group type enables expression of ABO fucosylated carbohydrates on the surface of the lung, gut, and in secretions.

(56) All three strains, SARS-CoV, SARS-CoV-2, and hCoV-NL63, use the ACE2 receptor for binding pulmonary mucosa. The ACE2 receptor is heavily glycosylated, an important feature that enables coronavirus binding to this receptor site. It is proposed that A, B, and AB antigens and other fucose-containing glycans are found more abundantly in the ACE2 receptors of individuals who are secretors vs non-secretors.

(57) It's further indicated, not intended to be limiting, that fucosylated receptors participate in or enhance binding of the respiratory pathogens, and that soluble fucosylated oligosaccharide structures can inhibit binding. Preliminary evidence from the PREVAIL cohort shows that the risk of hCoV-NL63 infection was increased two-fold in FUT2+ infants of histo-blood group types A, B, and O. But if the infant has a FUT2+ secretor mother and is being breast-fed, the risk of hCoV-NL63 was reduced 4-fold, indicating that the human milk oligosaccharides inhibit processes that result in infection.

(58) In conclusion, the current studies provide an understanding personal risk of respiratory pathogen infection. The risk in the O blood group type appears one-third lower than the A or B blood group type. In addition, since fucosylated receptors are a critical co-factor in viral binding and infection, similar soluble fucosylated oligosaccharides may be used to competitively inhibit such viral binding and infection.

(59) In an exemplary treatment, 2′-FL, or at least one of other HMOS, is given orally at 1-20 g/day to a subject to reduce infection of the lung. Oral 2′-FL may be administered either by itself in tablet or powder form, or it may be conveniently dissolved in a little water or another beverage, or it may be included in foodstuffs or in food or medical supplements. The preferred 2′-FL adult dose is 10 g per day, but anywhere between 0.2 g per day up to 20 g per day may be taken. 2′-FL is a natural component of human milk and as such it is non-toxic, however high doses (i.e. >10 g per day) may lead to mild gut discomfort and a reversible osmotic diarrhea. Examples of additional fucosylated hMOS which could be used in combination with 2′-FL in the reduction of infection by SARS-CoV-2 (COVID-19), SARS-CoV and other major respiratory pathogens include, but not limited to, 3-fucosyllactose (3-FL), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lactodifucotetraose (LDFT), Lacto-N-difucohexaose I (LDFH I), and Lacto-N-difucohexaose II (LDFH II).

(60) In another example, 2′-FL, or at least one of other HMOS, is given by inhalation of a powder or aerosol or lavage into the lungs to provide a competitive inhibitor of viral binding to receptor that can reduce cell-surface binding and infection.

(61) In other examples, 2′-FL, alone or mixed with other hMOs, is given by injection into the blood stream of the recipient.

(62) Further, 2′-FL, or at least one of other HMOS, is used to prevent infection (prophylaxis) during times when COVID-19 is prevalent in populations, or to reduce symptomatic infection, or to reduce risk of severe infection (secondary prevention), in a subject.

(63) Clinical trials are designed and performed to test oligosaccharides (such as 2′-FL), either alone or in combination, for their safety and/or effectiveness to improve the overall health of the recipient individual. The recruited individuals may be either healthy or have at least one symptom of diseases and disorders associated with infection by enteric respiratory pathogens (such as COVID-19). Exemplary diseases and disorders may include, at least, acute infectious diarrhea, respiratory tract infection, ear, nose, or throat (ENT) infection, an infectious complication in surgical and critically ill patients, etc. Different individual groups may be set up according to age, sex, health conditions, etc. for treatment.

(64) Various oligosaccharides, such as hMOS, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and lactulose, may be administered to the recruited individuals for the clinical trials. Different combinations, such as 2′-FL and GOS, may be administered together, or sequentially, to the recruited individuals. The attending physician may decide the appropriate amount and dosage regimen for such single, double, or multiple combinational administrations. For example, the oligosaccharide may be administered between 0.1 g and 10 g, between 0.5 g and 5 g, between 1 g and 5 g, between 1.5 g and 3 g, or in other reasonable or effective amounts according to the individual body weight, every day, every two days, every three days, every week, or in other time periods, for one week, two weeks, three weeks, one month, two months, three months, six months, one year, or any longer periods. The exemplary ratios of two oligosaccharides, such as 2′-FL and another oligosaccharide, for administration are 1:1, 1:2, 1:5, 1:10, 1:100, 100:1, 10:1, 5:1, 2:1, or any ratios found proper or resulting in preferred safety and/or effectiveness readout. Negative controls (e.g., no oligosaccharide or other placebos) and positive controls (e.g., glucose or other sugars) may be used for comparison.

(65) Administration routes may be by oral, anal, inhalation, injection, or other known or acceptable routes to the GI tract, lung, or blood stream of the individuals.

(66) The result of oligosaccharide treatments in the exemplary clinical trials may be gathered by collecting fecal samples of the treated individuals, as described herein or known or acceptable in the art, or other methods to detect the reduction or inhibition of viral infection or binding to cells or tissues of the individuals, or to detect the recovery from at least one symptom related to the viral infection. The readout of the treatment may involve an evaluation of the amount or function of the respiratory pathogen or the amount or function of a biomarker related to viral infection in the treated individual, or general healthy conditions of the treated individuals.

Other Embodiments

(67) While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

(68) The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

(69) While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.