U.S. patent application number 13/032568 was filed with the patent office on 2011-08-25 for oligosaccharide compositions and use thereof in the treatment of infection.
This patent application is currently assigned to Children's Hospital Medical Center. Invention is credited to Ardythe L. Morrow, David S. Newburg, Guillermo M. Ruiz-Palacios.
Application Number | 20110207659 13/032568 |
Document ID | / |
Family ID | 34676762 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110207659 |
Kind Code |
A1 |
Morrow; Ardythe L. ; et
al. |
August 25, 2011 |
OLIGOSACCHARIDE COMPOSITIONS AND USE THEREOF IN THE TREATMENT OF
INFECTION
Abstract
Oligosaccharides and oligosaccharides linked to backbones such
as proteins, methods for making such oligosaccharides and methods
for using them to treat and/or prevent various disorders are
described.
Inventors: |
Morrow; Ardythe L.;
(Cincinnati, OH) ; Newburg; David S.;
(Newtonville, MA) ; Ruiz-Palacios; Guillermo M.;
(Sanbuenaventura-Talpuente, MX) |
Assignee: |
Children's Hospital Medical
Center
Cincinnati
OH
University of Massachusetts
Boston
MA
Instituto Nacional de Ciencias Medicas Y Nutricion
Mexico
|
Family ID: |
34676762 |
Appl. No.: |
13/032568 |
Filed: |
February 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10581759 |
Jul 26, 2007 |
7893041 |
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PCT/US04/40882 |
Dec 6, 2004 |
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13032568 |
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60527591 |
Dec 5, 2003 |
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Current U.S.
Class: |
514/3.1 ;
435/192; 435/198; 435/201; 435/206; 435/254.2; 514/54;
536/23.4 |
Current CPC
Class: |
A61P 1/00 20180101; A23V
2002/00 20130101; A23L 33/17 20160801; A61P 31/12 20180101; A61K
45/06 20130101; A23L 33/40 20160801; A61K 38/00 20130101; A61P
31/00 20180101; A61P 3/02 20180101; Y02A 50/30 20180101; Y02A
50/475 20180101; A61P 31/04 20180101; A61K 35/20 20130101; A61P
1/12 20180101; Y02A 50/47 20180101; A61P 31/14 20180101; A61K
31/702 20130101; A23V 2002/00 20130101; A23V 2200/324 20130101;
A23V 2250/28 20130101; A23V 2250/54 20130101 |
Class at
Publication: |
514/3.1 ;
435/198; 435/254.2; 536/23.4; 514/54; 435/206; 435/192;
435/201 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C12N 9/20 20060101 C12N009/20; C12N 1/19 20060101
C12N001/19; C07H 21/04 20060101 C07H021/04; A61P 31/04 20060101
A61P031/04; A61K 31/715 20060101 A61K031/715; C12N 9/36 20060101
C12N009/36; C12N 9/08 20060101 C12N009/08; C12N 9/26 20060101
C12N009/26 |
Claims
1-9. (canceled)
10. A pharmaceutical composition, comprising a purified protein
that includes at least two different oligosaccharide groups
selected from 2'-Fucosyllactose; Lacto-N-fucopentaose I;
Lacto-N-fucopentaose II; 3-Fucosyllactose; Lacto-N-fucopentaose
III; Lacto-N-difucohexaose I; Lactodifucotetraose; LactoN-tetraose;
LactoN-neotetraose; 3'-Sialyllactose; 3'-Sialyllactosamine;
6'-Sialyllactose; 6'-Sialyllactosamine; Sialyllacto-N-neotetraose
c; Monosialyllacto-N-hexaose; Disialyllacto-N-hexaose I;
Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II
Disialyllacto-N-neohexaose Disialyllacto-N-tetraose;
Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a;
Disialyllacto-N-hexaose I; Sialyllacto-N-tetraose b;
3'-Sialyl-3-fucosyllactose; Disialomonofucosyllacto-N-neohexaose;
Monofucosylmonosialyllacto-N-octaose (sialyl Lea);
Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II; and
Monofucosyldisialyllacto-N-tetraose, or a variant thereof wherein a
sugar at the reducing end is GlcNAc.
11. (canceled)
12. The composition of claim 10, wherein the protein contains
multiple copies of each of the at least two different
oligosaccharide groups.
13. The composition of claim 10, wherein the protein is a human
milk protein.
14. The composition of claim 10, wherein the human milk protein is
selected from .kappa.-casein, .alpha.-lactalbumin, lactoferrin,
bile salt-stimulated lipase, lysozyme, serum albumin,
folate-binding protein, haptocorrin, lipoprotein lipase,
glycosaminoglycan, mucin, lactoperoxidase, and amylase.
15. The composition of claim 10, wherein the composition is
synthetic.
16. The composition of claim 10, wherein the composition is free of
mammalian milk.
17-29. (canceled)
30. A synthetic nutrition composition comprising a purified protein
that includes at least two oligosaccharide groups selected from
2'-Fucosyllactose; Lacto-N-fucopentaose I; Lacto-N-fucopentaose II;
3-Fucosyllactose; Lacto-N-fucopentaose III; Lacto-N-difucohexaose
I; Lactodifucotetraose; LactoN-tetraose; LactoN-neotetraose;
3'-Sialyllactose; 3'-Sialyllactosamine; 6'-Sialyllactose;
6'-Sialyllactosamine; Sialyllacto-N-neotetraose c;
Monosialyllacto-N-hexaose; Disialyllacto-N-hexaose I;
Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II
Disialyllacto-N-neohexaose Disialyllacto-N-tetraose; Disialyllacto
-N-hexaose II; Sialyllacto-N-tetraose a; Disialyllacto-N-hexaose I;
Sialyllacto-N-tetraose b; 3'-Sialyl-3-fucosyllactose;
Disialomonofucosyllacto-N-neohexaose;
Monofucosylmonosialyllacto-N-octaose (sialyl Lea);
Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II; and
Monofucosyldisialyllacto-N-tetraose or a variant thereof in which a
sugar at the reducing end is GlcNAc wherein the at least two
oligosaccharide groups are the same or different.
31. The composition of claim 30 wherein the at least two
oligosaccharide groups are different.
32-37. (canceled)
38. A method for reducing the risk of enteric disease in a patient,
the method comprising: (a) identifying the two most prevalent
agents capable of causing enteric disease in the geographic
location of the patient; and (b) administering to the patient a
composition containing a molecule that includes a first glycan
which interferes with the binding to epithelial cells of the first
of the two most prevalent agents and a second glycan which
interferes with the binding to epithelial cells of the second of
the two most prevalent agents wherein said composition is free of
breast milk.
39. A method for reducing the risk of enteric disease in a patient,
the method comprising: (a) identifying the two most prevalent
agents capable of causing enteric disease in the geographic
location of the patient, (b) administering to the patient a
composition comprising i) a first molecule containing a first
glycan which interferes with the binding to epithelial cells of the
first of the two most prevalent agents; and ii) a second molecule
containing a glycan which interferes with the binding to epithelial
cells of the second of the two most prevalent agents, wherein said
composition is free of breast milk.
40. A yeast cell harboring a recombinant vector, comprising a
nucleotide sequence encoding GDP-mannose 4, 6 dehydratase and a
nucleotide sequence encoding GDP-L-fucose synthetase.
41. The yeast cell of claim 40, wherein the GDP-mannose 4, 6
dehydratase is H. pylori GDP-mannose 4, 6 dehydratase.
42. The yeast cell of claim 40, wherein the GDP-L-fucose synthetase
is H. pylori GDP-L-fucose synthetase.
43. The yeast cell of claim 40, wherein the yeast cell harbors a
nucleic acid molecule encoding a GDP-fucose/GMP antiporter fusion
protein.
44. The yeast cell of claim 43, wherein the fusion protein
comprises a golgi-membrane location sequence.
45. The yeast cell of claim 43 wherein the golgi-membrane location
sequence is from Vrg4p.
46. An isolated nucleic acid molecule encoding a fusion protein,
said protein comprising a first portion and a second portion, the
first portion including the active domain of a GDP-fucose/GMP
antiporter and the second portion including a golgi localization
sequence.
47. The isolated nucleic molecule of claim 46 wherein the golgi
localization sequence is a yeast golgi localization sequence.
48. A yeast harboring the isolated nucleic acid molecule of claim
46.
49. The yeast of claim 48 further harboring a nucleic acid molecule
encoding a fucosyltransferase or a galactosyltransferase.
50. The yeast of claim 49, wherein the fucosyltransferase is
selected from Homo sapiens fucosyltransferase 1 (galactoside
2-alpha-L-fucosyltransferase, Bombay phenotype included) (FUT1);
Homo sapiens fucosyltransferase 2 (secretor status included)
(FUT2); Homo sapiens fucosyltransferase 3 (galactoside
3(4)-L-fucosyltransferase, Lewis blood group included) (FUT3); Homo
sapiens fucosyltransferase 4 (alpha (1,3) fucosyltransferase,
myeloid-specific) (FUT4); Homo sapiens fucosyltransferase 5 (alpha
(1,3) fucosyltransferase) (FUT5); Homo sapiens fucosyltransferase 6
(alpha (1,3) fucosyltransferase) (FUT6); Homo sapiens
fucosyltransferase 7 (alpha (1,3) fucosyltransferase) (FUT7); Homo
sapiens fucosyltransferase 8 (alpha (1,6) fucosyltransferase)
(FUT8); Homo sapiens fucosyltransferase 9 (alpha (1,3)
fucosyltransferase) (FUT9); and Homo sapiens protein
o-fucosyltransferase (POFUT1).
51. (canceled)
52. A pharmaceutical composition, comprising an oligosaccharide or
a glycoconjugate, wherein the oligosaccharide or the glycoconjugate
includes a molecule containing a fucose group in an .alpha.1,4
linkage to an N-acetylglucosamine group, a fucose group in an
.alpha. 1,3 linkage to an N-acetylglucosamine group, or a fucose
group in an .alpha. 1,3 linkage to a glucose group; and a
pharmaceutically acceptable carrier.
53. The composition of claim 52, wherein the molecule is selected
from the group consisting of lacto-N-difucohexaose I (LDFH-I),
lactodifucotetraose (LDFT), disialyllacto-N-neohexaose (DSLNnH),
lacto-N-fucopentaose II (LNF-II), lactodifucohexaose I (LDFH-I),
monosialyllacto-N-neohexaose II (MSLNnH-II),
monofucosylmonosialyllacto-N-octaose (MFMSLNO),
sialyllacto-N-fucohexaose II (SLNFH-II),
disialyllacto-N-fucopentaose II (DSLNFP-II),
monofucosyldisialyllacto-N-tetraose (MFDLNT), lacto-N-fucopentaose
III (LNF-III), monosialyllacto-N-neohexaose I (MSLNnH-I),
disialomonofucosyllacto-N-neohexaose (DSFLNH), 3-fucosyllactose
(3-FL), and 3'-sialyl-3-fucosyllactose (3'-S-FL), or a variant
thereof in which a sugar at the reducing end is GlcNAc.
54. The composition of claim 52, further comprising a molecule
including a fucose group in an .alpha. 1,2 linkage to a galactose
group.
55. The composition of claim 52, wherein the glycoconjugate is a
glycan, a glycolipid, or a glycoprotein.
56. The composition of claim 52, wherein the glycoconjugate further
includes a fucose group in an .alpha. 1,2 linkage to a galactose
group.
57. A method for treating or reducing the risk of infection, the
method comprising administering to a subject in need thereof an
effective amount of a composition containing an oligosaccharide or
a glycoconjugate, wherein the oligosaccharide or the glycoconjugate
includes a fucose group in an .alpha.1,4 linkage to an
N-acetylglucosamine group, a fucose group in an .alpha. 1,3 linkage
to an N-acetylglucosamine group, or a fucose group in an .alpha.
1,3 linkage to a glucose group.
58. The method of claim 57, wherein the composition further
contains an oligosaccharide including a fucose group in an .alpha.
1,2 linkage to a galactose group.
59. The method of claim 57, wherein the glycoconjugate further
includes an oligosaccharide including a fucose group in an .alpha.
1,2 linkage to a galactose group.
60. The method of claim 57, wherein the infection is caused by V.
cholerea or C. jejuni.
61. The method of claim 57, wherein the infection is an enteric
infection.
62. The method of claim 57, wherein the glycoconjugate is a glycan,
a glycolipid, or a glycoprotein.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/581,759, filed Jun. 5, 2006, which is a U.S. National Phase
of PCT Application No. PCT/US2004/040882, filed Dec. 6, 2004, which
claims priority under 35 U.S.C. .sctn.119(e)(1) from U.S.
Application No. 60/527,591, filed Dec. 5, 2003. The entire contents
of all three applications are hereby incorporated by reference.
BACKGROUND
[0002] Consumption of human milk is one of the most cost-effective
strategies known to medicine for protecting infants against
morbidity and mortality due to infectious disease. Human milk may
be considered a natural and efficacious "nutriceutical," i.e., a
model food that conveys immunologic benefits. Protection against
infectious diseases occurs through a variety of complementary
mechanisms found in human milk, including oligosaccharides and
their related glycoconjugates. Significantly enhanced immunologic
protection by breastfeeding has been demonstrated for diarrheal
diseases, respiratory tract illnesses, bacteremia, meningitis, and
necrotizing enterocolitis. Protection by breastfeeding is
especially efficacious against diarrheal disease.
[0003] Milk oligosaccharide structures are thought to serve as
receptor analogs that can inhibit pathogen binding to host ligands
(1-3). It appears that certain .alpha.1,2-linked fucosylated
oligosaccharides in human milk are associated with protection
against diarrhea due to campylobacter (2,4), caliciviruses (3-5),
and stable toxin (ST)-associated Escherichia coli (1,6,7).
[0004] Oligosaccharides and their related glycoconjugates are major
components of the innate defense system found in human milk.
Oligosaccharides, which vary from 3 to 32 sugars in size,
constitute the third-most common solid component of human milk
after lactose and lipid, but their role is immunologic rather than
nutritive. Oligosaccharides appear to have several different
immunologic functions. Several types of oligosaccharides, including
fucosyloligosaccharides, sialylated oligosaccharides, and
non-fucosylated non-sialylated oligosaccharides in human milk, have
prebiotic properties, i.e., selective stimulation of the growth of
beneficial bacteria in the intestine. Importantly, protection
against specific pathogens has been described for both fucosylated
and sialylated human milk oligosaccharides (5, 6, 24, 27, 28). Both
the fucosylated oligosaccharides and the sialylated
oligosaccharides may have structural homology to cell receptors for
enteropathogens and inhibit pathogen binding by blocking binding to
relevant cell receptors (24, 29, 30). Certain pathogens are thought
to bind to sialic acid- and fucose-containing receptors, including
enteropathogenic Escherichia coli (EPEC), rotavirus, Haemophilus
influenzae and other pathogens (30-33). In addition to the unbound
oligosaccharides, protection by glycoconjugated substances in human
milk has been demonstrated by in vitro studies and/or animal models
against labile toxin and cholera toxin, heat-stable enterotoxin of
E. coli (ETEC), campylobacter, shiga toxin, Streptococcus
pneumoniae and rotavirus (16, 17, 24, 34). Lactadherin, a 46-kDa
glycoprotein, has been found to vary in concentration in human
milk, and significant protection against symptomatic rotavirus
infection is associated with increasing concentrations of
lactadherin in maternal milk (34).
[0005] The fucose terminus of oligosaccharide structures may be
connected by an .alpha.1,2 linkage catalyzed by a
fucosyltransferase produced by the secretor gene (FUT2) or by the
fucosyltransferase I gene (FUT1), or by an .alpha.1,3 or .alpha.1,4
linkage catalyzed by fucosyltransferases produced by the Lewis gene
(FUT2) family. Polymorphisms of the secretor and Lewis genes are
known to determine expression of the Lewis blood group type,
fucosylated oligosaccharide patterns in human milk, and histo-blood
group antigens on human epithelial cell surfaces (21, 22, 35). Some
individuals are non-secretors (i.e., homozygous recessive for the
secretor gene) who do not synthesize .alpha.1,2-linked
fucosyloligosaccharides in their secretions. In Indo-European and
African populations, the prevalence of non-secretors is
approximately 20%, while in some other populations, such as
Mexicans of indigenous ancestry, non-secretors are much less common
(36-38). In certain cases it has been shown that this heterogeneity
of expression is associated with differential risk of infectious
diseases in individuals and populations (5, 6, 28, 37, 39-44).
Moreover, variation in concentration of protective oligosaccharides
in human milk may result in breastfed infants with differing levels
of protection against specific infectious diseases (21, 22, 36, 39,
45).
[0006] The most common oligosaccharides of human milk include four
.alpha.1,2-linked fucosylated oligosaccharides
(lacto-N-fucopentaose I [LNF-I], 2-fucosyllactose [2'-FL],
lacto-N-difucohexaose I [LDFH-I] and lactodifucotetraose [LDFT]);
three fucosylated oligosaccharides that lack 2-linked fucose
(lacto-N-fuco-pentaose II [LNF-II], 3-fucosyllactose [3-FL], and
lacto-N-fucopentaose III [LNF-III]); and their two precursors
(lacto-N-tetraose [LNT ] and lacto-N-neotetraose [LNneoT]). These
nine oligosaccharides are homologs of the Lewis histo-blood group
antigens, respectively: H-1, H-2, Le.sup.b, Ley, Le.sup.a,
Le.sup.x, type 1 precursor, and type 2 precursor. The most commonly
occurring specific .alpha.1,2-linked fucosylated oligosaccharide in
human milk is 2'-FL (H-2 epitope). Comparing the composition of
milks from many different mammalian species, 2'-FL is also the most
conserved oligosaccharide structure, suggesting its importance in
evolutionary biology (46). 2'-FL is absent, however, from the milk
of some species, including cow's milk.
SUMMARY
[0007] The invention features a pharmaceutical composition
comprising a molecule comprising a fucose group in an .alpha.1,2
linkage, .alpha.1,3 linkage, or .alpha.1,4 linkage to a galactose
group and a pharmaceutically acceptable carrier. The fucose can be
is contained within an LNF-I group, an 2'FL group, an LDFH-I group
or a LDFT group. In some situations the molecule is a glycan, a
glycolipid, a glycoprotein, a glycosaminoglycan or a mucin. Thus,
the fucose group can be directly or indirectly linked to a protein.
The protein or other backbone molecule can contain at least two
(three or four) different groups selected from an LNF-I group, and
2'FL group, an LDFH-I group and a LDFT group. The protein or other
backbone molecule can bear multiple copies of two or more different
groups. The composition does not contain a mammalian milk (e.g., it
does not contain human milk).
[0008] The compositions can be used as a probiotic agent, i.e., an
indigestible agent which induces or promotes colonization of the
gut by beneficial microorganisms, e.g., bacteria that improve
health or prevent disease.
[0009] In another aspect the invention features a pharmaceutical
composition comprising a purified protein modified to include at
least two (three, four, five, six, seven, eight, nine , ten or
more) different groups selected from: [0010] 2'-Fucosyllactose;
[0011] Lacto-N-fucopentaose I; [0012] Lacto-N-fucopentaose II;
[0013] 3'-Fucosyllactose; [0014] Lacto-N-fucopentaose II; [0015]
Lacto-N-difucohexaose I; [0016] Lactodifucotetraose; [0017]
LactoN-tetraose; [0018] LactoN-neotetraose; [0019]
3'-Sialyllactose; [0020] 3'-Sialyllactosamine; [0021]
6'-Sialyllactose; [0022] 6'-Sialyllactosamine; [0023]
Sialyllacto-N-neotetraose c; [0024] Monosialyllacto-N-hexaose;
[0025] Disialyllacto-N-hexaose I; [0026]
Monosialyllacto-N-neohexaose I; [0027] Monosialyllacto-N-neohexaose
II [0028] Disialyllacto-N-neohexaose [0029]
Disialyllacto-N-tetraose; [0030] Disialyllacto -N-hexaose II;
[0031] Sialyllacto-N-tetraose a; [0032] Disialyllacto-N-hexaose I;
[0033] Sialyllacto-N-tetraose b; [0034] 3'-Sialyl-3-fucosyllactose;
[0035] Disialomonofucosyllacto-N-neohexaose; [0036]
Monofucosylmonosialyllacto-N-octaose (sialyl Lea); [0037]
Sialyllacto-N-fucohexaose II; [0038] Disialyllacto-N-fucopentaose
II; and [0039] Monofucosyldisialyllacto-N-tetraose.
[0040] The protein can be modified to contain multiple copies (two,
three, fours, five, six, seven, eight, nine, 10, 15, 20, 25 or
more) of each of the different groups. The protein itself can be,
for example a human milk protein (e.g., .kappa.-casein,
.alpha.-lactalbumin, lactoferrin, bile salt-stimulated lipase,
lysozyme, serum albumin, folate-binding protein, haptocorrin,
lipoprotein lipase, glycosaminoglycan, mucin, lactoperoxidase, or
amylase) or some other protein, e.g., BSA. The composition is most
often a synthetic composition that is not a mammalian milk,
although in use it might be mixed with a mammalian milk such as
cows milk or human milk. The composition can contain: at least one
vitamin; at least one mineral; at least one edible fat; and other
nutritional components.
[0041] The invention also includes a pharmaceutical composition
comprising a purified protein modified to include at least two
different groups selected from: [0042] 2'-Fucosyllactose; [0043]
Lacto-N-fucopentaose I; [0044] Lacto-N-fucopentaose II; [0045]
3'-Fucosyllactose; [0046] Lacto-N-fucopentaose II; [0047]
Lacto-N-difucohexaose I; [0048] Lactodifucotetraose; and [0049]
2'-FLNac, wherein the protein is not modified to contain any other
oligosaccarides.
[0050] In another aspect the invention includes a synthetic
nutritional composition comprising a glycan, a glycolipid, a
glycoprotein, a glycosaminoglycan or a mucin that comprises at
least two different groups selected from an LNF-I group, and 2'FL
group, an LDFH-I group and a LDFT group. The molecule can include
at three different groups selected from an LNF-I group, an 2'FL
group, an LDFH-I group and a LDFT group and can include multiple
copies (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the same
group.
[0051] The invention includes a synthetic nutrition composition
comprising a purified protein modified to include at least two (3,
4, 5, 6, or 7) groups selected from: a Lacto-N-fucopentaose I
group, a Lacto-N-fucopentaose II group, a 2-Fucosyllactose group, a
3-Fucosyllactose group, a Lacto-N-fucopentaose II group, a
Lacto-N-difucohexaose I group, and a Lactodifucotetraose group.
[0052] The invention includes a synthetic nutrition composition
comprising a purified protein modified to include at least two (3,
4, 5, 6, or 7 or more) groups selected from: [0053]
Lacto-N-fucopentaose I; [0054] Lacto-N-fucopentaose II; [0055]
3'-Fucosyllactose; [0056] Lacto-N-fucopentaose II; [0057]
Lacto-N-difucohexaose I; [0058] Lactodifucotetraose; [0059]
LactoN-tetraose; [0060] LactoN-neotetraose; [0061]
3'-Sialyllactose; [0062] 3'-Sialyllactosamine; [0063]
6'-Sialyllactose; [0064] 6'-Sialyllactosamine; [0065]
Sialyllacto-N-neotetraose c; [0066] Monosialyllacto-N-hexaose;
[0067] Disialyllacto-N-hexaose I; [0068]
Monosialyllacto-N-neohexaose I; [0069] Monosialyllacto-N-neohexaose
II [0070] Disialyllacto-N-neohexaose [0071]
Disialyllacto-N-tetraose; [0072] Disialyllacto -N-hexaose II;
[0073] Sialyllacto-N-tetraose a; [0074] Disialyllacto-N-hexaose I;
[0075] Sialyllacto-N-tetraose b; [0076] 3'-Sialyl-3-fucosyllactose;
[0077] Disialomonofucosyllacto-N-neohexaose; [0078]
Monofucosylmonosialyllacto-N-octaose (sialyl Lea); [0079]
Sialyllacto-N-fucohexaose II; [0080] Disialyllacto-N-fucopentaose
II; and [0081] Monofucosyldisialyllacto-N-tetraose.
[0082] In another aspect the invention features a method for
treating or reducing the risk of infection (e.g., a respiratory or
enteric infection such as infection by V. cholerea or C. jejuni),
the method comprising administering (to an infant, child or adult)
a composition comprising a molecule comprising a fucose group in an
I1,2 linkage to a galactose group wherein said composition is not a
mammalian milk. Thus, any of the pharmaceutical compositions noted
herein can be administered in this method.
[0083] The invention also features a method for reducing the risk
of enteric disease in a patient, the method comprising: (a)
identifying the two most prevalent agents capable of causing
enteric disease in the geographic location of the patient; (b)
administering to the patient a composition comprising a molecule
comprising a first glycan which interferes with the binding to
epithelial cells of the first of the two most prevalent agents and
a second glycan which interferes with the binding to epithelial
cells of the second of the two most prevalent agents wherein said
composition is not breast milk.
[0084] The invention also features a method for reducing the risk
of enteric disease in a patient, the method comprising: (a)
identifying the two most prevalent agents capable of causing
enteric disease in the geographic location of the patient; (b)
administering to the patient composition comprising: i) a first
molecule comprising a first glycan which interferes with the
binding to epithelial cells of the first of the two most prevalent
agents; and ii) a second molecule comprising a glycan which
interferes with the binding to epithelial cells of the second of
the two most prevalent agents; wherein the composition is not
breast milk.
[0085] The invention also includes a yeast cell harboring a
recombinant vector comprising a nucleotide sequence encoding
GDP-mannose 4, 6 dehydratase and a nucleotide sequence encoding
GDP-L-fucose synthetase. The yeast cell can further harbor a
nucleic acid molecule encoding a GDP-fucose/GMP antiporter fusion
protein (e.g., a fusion protein that comprises a golgi-membrane
location sequence (e.g., yeast Vrg4p).
[0086] Also within the invention is an isolated nucleic acid
molecule encoding a fusion protein comprising at least a first
portion and a second portion, the first portion comprising the
active domain of a GDP-fucose/GMP antiporter and the second portion
comprising a golgi localization sequence. The golgi localization
sequence can be a yeast golgi localization sequence. The invention
also includes yeast harboring this isolated nucleic acid molecule
and optionally a nucleic acid molecule encoding a
fucosyltransferase or a galactosyltransferase, e.g., a
fucosyltransferase is selected from: [0087] Homo sapiens
fucosyltransferase 1 (galactoside 2-alpha-L-fucosyltransferase,
Bombay phenotype included) (FUT1); [0088] Homo sapiens
fucosyltransferase 2 (secretor status included) (FUT2); [0089] Homo
sapiens fucosyltransferase 3 (galactoside
3(4)-L-fucosyltransferase, Lewis blood group included) (FUT3);
[0090] Homo sapiens fucosyltransferase 4 (alpha (1,3)
fucosyltransferase, myeloid-specific) (FUT4); [0091] Homo sapiens
fucosyltransferase 5 (alpha (1,3) fucosyltransferase) (FUT5);
[0092] Homo sapiens fucosyltransferase 6 (alpha (1,3)
fucosyltransferase) (FUT6); [0093] Homo sapiens fucosyltransferase
7 (alpha (1,3) fucosyltransferase) (FUT7); [0094] Homo sapiens
fucosyltransferase 8 (alpha (1,6) fucosyltransferase) (FUT8);
[0095] Homo sapiens fucosyltransferase 9 (alpha (1,3)
fucosyltransferase) (FUT9); and [0096] Homo sapiens protein
o-fucosyltransferase (POFUT1).
[0097] In one embodiment the invention features a nucleic acid
molecule (e.g., a recombinant or isolated nucleic acid molecule
encoding a fusion protein comprising a yeast golgi localization
sequence, e.g., the golgi localization sequence of VRG4, fused to
human GDP-fucose transporter or a functional fragment thereof. In
other embodiments the invention features protein comprising,
consisting of or consisting essentially of a yeast golgi
localization sequence, e.g., the golgi localization sequence of
VRG4, fused to human GDP-fucose transporter or a functional
fragment thereof. The protein can be purified and the purified
protein can further include a heterologous amino acid sequence,
e.g., an amino-terminal or carboxy-terminal sequence. Also featured
are purified fragments of the aforementioned protein, e.g., a
fragment of at least about 75, 85, 104, 106, 113 150, 200, 250,
300, 350, 400, or 450 amino acids. The protein or fragment thereof
can be modified, e.g., processed, truncated, modified (e.g. by
glycosylation, phosphorylation, acetylation, myristylation,
prenylation, palmitoylation, amidation, addition of
glycerophosphatidyl inositol), or any combination of the above.
[0098] In another aspect, the invention features a vector, e.g., a
vector containing an aforementioned nucleic acid. The vector can
further include one or more regulatory elements, e.g., a
heterologous promoter or elements required for translation in
yeast. The regulatory elements can be operably linked to the fusion
protein in order to express the fusion protein. In yet another
aspect, the invention features an isolated recombinant cell, e.g.,
a yeast cell. containing an aforementioned nucleic acid molecule or
vector. The nucleic acid sequence can be optionally integrated into
the genome.
[0099] A "purified protein", as used herein, refers to a protein
that has been separated from other proteins, lipids, and nucleic
acids with which it is naturally associated. The protein can
constitute at least 10, 20, 50 70, 80 or 95% by dry weight of the
purified preparation.
[0100] An "isolated nucleic acid" is a nucleic acid, the structure
of which is not identical to that of any naturally occurring
nucleic acid, or to that of any fragment of a naturally occurring
genomic nucleic acid spanning more than three separate genes. The
term therefore covers, for example: (a) a DNA which is part of a
naturally occurring genomic DNA molecule but is not flanked by both
of the nucleic acid sequences that flank that part of the molecule
in the genome of the organism in which it naturally occurs; (b) a
nucleic acid incorporated into a vector or into the genomic DNA of
a prokaryote or eukaryote in a manner such that the resulting
molecule is not identical to any naturally occurring vector or
genomic DNA; (c) a separate molecule such as a cDNA, a genomic
fragment, a fragment produced by polymerase chain reaction (PCR),
or a restriction fragment; and (d) a recombinant nucleotide
sequence that is part of a hybrid gene, i.e., a gene encoding a
fusion protein. Specifically excluded from this definition are
nucleic acids present in mixtures of different (i) DNA molecules,
(ii) transfected cells, or (iii) cell clones in a DNA library such
as a cDNA or genomic DNA library. Isolated nucleic acid molecules
according to the present invention further include molecules
produced synthetically, as well as any nucleic acids that have been
altered chemically and/or that have modified backbones.
[0101] Although the phrase "nucleic acid molecule" primarily refers
to the physical nucleic acid molecule and the phrase "nucleic acid
sequence" refers to the sequence of the nucleotides in the nucleic
acid molecule, the two phrases can be used interchangeably.
[0102] The term "substantially pure" as used herein in reference to
a given polypeptide means that the polypeptide is substantially
free from other biological macromolecules. The substantially pure
polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%)
pure by dry weight. Purity can be measured by any appropriate
standard method, for example, by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
[0103] A "heterologous promoter", when operably linked to a nucleic
acid sequence, refers to a promoter which is not naturally
associated with the nucleic acid sequence.
[0104] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0105] FIG. 1 schematically depicts the Lewis synthesis pathway
applied to human milk oligosaccharide structures.
[0106] FIG. 2 schematically depicts a method for chemically
synthesizing certain oligosaccharides.
[0107] FIG. 3 schematically depicts a partially in vivo approach to
synthesizing certain oligosaccharides.
[0108] FIG. 4 depicts a synthetic scheme for milk
oligosaccharides.
[0109] FIG. 5 schematically depicts the constuction of a vector
useful for producing oligosaccharides in yeast
[0110] FIGS. 6A, 6B, and 6C are a series of graphs illustrating the
incidence of C. jejuni diarrhea, calicivirus diarrhea and moderate
to severe diarrhea of all causes in study children whose mother's
milk contains low, intermediate, or high relative amounts of (FIG.
6A) 2'-FL, (FIG. 6B) LDFH-I, and (FIG. 6C) total 2-linked
fucosylated oligosaccharide as a percent of milk oligosaccharide.
The bars indicate the cause-specific incidence rates of diarrhea in
each group; the vertical lines indicate the standard error. The
low, intermediate, and high groups each represent the
oligosaccharide values of a tertile (n=31) of the study population.
For 2'-FL, the percent of milk oligosaccharide values by group: low
(<0.29), intermediate (0.29-0.36), and high (>0.37). Compared
to the low group, campylobacter incidence in the intermediate and
high groups were both significantly (P<0.01) less. For LDFH-I,
the percent of milk oligosaccharide values by group: low
(<0.07), intermediate (0.07-0.11), and high (>0.12). Compared
to the low group, calicivirus incidence in the high group was
significantly (P=0.02) less. For total 2-linked fucosylated
oligosaccharide, the percent of milk oligosaccharide values in each
group: low (<0.72), intermediate (0.72-0.77) and high
(>0.77). Compared to the low group, incidence of
moderate-to-severe diarrhea in the intermediate and high groups
were both significantly (P<0.01) less.
[0111] FIG. 7 is a graph presenting the results of a study showing
the inhibition of Campylobacter binding to FUT1- CHO cells by H-2
ligands and H-2 mimetics and human milk oligosaccharides.
[0112] FIG. 8 is a chart presenting the results of a study showing
that cell agglutination is induced by invasive Campylobacter strain
287ip on transfected CHO cells carrying FUT1 (1,2 Fuc), FUT3
(1,3/1,4 Fuc), and FUT4 (1,3 Fuc) gene.
[0113] FIGS. 9A and 9B are a pair of graphs depicting the results
of an study showing inhibition of Campylobacter colonization in
BALB/c mice fed with 2 mg of milk fucosylated oligosaccharides
given during challenge with 10.sup.4 and 10.sup.8 CFU of bacteria
(left). Ex vivo assays of inhibition of human gut colonization of
Campylobacter with 2'-fucosyllactose (2'-FL) and milk fucosylated
oligosaccharides (OS).
[0114] FIGS. 10A and 10B are a graph and a chart depicting the
results of a study of Campylobacter colonization in transgenic mice
carrying the FUT1 gene with the WAP promoter that directs the
expression of H antigens primarily to lactating mammary gland. Pups
fed from transgenic mice cleared colonization 5 to 9 days after
challenge with Campylobacter. Control pups from non-transgenic mice
are unable to clear Campylobacter colonization. CFU=colony forming
units.
DETAILED DESCRIPTION
[0115] Specific oligosaccharides and specific combinations of
oligosaccharides can be used to treat and/or prevent infection by
various infectious agents, e.g., infectious agents associated with
enteric disorders, respiratory infections, vaginal infections,
urinary tract infections, ocular infections, or infections of the
oral cavity. Thus, specific combinations of oligosaccharides are
expected to be effective in treating and preventing cholera,
Campylobacter diarrhea, calicivirus diarrhea, Candida albicans
infection, HIV infection, and other disorders. Oligosaccharides can
be administered in monovalent or polyvalent forms, or in
combinations thereof. In the monovalent form, free oligosaccharides
can be administered singly or in combination. In polyvalent forms
two or more oliogosaccharides, which can be the same or different,
are attached to a backbone such as a mucin, bile salt stimulated
lipase, or bovine serum albumin. The oligosaccharides can be
synthesized in vivo by methods described herein. In addition,
polyvalent forms of oligosaccharides can be prepared as described
herein either partially or entirely in vivo. Also described herein
are diagnostic methods for determining which oligosaccharide or
combination of oligosaccharides is most likely to be protective for
a given individual.
[0116] Oligosaccharides
[0117] The oligosaccharides that can be used individually or in
combination, all of which are depicted below, include: fucosyl
oligosaccharides (i.e., Lacto-N-fucopentaose I;
Lacto-N-fucopentaose II; 2-Fucosyllactose; 3-Fucosyllactose;
Lacto-N-fucopentaose II; Lacto-N-difucohexaose I; and
Lactodifucotetraose); non-fucosylated, non-sialylated
oligosaccharides (i.e., Lacto-N-tetraose and Lacto-N-neotetraose);
sialyl oligosaccharides (i.e., 3'-Sialyl-3-fucosyllactose;
Disialomonofucosyllacto-N-neohexaose;
Monofucosylmonosialyllacto-N-octaose (sialyl Le.sup.a);
Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II;
Monofucosyldisialyllacto-N-tetraose); and sialyl fucosyl
oligosaccharides (i.e., 3'-Sialyllactose; 3'-Sialyllactosamine;
6'-Sialyllactose; 6'-Sialyllactosamine; Sialyllacto-N-neotetraose
c; Monosialyllacto-N-hexaose; Disialyllacto-N-hexaose I;
Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II;
Disialyllacto-N-neohexaose; Disialyllacto-N-tetraose;
Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a;
Disialyllacto-N-hexaose I; and Sialyllacto-N-tetraose b). Also
useful are variants in which the glucose (Glc at the reducing end
is replaced by N-acetylglucosamine (e.g.,
2'-fucosyl-N-acetylglucosamine (2'-FLNac) is such a variant to
2'-FL). These oligosaccharides or their non-reducing terminal
moieties can be linked to proteins, mucins, lipids, or
carbohydrates in various combinations to yield monovalent or
polyvalent glycoconjugated molecules. In the case of polyvalent
glycoconjugates two or more oligosaccharides are linked to a
backbone either directly or via a linker. The two or more
oligosaccharides can be the same or different. For example, one or
more 2'-FL and one or more 2'-FLNAc can be linked to human serum
albumin.
[0118] The oligosaccharides noted above are commonly found in human
milk. FIG.1 schematically depicts the Lewis synthesis pathway
applied to human milk oligosaccharide structures. The core type 1
structure, lacto-N-tetraose (LNT), is Gal.beta.1,3GlcNAc on the
terminal end of lactose (-R). The core for the most abundant type 2
structures in milk includes lactose (for 2'-FL, 3-FL, and LDFT),
lacto-N-neo-tetraose (for LNneoT), and Gal.beta.31,4GlcNAc on a
lactose terminus (for LNF-III). Lewis structural moieties are based
on a backbone ending in Gal-GlcNAc; however, the most prevalent
type 2 structures in human milk contain lactose (Gal-Glc) and
therefore are defined as the glucose analogs (ga) to the type 2
Lewis structures, where --R.sub.1 is --OH and -R.sub.2 is --H. True
Lewis structures, such as LNF-III, have an R1 of N-acetyl and an R2
of lactose or lactosamine. Abbreviations for the fucosyltransferase
genes are: Se (secretor gene, FUT2), Le (Lewis gene, FUT3), and
FUT4,5,6,7,9 (Lewis gene family of 3-fucosyltransferases). Blood
group A and B structures, synthesized from H-1 and H-2 antigens,
have been reported but are not major components of milk
oligosaccharides.
TABLE-US-00001 Fucosyl oligosaccharides 2'FL 2'-Fucosyllactose
Fuc.alpha.1,2Gal.beta.1,4Glc LNF-I Lacto-N-fucopentaose I
Fuc.alpha.1,2Gal.beta.1,3GlcNAc.beta.1,3Gal.beta.1,4Glc LNF-II
Lacto-N-fucopentaose II ##STR00001## 3FL 3-Fucosyllactose
##STR00002## LNF-III Lacto-N-fucopentaose III ##STR00003## LDFH-I
Lacto-N-difucohexaose I ##STR00004## LDFT Lactodifucotetraose
##STR00005##
TABLE-US-00002 Nonfucosylated, nonsialylated oligosaccharides LNT
LactoN-tetraose Gal.beta.1,3GlcNAc.beta.1,3Gal.beta.1,4Glc LNneoT
LactoN-neotetraose Gal.beta.1,4GlcNAc.beta.1,3Gal.beta.1,4Glc
TABLE-US-00003 Sialyl milk oligosaccharide structures 3'-SL
3'-Sialyllactose NANA.alpha.2,3Gal.beta.1,4Glc 3'-SLN
3'-Sialyllactosamine NANA.alpha.2,3Gal.beta.1,4GlcNAc 6'-SL
6'-Sialyllactose NANA.alpha.2,6Gal.beta.1,4Glc 6'-SLN
6'-Sialyllactosamine NANA.alpha.2,6Gal.beta.1,4GlcNAc SLNT-c
Sialyllacto-N-neotetraose c
NANA.alpha.2,6Gal.beta.1,4GlcNAc.beta.1,3Gal.beta.1,4Glc MSLNH
Monosialyllacto-N-hexaose ##STR00006## DSLNH-I
Disialyllacto-N-hexaose I ##STR00007## MSLNnH-I
Monosialyllacto-N-neohexaose I ##STR00008## MSLNnH-II
Monosialyllacto-N-neohexaose II ##STR00009## DSLNnH
Disialyllacto-N-neohexaose ##STR00010## DSLNT
Disialyllacto-N-tetraose ##STR00011## DSLNH-II
Disialyllacto-N-hexaose II ##STR00012## SLNT-a
Sialyllacto-N-tetraose a
NANA.alpha.2,3Gal.beta.1,3GlcNAc.beta.1,3Gal.beta.1,4Glc DSLNH-I
Disialyllacto-N-hexaose I ##STR00013## SLNT-b
Sialyllacto-N-tetraose b ##STR00014##
TABLE-US-00004 Sialyl fucosyl oligosaccharides 3'-S-3FL
3'-Sialyl-3-fucosyllactose ##STR00015## DSFLNH
Disialomonofucosyllacto-N- neohexaose ##STR00016## MFMSLNO
Monofucosylmonosialyllacto- N-octaose (sialyl Le.sup.a)
##STR00017## SLNFH-II Sialyllacto-N-fucohexaose II ##STR00018##
DSLNFP-II Disialyllacto-N-fucopentaose II ##STR00019## MFDLNT
Monofucosyldisialyllacto-N- tetraose ##STR00020##
[0119] Oligosaccharides for Linking to Proteins
[0120] As discussed in greater detail below, the above described
milk oligosaccharides can be covalently attached to a protein to
create O-linked (to serine or threonine) or N-linked (to
asparagines) oligosaccharides. When the milk oligosaccarides are
directly linked to a protein the Glc at the reducing end of
oligosaccharide must be replacaced by GlcNAc to create N-Acetyl
glucosamine versions of the oligosaccharides as shown below.
TABLE-US-00005 Fucosyl oligosaccharides for covalent attachment to
proteins 2'FL-N Fuc.alpha.1,2Gal.beta.1,4GlcNAc LNF-I-N
Fuc.alpha.1,2Gal.beta.1,3GlcNAc.beta.1,3Gal.beta.1,4GlcNAc LNF-II-N
##STR00021## 3'FL-N ##STR00022## LNF-III-N ##STR00023## LDFH-I-N
##STR00024## LDFT-N ##STR00025##
TABLE-US-00006 Non-fucosylated, nonsialylated oligosaccharides for
attachment to proteins LNT-N
Gal.beta.1,3GlcNAc.beta.1,3Gal.beta.1,4GlcNAc LNneoT-N
Gal.beta.1,4GlcNAc.beta.1,3Gal.beta.1,4GlcNAc
TABLE-US-00007 Sialyl milk oligosaccharide for attachment to
proteins 3'-SL-N NANA.alpha.2,3Gal.beta.1,4GlcNAc 6'-SL-N
NANA.alpha.2,6Gal.beta.1,4GlcNAc SLNT-c-N
NANA.alpha.2,6Gal.beta.1,4GlcNAc.beta.1,3Gal.beta.1,4GlcNAc MSLNH-
N ##STR00026## DSLNH-I- N ##STR00027## MSLNnH- I-N ##STR00028##
MSLNnH- II-N ##STR00029## DSLNnH- N ##STR00030## DSLNT-N
##STR00031## DSLNH- II-N ##STR00032## SLNT-a-N
NANA.alpha.2,3Gal.beta.1,3GlcNAc.beta.1,3 Gal.beta.1,4GlcNAc DSLNH-
I-N ##STR00033## SLNT-b-N ##STR00034##
TABLE-US-00008 Sialyl fucosyl oligosaccharides for attachment to
proteins 3'-S-3FL-N ##STR00035## DSFLNH-N ##STR00036## MFMSLNO-N
##STR00037## SLNFH-II-N ##STR00038## DSLNFP-II-N ##STR00039##
MFDLNT- N ##STR00040##
[0121] Also useful are variants of LNT, LNneoT, LNT-N, and LNneoT-N
having multiple copies (2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
40, 45, or 50 or more) of the internal Gal residue.
[0122] Infectious Agents
[0123] The compositions and methods described herein can be used to
treat and/or prevent infection by a variety of infectious agents
that recognize oligosaccharides. The compositions can be used to
treat and/or prevent infection by Campylobacter, V. cholerae, EPEC,
ETEC, EHEC, Shigella, Listeria, Candida albicans, HIV, Noroviruses,
rotavirus, Helicobacter pylori, and other infectious agents of the
respiratory tract, alimentary canal, vaginal tract, urinary tract,
and eye.
[0124] Campylobacter
[0125] Campylobacter strains are among the most common human and
veterinary pathogens worldwide (47-54). Although diarrhea is the
most frequent clinical presentation associated with campylobacter,
a broad clinical spectrum is observed with this infection,
including bacteremia, localized infection, and Guillain-Barre
Syndrome, a severe immunoreactive complication (47,48). In the
United States, the estimated incidence of campylobacter is two
million symptomatic infections per year, approximately 1% of the
U.S. population (49). Population-based studies in England, the
U.S., and Sweden have shown a bimodal distribution, with a peak of
illness in children less than 5 years of age and a second peak in
adolescents and young adults 15 to 29 years old (50-54). The
highest isolation rate (15 cases per 100,000) occurs in the first
year of life (51). In endemic areas of developing countries, the
isolation rate among children with diarrhea is 8% to 45%, with a
similar rate of isolation among asymptomatic children (55,56). The
annual incidence of campylobacter infections can be as high as 2.1
episodes per child-year. Foodborne infections are an emerging
concern affecting millions of individuals every year. Campylobacter
is the second most common cause of foodborne infection after
calicivirus (49,50). The alarming increase in multiply antibiotic
resistant strains of campylobacter being isolated probably results
from the use of quinolones in veterinary medicine and as animal
food supplements (57).
[0126] Children living in endemic areas develop effective natural
immunity to campylobacter infection as the result of an intense
early exposure to the organism (55,56). While immunoglobulins in
human milk provide important protection against campylobacter as
well as other causes of respiratory and gastrointestinal tract
infections (58-60), non-immunoglobulin components in human milk
also appear to play a dominant role (61-63). Among the
non-immunoglobulin protective factors in human milk, the
oligosaccharides and glycoconjugates appear to be the most
important (30,35,45). It appears that the initial steps of
attachment of campylobacter to the host cell surface, critical to
infection, involve binding to epithelial cell surface
glycoconjugates (2,28,64). Human milk oligosaccharides with
structural homology to these ligands may inhibit binding by the
pathogen (2). Thus, variable expression of these oligosaccharides
in milk due to maternal genetic heterogeneity may influence the
risk of infection in breastfed infants (42,65,66).
[0127] Recent advances in understanding the pathogenesis of
campylobacter infection have followed the sequencing of its
complete genome (67). The ability of campylobacter to adhere to and
invade the epithelial cells of the ileum and cecum is well known
(68-73). Motility and chemotaxis play a major role in the
localization of bacteria in the lower part of the intestine
(74-78). Studies of the chemotactic behavior of campylobacter have
shown a positive response to the presence of fucose, but not other
sugars, as well amino acids such as aspartate, cysteine, glutamate
and serine (76). L-Fucose is an important constituent of both bile
and mucin. These may be important factors for the affinity of the
organism for the gall bladder and the lower intestinal tract.
Environmental and chemotactic stimuli specifically upregulate the
C. jejuni flaA sigma 28 promoter (77). High pH, osmolarity, and
bile salts, including deoxycholate, also upregulate the fla
promoter while high viscosity results in downregulation of the fla
promoter. Considering that bile and mucin are mixed together in the
intestine, and that C. jejuni colonization of the mucin layer is a
prerequisite for pathogenesis in vivo, the overall response would
be an increase in flaA synthesis and chemotaxis towards the mucin
layer. These data explain the importance of fucose in the
pathogenesis of campylobacter infection in the gastrointestinal
tract (79).
[0128] Early studies demonstrated inhibition of campylobacter
adherence to intestinal epithelial cells by L-fucose (80).
Fucosylated human milk oligosaccharides inhibit cell adherence in
vitro and colonization of gut mucosa in vivo by (28).
Characterization of these human milk carbohydrate residues showed
that .alpha.1,2-fucosylated oligosaccharides are the main active
components, and that these oligosaccharides, particularly those
containing H-2 epitopes, can inhibit campylobacter adherence to its
host receptor. The specificity of binding to .alpha.1,2-fucosyl
moieties was confirmed by transfecting Chinese hamster ovary (CHO)
cells with the human gene for human .alpha.1,2-fucosyltransferase
whose expression product catalyzes the final step of H antigen
synthesis (81). While parental non-transfected CHO cells (which do
not express H antigen) are not infected with invasive
campylobacter, transfected cells are susceptible to adherence and
invasion by campylobacter. The differential expression of blood
group antigen H-2 at different sites of the gastrointestinal tract
could explain the essential features of the pathology of
campylobacter diarrhea, and likewise, the localization of
infection. Mice transfected with the FUT1 gene, flanked by the
murine whey acidic protein promoter, specifically express FUT1 in
milk during lactation (82). These transfected mice produce large
amounts of H-2 antigens in milk, whereas the wild type mice produce
none. Pups nursing these transfected dams were protected against
intestinal colonization by campylobacter. These data support the
concept that H antigens are the intestinal ligands essential for
the binding of campylobacter to the intestinal tract. In milk,
soluble ligands containing H-2 epitopes can serve as receptor
analogs that protect infants from campylobacter infection, and they
may represent an important component of the innate immune system of
human milk (63).
[0129] Cholera
[0130] Susceptibility to cholera appears to be related to the
ABH(O) tissue-blood group antigens (83, 84). Studies of immunity to
experimental cholera in human volunteers showed that blood group O
was significantly more frequent in volunteers who developed severe
cholera (stool volume >5.0 L). A large epidemiological study
done in Bangladesh demonstrated that patients with cholera were
twice as likely to have O blood group as community controls (44).
This study also showed that individuals with the most severe type
of diarrhea were most likely to be of blood group O (68% versus
31%; P<0.01). One possible explanation for the increased
severity of cholera in persons of blood group O is that an enhanced
adherence of vibrios to the intestinal mucosa may occur in such
individuals. Because of this increased susceptibility of
individuals of O blood group to develop severe cholera, the
immunogenicity and protective efficacy of a cholera vaccine in
persons of this group have also been examined. Randomized,
double-blind, placebo controlled studies with an attenuated cholera
vaccine showed a stronger immune response in persons of the O blood
group type, with significantly higher reciprocal geometric mean
titers than the non-O group vaccinees (85); this information
supports the concept that Vibrio cholerae adheres more avidly to
intestinal mucosa of persons of blood group O, inducing a
heightened vibrocidal response. However, the biological and
molecular basis for this genetically related protection has not yet
been elucidated. Previous studies have demonstrated that
hemagglutination produced by V. cholerae with human 0 erythrocytes
can be inhibited with L-fucose (86). The human intestinal
epithelium is rich in glycolipids and glycoproteins of the ABH(O)
and Lewis histo-blood group antigens (87). The H(O) antigen
consists of a backbone of fucose .alpha.1,2DGal.beta.1. It is
therefore conceivable that the H antigen serves as a receptor for
V. cholerae. As we have previously shown in campylobacter, V.
cholerae also binds in vitro to ABH-Lewis neoglycoproteins and also
attaches preferentially to .alpha.1,2 fucose determinants expressed
on the surface of FUT1-transfected CHO cells (69). There is also
recent evidence that the B subunit of cholera toxin and the labile
toxin (LT) of enterotoxigenic Escherichia coli not only binds with
high affinity to GM1 ganglioside, but LTB also interacts with
N-acetyl lactosamine-terminated glycoconjugates (87,88).
[0131] Other Pathogens
[0132] An association between Lewis and secretor histo-blood group
genotypes appears to be associated with a number of different
pathogens. For example, Ikehara and others have reported that Lewis
and secretor histo-blood group genotypes are associated with
differing risk of infection with Helicobacter pylori (42). Huang et
al and have reported that secretor blood group individuals have
increased susceptibility to several strains of caliciviruses (3).
Influenza virus binding has been shown to vary in relation to host
Lewis blood group antigens (89). Further, Raza et al reported that
secretor children have increased risk of hospitalization for
respiratory infections due to influenza viruses A and B,
rhinoviruses, respiratory syncytial virus, and echoviruses
(90).
[0133] Resistance to ST-associated E. coli (27, 6), and several
strains of caliciviruses is associated with inhibition by
.alpha.1,2-linked fucosylated oligosaccharide structures. A human
milk fucosyl oligosaccharide inhibits the ability of ST to induce
diarrehea in vivo. Studies with caliciviruses have shown that
Norwalk virus-like particles bind to tissue sections of the
gastro-duodenal junction from secretors but not from nonsecretors
(5), and that binding is blocked by milk from a secretor (91).
Volunteers challenged with Norwalk virus become symptomatically
infected only if they are secretors. Moreover, it appears that
Le.sup.b epitopes and other 2-linked fucosylated oligosaccharide
structures inhibit binding by major strains of caliciviruses.
[0134] Synthesis of Oligosaccharides and Fucosylated Glycans
[0135] Described below are methods, employing recombinant yeast and
bacteria, for the synthesis of oligosaccharides and various
fucosylated glycans.
[0136] Conventionally, oligosaccharides are isolated from milk,
e.g., human milk, or chemically synthesized. Oligosaccharides
isolated human milk are generally very expensive and may be
contaminated with infectious agents. Sophisticated methods for
chemically synthesizing oligosaccharides are available. Chemical
synthesis of oligosaccharides involves the differential
derivatization of the hydroxyl groups of each sugar that is added
in sequence to form the desired structure. The hydroxyl group that
participates in each linkage must be protected by a different
protecting group than the hydroxyl groups that are not involved in
linkage. Thus, synthesizing complex structures involves the use of
many blocking agents. Over the past 20 years suitable blocking
agents have been developed to allow the complete chemical synthesis
of complex glycan structures that were heretofore not feasible.
[0137] More recently, the cloning of certain biosynthetic genes has
made available certain of the components required chemienzymatic
synthesis of oligosaccharides. In this approach, genes encoding
enzymes that catalyze critical steps in the formation of essential
precursors or enzymes that use these precursors to make the desired
product are inserted into the appropriate plasmids and transfected
into a well-defined bacterium such as E. coli. The enzyme is
isolated and purified and attached to a solid-phase, which is then
packed into a column. The precursors for each reaction are put
through the column, and the product is isolated from the eluate.
Examples of this technology includes the conversion of the
GDP-mannose to GDP-fucose, and the subsequent transfer of the
fucose from GDP-fucose onto lactose to form 2'-FL (92). This
approach readily allows scale up of the reaction to produce gram-
and kilogram quantities of both 2'-FL and 2'-FLNAc.
[0138] Production of Oliogosaccharides in Recombinant Yeast
[0139] Suitable yeast include those belonging to the genera Candida
(e.g., Candida albicans), Debaryomyces, Hansenula, Kluyveromyces,
Pichia and Saccharomyces (e.g., Saccharomyces cerevisae and Pichia
pastori). However, other organisms can be used for in vivo
production of oligosaccharides, e.g., E. coli and baculovirus
systems.
[0140] In generating the vectors and engineered yeast described
below any of a variety of promoters, expression control elements
and termination sequences can be employed. Any promoter that is
generally used for a yeast expression system and allows expression
of a gene in yeast can be used. Examples of such a promoters
include PGK, GAP, TPI, GAL1, GAL1O, ADH2, PH05 and CUP1. Useful
terminators include ADH1, TDH1, TFF and TRPS.
[0141] A nucleic acid sequence introduced into yeast can be
integrated into the yeast genome or can be carried on a vector,
e.g., a vector containing the yeast 2T sequence that allows
autonomous replication. A number of autonomous vector for
expression in yeast are know, including: YEp51, pYES2, YEp351,
YEp352 or the like. Often the vectors include a selectable maker
such as HIS3, TRP1, LEU2, URA3, ADE2, SUC2, or LYS2.
[0142] Vectors and expression systems suitable for expressing
proteins in yeast are well known to those of ordinary skill in the
art. Such vectors and expression systems are described in U.S.
Published Patent Application 20010012630; U.S. Pat. Nos. 6,312,923;
6,306,625; 6,300,065; 6,258,566; 6,172,039; 6,165,738; 6,159,705;
6,114,147; 6,100,042; 6,083,723; 6,027,910; 5,876,951; 5,739,029;
5,602,034; 5,482,835; 5,302,697; and RE 37,343.
[0143] Production of 2'-FL and 2'-FLNAc in vitro
[0144] 2'-FL and 2'-FLNAc can be produced by: 1) providing a
genetically engineered yeast (e.g., Saccharomyces cerevisiae) that
produces GDP-fucose from GDP-mannose, 2) obtaining a fraction from
the yeast that contains GDP-fucose and 3) exposing the fraction to
the appropriate fucosyltransferase and substrate to produce, e.g.,
2'-FL or 2'-FLNAc. Yeast which produce GDP-fucose can be created by
transforming yeast with: 1) a nucleic acid molecule encoding a
GDP-mannose 4, 6 dehydratase (e.g., H. pylori GDP-mannose 4, 6
dehydratase; Genbank.RTM. Accession No. AAD05625.1 GI:4154547; SEQ
ID NO:6 or E. coli GMD), an enzyme which converts GDP-mannose to
GDP-4-keto-6-D-mannose, and 2) a nucleic acid molecule encoding
GDP-4-keto-6-deoxy-alpha-D-mannose 3,5-epimerase-4-reductase (e.g.,
H. pylori GDP-L-fucose synthetase; GenBank.RTM. Accession No:
AAL33678.1 GI:17017466; SEQ ID NO:7 or E. coli GMER(FX)), an enzyme
which converts GDP-4-keto-6-D-mannose to GDP-L-fucose via
epimerization and reduction. One approach for engineering yeast to
produce GDP-mannose has been described previously (93). GDP-fucose
can be partially or completely purified from the genetically
engineered yeast that have been cultured under conditions which
permit the synthesis of GDP-fucose. The fully or partially purified
GDP-fucose can be converted to 2'-FL using purified H. pylori
alpha-1,2-fucosyltransferase (FucT2; GenBank Accession No. AAC99764
GI:4093139; SEQ ID NO:8) with lactose essentially as described
previously (92).
[0145] In one embodiment, GDP-fucose is produced in yeast
expressing E. coli GDP-D-mannose-4,6 dehydratase (encoded by the
gmd-gene) and E. coli GDP-4-keto-6-deoxy-D-mannose
epimerase/reductase (encoded by the wcaG-gene). The E. coli genes
can be transformed into yeast using a vector referred to as the
pESC-leu/gmd/wcaG vector. To create this vector, the E. coli
gmd-gene and wcaG-gene, i.e., GMER (FX) is inserted into
pESC-leu-vector under GAL1 and GAL10 promoters, respectively. The
gmd gene was inserted in frame with c-myc-epitope and the wcaG gene
was inserted in frame with the FLAG-epitope. S. cerevisiae
transfected with this vector can produce approximately 0.2 mg/L of
GDP-fucose without addition of any external GDP-mannose (93). As
explained above, the synthesis of 2'-FL and 2'-FLNAc can be carried
out using fucosyltransferase enzymes expressed in E. coli, e.g.,
human (FUT1 and FUT2) or H. pylori (FucT2). These
fucosyltransferase genes are inserted into E. coli an appropriate
vector such as pGEX4T-1, and overexpressed. The fusion protein is
purified by affinity chromatography on a GSTrap-column
(Pharmacia/Amersham Biosciences). The purified protein can be
covalently linked to sepharose through the solid phase reductive
amidation. GDP-fucose and lactose in a suitable buffer (e.g., PBS)
is passed over the column to produce 2'-FL. GDP-fucose and
N-acetylactosamine are passed over the column to produce 2'-FLNAc.
Excess GDP-fucose and nucleotide phosphate are be removed by ion
exchange. The fucosylated oligosaccharides are separated from their
starting materials by passing them through a Ulex europaeus
affinity column. The yield and purity of these products is assessed
by HPLC analysis of the resulting oligosaccharides.
[0146] Production of 2'-FL and 2'-FLNAc in vivo using Cell Wall
Expressed Transferases
[0147] Described below is an improved method for synthesis of
oligosaccharides. The method entails the use of yeast that have
been genetically engineered to: 1) convert GDP-mannose to
GDP-fucose; 2) contain a fucosyltransferase so that they can
produce 2'-FL or another fucosylated products of interest in
vivo.
[0148] For coversion of GDP-mannose to GDP-fucose in yeast, the
cells are engineered to express a suitable GDP-mannose 4, 6
dehydratase gene. For example, H. pylori GDP-mannose 4, 6
dehydratase (Genbank Accession No. AAD05625.1 GI:4154547; SEQ ID
NO:6). This enzyme converts GDP-mannose to GDP-4-keto-6-D-mannose.
The yeast is also engineered to express a suitable
GDP-4-keto-6-deoxy-alpha-D-mannose 3,5-epimerase-4-reductase (e.g.,
H. pylori GDP-L-fucose synthetase; GenBank.RTM. Accession No:
AAL33678.1 GI:17017466; SEQ ID NO:7), which converts
GDP-4-keto-6-D-mannose to GDP-L-fucose via epimerization and
reduction.
[0149] In this method fucosyltransferase is expressed on the
extracellular side of the yeast cell wall in order to effectively
produce fucosylated glycans. This is accomplished by creating a
fusion protein in which all or a functional portion of the yeast
cell wall protein, PIR, is fused to the amino terminus of a
fucosyltransferase such as H. pylori FucT2 or human
I-1,3,fucosyltransferase (FucT; encoded by the FUT6 gene) . The
generation of nucleic acid molecules encoding such proteins is
described in Abe et al. (FEMS Yeast Research 4:417, 2004) and Abe
et al. U.S. Published Application 20030059872.
[0150] Useful PIR include S. cerevisiase PIR1 (SEQ ID NO:1;
GenBank.RTM. Accession No. Q03178 GI:417492). In this 341 amino
acid (aa) protein, aa 1-18 is the signal sequence, aa 19-63 is the
propeptide, 61-341 is the mature protein, within which aa 83-101,
102-125, 126-144, 145-163, 164-182, 183-201 and 202-220 are
repetitive regions. S. cerevisiase PIR2 (SEQ ID NO:2; GenBank.RTM.
Accession No. BAA02886.1 GI:218459); and PIR3 (SEQ ID NO:3;
GenBank.RTM. Accession No. 537788 GI:481107).
[0151] Fucosyltransferases which can be fused include human
galactoside 2-alpha-L-fucosyltransferase 2 (fucosyltransferase 2;
FUT2; GenBank.RTM. Accession No. Q10981; GI:1730125; SEQ ID NO:4).
This 343 aa protein has a transmembrane domain that extends from aa
15 to aa 28. Since transmembrane domain and sequences amino
terminal to the transmembrane domain are not required in the
PIR-FUT2 fusion protein, the PIR-FUT2 fusion protein can include aa
29-343 of SEQ ID NO:4 and need not include aa 1-28 of SEQ ID NO:4.
Other useful fucosyltransferases include human
alpha-(1,3)-fucosyltransferase (galactoside 3-L-fucosyltransferase;
fucosyltransferase 6; FUT6; GenBank.RTM. Accession No. P51993
GI:1730136; SEQ ID NO:5). This 359 aa protein has transmembrane
domain that extends from aa 15 to aa 34. Since transmembrane domain
and sequences amino terminal to the transmembrane domain are not
required in the PIR-FUT6 fusion protein, the PIR-FUT6 fusion
protein can include aa 35-359 of SEQ ID NO:5 and need not include
aa 1-34 of SEQ ID NO:5. Fucosyltransferases from other species,
such Helicobacter pylori as alpha-(1,3)-fucosyltransferase
(GenBank.RTM. Accession No. AAB93985.1 GI:2240202; SEQ ID NO:6) can
be used.
[0152] A number of glycosyltransferases, including
fucosyltransferases, galactosyltransferases, glucosyltransferases,
mannosyltransferases, galactosaminyltransferases,
sialyltransferases and N-acetylglucosaminyltransferases are known
and can be used in the above-described method. The sequence and
activity of glycosyltransferases are described in, for example,
U.S. Pat. Nos. 6,291,219; 6,270,987; 6,238,894; 6,204,431;
6,143,868; 6,087,143; 6,054,309; 6,027,928; 6,025,174; 6,025,173;
5,955,282; 5,945,322; 5,922,540; 5,892,070; 5,876,714; 5,874,261;
5,871,983; 5,861,293; 5,859,334; 5,858,752; 5,856,159; 5,545,553;
and RE 37,206. Additional transferases are noted below.
[0153] Production of Glycans in Yeast using a GDP-fucose/fucosee
Antiporter
[0154] Described below is a method for producing fucosylated
glycans in yeast. This general approach can be used to produce
other glycans in yeast. As noted above, yeast, a common food
ingredient, is a rich natural source of GDP-mannose that with the
addition of the genes for two enzymes will produce GDP-fucoses,
which is the direct precursor for the synthesis of fucosylated
oligosaccharides and fucosylated glycans. With insertion of
fucosyltransferase genes, fucosylated glycans can be
synthesized.
[0155] The method of the invention improves the production of
GDP-fucose and fucosylated glycan by placing all of the the all
needed genes in one cassette and the providing an antiporter for
GDP-fucose/GMP (160). This antiporter shuttles GDP-fucose from the
cytoplasm, its site of synthesis, to the lumen of the Golgi, the
site of fucosylation (driving the synthesis of more GDP-fucose in
the cytoplasm), while shuttling GMP from the lumen of the Golgi
(driving fucosylation) to the cytoplasm, where it would be recycled
into more GDP-fucose (161).
[0156] Production of 2'-fucosyllactose (2'-FL) in Yeast
[0157] Both Saccharomyces cerevisiae and Pichia pastoris can be
used to produce fucosylated glycans. First, a single cassette is
used to introduce the two enzymes (GDP-mannose 4,6 dehydratase, and
GDP-L-fucose synthetase from, for example, H. pylori: 162)
necessary to produce GDP- L-fucose in situ. A suitable cassette can
be produced as follows. First, a plasmid is constructed by
replacing the smaller EcoRI/Xba I fragment in pPIC9K with the
smaller EcoRI/XbaI fragment from pAO815. The integrative plasmid
pPIC9K contains the bacterial kanamycin-resistance gene between
HIS4 (the histidinol dehydrogenase gene) and 3' AOX1 (the alcohol
oxidase gene) for screening the multi-copy gene transformants. The
coding sequences for GDP-D-mannose 4, 6-dehydratase (GMD) and
GDP-L-fucose synthetase (GFS) are amplified from plasmid DNA
pET15b-GMD and pET15b-GFS by PCR using primers incorporating 5'
EcoRI sites, and subcloned into the EcoRI site of pPIC9K to
generate the plasmids pPIC9K/GMD and pPIC9K/GFS respectively. The
GFS expression cassette is removed from pPIC9K/GFS with Bgl II/BamH
I and subcloned into the BamH I site of pPIC9K/GMD to generate the
co-expression vector, which contains expression cassettes for GMD
and GFS (163). The genes, now incorporated onto one plasmid, are
integrated into the P. pastoris chromosome by electroporation with
the co-expression plasmid digested with the restriction
endonuclease Sal I; transformants are isolated on kanamicin medium.
Each gene has its own methanol-inducible alcohol oxidase 1 promoter
and transcription terminator on the chromosomal DNA of P. pastoris
strain GS 115 his4. The proteins are co-expressed intracellularly
under methanol induction. The fermentation process consists of the
three distinctive phases; glycerol batch phase for initial cell
growth, glycerol fed-batch phase for AOX1 derepression and high
cell density, and induction phase for expression of these enzymes
(163)The production of sugar nucleotides is monitored by capillary
electrophoresis (164, 165).
[0158] The recombinant S. cerevisiae for production of GDP-fucose
is constructed through mating. First individual expression vectors
are created using the plasmid pGLD, which contains the
glyceraldehyde-3-phosphate dehydrogenase (GLDp) promoter and
phosphoglycerol kinase (PGKt) terminator. Fragments of GMD and GFS
genes are inserted into the plasmids..sup.54 These plasmids are
inserted by electroporation into S. cerevisiae ATCC60729 (Mata;
his, trp1, leu2, ura3) and ATCC60729 (Mata; his, trp1, leu2, ura3).
The two kinds of mating types in yeast cells, .alpha.-type and
.alpha.-type, can make a diploid, which carries genes originating
from .alpha.-type and .alpha.-type. Diploids containing GMD and GFS
genes are formed and produce the enzymes.
[0159] The fermentation process consists of the two phases: 1)
batch phase for initial cell growth and expression of these enzymes
and 2) fed-batch phase for high cell density and high expression of
these enzymes. In the fed-batch phase, the feed medium (200 ml of
80% sucrose solution) is fed using a DO-stat of 50% air saturation.
Yeast constructs grown under these conditions are assessed for
enzyme expression, stability of plasmids, and overall yield. The
production of sugar nucleotides is monitored by capillary
electrophoresis (164, 165).
[0160] The high capacity for production of GDP-mannose in yeast
provides potential capacity for the transformants to produce large
amount of GDP-fucose. GDP-mannose production could be further
channeled toward GDP-fucose by inhibiting the production of
high-mannose structures in the yeast (166, 167). This can be
achieved, for example, by using Ochlp (alfa 1,6
manosyltransferase)deletion mutants or by including of heterologous
genes coding for an alpha 1,2 manosidase, a GlcNac transferase, and
a UDP-GlcNac transporter. (Hamilton et al. 2003 Science
301:1244)
[0161] Conversion of GDP-mannose to GDP-fucose can be inhibited by
moderate build up of GDP-fucose (168, 169). In mammals, this is
overcome by an antiporter, a Golgi membrane spanning transporter
that shuttles GDP-fucose from the cytoplasm to the lumen of the
smooth ER/Golgi, where it is utilized for fucosylation, releasing
free GDP, which is returned by the antiporter to the cytoplasm
(161). Thus, in cytoplasm, GDP-fucose can be produced as fast as it
is consumed. This elegant control mechanism allows extensive
fucosylation in mammals, especially in human mammary epithelial
cells, which can be introduced into yeast by inserting the
antiporter gene responsible for GDP-fucose movement across ER/Golgi
membranes (160). For this antiporter to be useful, it must insert
itself into the membrane of the Golgi. GDP-mannose, the principal
nucleotide sugar for glycosylation in the Golgi of Saccharomyces
cerevisiae, is transported into the lumen by the antiporter VRG4
gene product (GenBank.RTM. Accession No. P40107 GI:72961 1; SEQ ID
NO:9). Mutant Vrg4 proteins lacking the N-terminal cytosolic tail
do not localize to the Golgi membrane, while fusion of the N
terminus of Vrg4p to non-Golgi membrane proteins promotes their
transport to the Golgi; thus, the N terminus navigates protein
expression specifically to the Golgi (170). By fusing the N
terminus of the Vrg4p yeast antiporter (amino acids 1-53 or 21-53
or 31-53) to the human GDP-fucose transporter (GenBank Accession
No. AAK50397.1 GI:13940506; SEQ ID NO:10; AAK51705.1 GI:14009667;
SEQ ID NO:11), it can be directed into the yeast Golgi membrane
(171).
[0162] Any suitable fucose transporter can be fused to a golgi
localization sequence in order to create a fuccose transporter that
will shuttle fucose to the lumen of smooth ER and golgi. The
following GDP-fucose transporters may be used. [0163] 1:
Genbank.RTM. Accession No. Q968A5; gi|20138279| [0164] 2:
Genbank.RTM. Accession No. XP.sub.--508388; gi|55635789| [0165] 3:
Genbank.RTM. Accession No. Q9VHT4; gi|20138437| [0166] 4:
Genbank.RTM. Accession No. NP.sub.--997597; gi|46877098| [0167] 5:
Genbank.RTM. Accession No. NP.sub.--665831; gi|22003876| [0168] 6:
Genbank.RTM. Accession No. EAL38393; gi|54659831| [0169] 7:
Genbank.RTM. Accession No. EAL38067; gi|54659493| [0170] 8:
Genbank.RTM. Accession No. NP.sub.--060859; gi|37059731| [0171] 9:
Genbank.RTM. Accession No. AAS46733; gi|144151600| [0172] 10:
Genbank.RTM. Accession No. XP.sub.--421127; gi|50748147| [0173] 11:
Genbank.RTM. Accession No. NP.sub.--732412; g1|124648166| [0174]
12: Genbank.RTM. Accession No. NP.sub.--723268; g1|24582476| [0175]
13: Genbank.RTM. Accession No. NP.sub.--476859; gi|24639454| [0176]
14: Genbank.RTM. Accession No. NP.sub.--525033; gi|18079265| [0177]
15: Genbank.RTM. Accession No. NP.sub.--524191; gi|17737703| [0178]
16: Genbank.RTM. Accession No. NP.sub.--523502; gi|17648113| [0179]
17: Genbank.RTM. Accession No. NP.sub.--477264; g1|17137388| [0180]
18: Genbank.RTM. Accession No. BAC40223; gi|26353186| [0181] 19:
Genbank.RTM. Accession No. BAC38127; gi|26348975| [0182] 20:
Genbank.RTM. Accession No. BAC34181; gi|26341038| [0183] 21:
Genbank.RTM. Accession No. BAC32554; gi|26337737| [0184] 22:
Genbank.RTM. Accession No. BAC30770; gi|26334105| [0185] 23:
Genbank.RTM. Accession No. BAC30595; gi|26333755| [0186] 24:
Genbank.RTM. Accession No. BAC28226; gi|26328975| [0187] 25:
Genbank.RTM. Accession No. BAC27199; gi|26326911| [0188] 26:
Genbank.RTM. Accession No. Q96A29; gi|20138280| [0189] 27:
Genbank.RTM. Accession No. NP.sub.--200520; gi|15242035| [0190] 28:
Genbank.RTM. Accession No. NP.sub.--566487; gi|18400381| [0191] 29:
Genbank.RTM. Accession No. AAO50954; gi|28828290| [0192] 30:
Genbank.RTM. Accession No. NP.sub.--502550; gi|25150865| [0193] 31:
Genbank.RTM. Accession No. NP.sub.--505467; gi|25150188| [0194] 32:
Genbank.RTM. Accession No. NP.sub.--741360; gi|25148765| [0195] 33:
Genbank.RTM. Accession No. NP.sub.--503604; gi|25146317| [0196] 34:
Genbank.RTM. Accession No. NP.sub.--500371; gi|17538248| [0197] 35:
Genbank.RTM. Accession No. XP.sub.--230292; gi|34856560| [0198] 36:
Genbank.RTM. Accession No. AAL62491; gi|18252816| [0199] 37:
Genbank.RTM. Accession No. AAK51705; gi|14009667| [0200] 38:
Genbank.RTM. Accession No. AAK50397; gi|13940506| [0201] 39:
Genbank.RTM. Accession No. AAK50396; gi|13940504| [0202] 40:
Genbank.RTM. Accession No. AAK49910; gi|13936720| [0203] 41:
Genbank.RTM. Accession No. AAK49909; gi|13936718| [0204] 42:
Genbank.RTM. Accession No. AAK49908; gi|13936716|
[0205] There are a number of sources of suitable golgi localization
sequences, including:
[0206] The golgi localization sequence from the S. cerevisae
GDP-Gal porter (GenBank Accession No. AAT92855.1 GI:51013123) can
also be used.
[0207] Yeast Producing 2'-FL, 3-fucosyllactose (3-FL),
lactodifucotetraose (LDFT), lacto-N-fucopentaose I,
lacto-N-fucopentaose II, or lacto-N-hexaose
[0208] Many useful oligosaccharides contain lactose at the reducing
end and fucose at the nonreducing end. Oligosaccharides of type 1
structure may have fucosyl .alpha.1,4 linked to
N-acetylglucosamine, whereas those of type 2 structure may have
fucosyl .alpha.1,3 linked to N-acetylglucosamine or glucose. Either
type may contain fucosyl .alpha.1,2 linked to galactose. The
addition of fucose to an oligosaccharide by a .alpha.1,2 linkage is
catalyzed by fucosyltransferases produced by FUT2. The addition of
fucose by an .alpha.1,3 or .alpha.1,4 linkage is catalyzed by
fucosyltransferases produced by FUT3 or other genes of this family.
The synthesis of milk oligosaccharides by pathways defined by these
enzymes is described in FIG. 4. Thus, in addition to the two enzyme
genes GMD and GFS, the development of yeasts as biocatalysts for
synthesis of human milk oligosaccharides requires the co-production
of FUT2, FUT3, or both, in addition to a GDP-fucose anti-porter
expressed on the golgi. Plasmid construction of FUT2 and FUT3, and
the transformation of yeast (P. pastoris and in S. cerevisiae) is
performed as discussed above. For example, the insertion of the
FUT2 (or FUT3, or both) genes into P. pastoris can be accomplished
by transformation with a vector constructed as shown in FIG. 5.
When the recombinant yeasts are fed lactose, they will produce
2'-FL, 3-fucosyllactose (3-FL), or lactodifucotetraose (LDFT). When
the yeasts are fed lacto-N-tetraose, they will produce the type 1
Lewis epitopes, lacto-N-fucopentaose I, lacto-N-fucopentaose II, or
lacto-N-hexaose.
[0209] Fucosyltransferases and Galactosyltransferases
[0210] Various desired oligosaccharides can be produced by
expressing the proper transferase in the cell. Suitable
fucosyltransferases include: [0211] Genbank.RTM. Accession No. NM
000148
[0212] Homo sapiens fucosyltransferase 1 (galactoside
2-alpha-L-fucosyltransferase, Bombay phenotype included) (FUT1)
[0213] Genbank.RTM. Accession No. NM 000511
[0214] Homo sapiens fucosyltransferase 2 (secretor status included)
(FUT2) [0215] Genbank.RTM. Accession No. NM 000149
[0216] Homo sapiens fucosyltransferase 3 (galactoside
3(4)-L-fucosyltransferase, Lewis blood group included) (FUT3)
[0217] Genbank.RTM. Accession No. NM 002033
[0218] Homo sapiens fucosyltransferase 4 (alpha (1,3)
fucosyltransferase, myeloid-specific) (FUT4) [0219] Genbank.RTM.
Accession No. NM 002034
[0220] Homo sapiens fucosyltransferase 5 (alpha (1,3)
fucosyltransferase) (FUT5) [0221] Genbank.RTM. Accession No. XM
012800
[0222] Homo sapiens fucosyltransferase 6 (alpha (1,3)
fucosyltransferase) (FUT6) [0223] Genbank.RTM. Accession No. XM
056659
[0224] Homo sapiens fucosyltransferase 7 (alpha (1,3)
fucosyltransferase) (FUT7) [0225] Genbank.RTM. Accession No. NM
004480
[0226] Homo sapiens fucosyltransferase 8 (alpha (1,6)
fucosyltransferase) (FUT8) [0227] Genbank.RTM. Accession No. NM
006581
[0228] Homo sapiens fucosyltransferase 9 (alpha (1,3)
fucosyltransferase) (FUT9) [0229] Genbank.RTM. Accession No.
AF375884
[0230] Homo sapiens protein o-fucosyltransferase (POFUT1)
[0231] Suitable Gal.beta.1,3 transferase galactosyltransferases
include: .beta.1,3Gal T core (Genbank Accession No..RTM. AF155582);
.beta.1,3GalT1 (Genbank Accession No..RTM. AF117222);
.beta.1,3GalT2 (Genbank Accession No..RTM. AF288390);
.beta.1,3GalT3 (Genbank Accession No..RTM. AF132731);
.beta.1,3GalT4 (Genbank Accession No..RTM. AB026730);
.beta.1,3GalT5 (Genbank Accession No..RTM. AF145784); and
.beta.1,3GalT6 (Genbank Accession No..RTM. AY050570).
[0232] Suitable Gal.beta.1,4 transferase galactosyltransferases
include: .beta.1,4GalT1(Genbank Accession No..RTM. D29805);
.beta.1,4GalT2 (Genbank Accession No..RTM. AB024434);
.theta..beta.1,4GalT3 (Genbank Accession No..RTM. AB024435);
.beta.1,4GaltT4 (Genbank Accession No..RTM. AF022367 Lc2 synthase);
.beta.1,4GalT5 (Genbank Accession No..RTM. AB004550);
.beta.1,4GalT6 (Genbank Accession No..RTM. AB024742); and
.beta.1,4GalT7 (Genbank Accession No..RTM. AB028600).
[0233] A suitable blood group B Gal.alpha.1,3 transferase
galactosyltransferase is Blood group B Gal.alpha.1,3 T (Genbank
Accession No..RTM. AF134414).
[0234] Generally, human FUT1 and FUT2 are useful for adding I1,2
linkages; human FUT3 is useful for adding I1,4 linkages; human
FUT5, FUT6, FUT7, and FUT9 are useful for adding I1,3 linkages; and
FUT8 is useful for adding I1,6 linkages.
[0235] Yeast Producing lacto-N-tetraose (LNT) and
lacto-N-neo-tetraose (LNneoT)
[0236] The human milk oligosaccharides include two tetraoses, LNT
(Gal.beta.1,3 GlcNAc.beta.1,3Gal .beta.1,4 Glc) and LNneoT
(Gal.beta.1,4 GlcNAc.beta.1,3 Gal.beta.1,4Glc) that are precursors
to common milk fucosyloligosaccharides. To synthesize
lacto-N-tetraose, .beta.1,3GlcNAc transferase transfers GlcNAc from
UDP-GlcNAc to lactose to synthesize lacto-N-triose II; then
galactose is transferred to lacto-N-triose II by GlcNAc.beta.1,3Gal
transferase. If the genes for this synthesis are inserted into
wild-type yeast, LNT will be produced; if inserted into a FUT2
construct, LNF-I will result; if inserted into a FUT3 construct,
LNF-II will result; if inserted into a FUT2/FUT3 construct,
lacto-N-difucohexaose (LDFH-I) would be the product. Similarly,
LNneoT will be synthesized in yeast that has been transformed with
a Gal.beta.1,4GlcNAc transferase and GlcNAc.beta.3,4Gal
transferase. These yeasts, when fed lactose, will produce LNneoT.
If these genes are inserted into a FUT3 construct, LNF-III would be
produced. These transformants arise through integration of the
plasmid into the chromosome. The level of geneticin resistance
indicates the relative copy number of the integrated plasmid, since
resistance is conferred by the kanamycin marker on the plasmid.
Generally, high-copy-number integrants are preferred for obtaining
a high level of heterologous protein production.
[0237] Chemical Synthesis
[0238] As an alternative, chemical synthesis methods can be used to
prepare oligopeptides that are useful in the various methods and
compositions described herein.
[0239] The choice of glycosyl donors and acceptors, protective
group strategies, and coupling conditions for formation of
.alpha.-L-fucosyl (cis), and .beta.-D-galactopyranosyl (trans)
linkages will be according to the established methods of modern
synthetic carbohydrate chemistry (103-105). One approach is
depicted in FIG. 2 and described below.
[0240] Glycosyl donor for .alpha.-L-fucosyl residue. The critical
requirement is for a donor with a "non-participating" group at O-2
(106). For this purpose 2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl
bromide is be employed, under conditions of halide ion catalysis, a
method that has been successfully employed in many syntheses of
.alpha.-L-fucosyl derivatives (107-111). The use of benzyl ether
groups as persistent protective groups (112,113) has the additional
advantage that after coupling, deprotection of donor and acceptor
residues in the target compounds can be achieved at the same time
by catalytic hydrogenolysis. Alternative donors, such as 1-thio,
fluoride, trichloroacetimidate, or 4-pentenyl glycosides (114) can
also be used.
[0241] 2-Acetamido-2-deoxy-D-glucopyranose acceptors (GlcNAc
acceptors). Initially, the compounds are be benzyl,
4,6-benzylidene, or allyl ether derivatives of benzyl glycosides
(see FIG. 2), but the groups are manipulated so that primarily
benzyl ethers will remain at the end of the synthesis. A final step
of catalytic hydrogenolysis is deprotection of residues derived
from both acceptor and fucosyl donor. The regioselectivity of the
reductive ring opening of benzyl
2-acetamido-4,6-O-benzylidene-2-deoxy-D-glucopyranoside derivatives
with lithium aluminum hydride/aluminum chloride is dependant on the
steric bulk of the substituent at O-3 (115).
[0242] .beta.-D-Galactopyranosyl donor. The primary requirement is
for a "participating" group at O-2 (105). At O-1 a temporary
protective group must allow introduction of a halogen atom as a
leaving group in the glycosidation reaction. Bromine is preferable
because of its greater reactivity (105), and the introduction
should be under the mildest conditions practical. Therefore the O-1
substituent will normally be p-nitrobenzoyl, to be reacted with
hydrogen bromide in dichloromethane (116). At O-2, a
"participating" group is necessary for 1,2-trans-glycoside
formation. O-benzoyl is favored over O-acetyl because the O-benzoyl
group is less labile to basic conditions, less prone to migration,
less likely to undergo unwanted ortho ester formation during
coupling reactions, and easier to introduce selectively (112). At
the O-2 position where linkage is desired for 2'-FL and 2'-FLNAc, a
temporary protective group is necessary that can be removed after
the first glycosidation without affecting other linkages or groups;
the benzoyl group will perform both functions. The remaining two
positions must be occupied by persistent groups (benzyl), to be
removed only at the end of the synthesis.
[0243] The coupling reaction between the specially protected
"internal" galactosyl donor and the protected glucosamine acceptor
will employ silver triflate as promoter, in the presence of acid
scavengers (collidine or tetramethylurea), and molecular sieves,
i.e., standard conditions for trans glycoside coupling (117-119).
Helferich conditions (120) (mercuric cyanide/mercuric bromide
promoter) or Koenigs-Knorr conditions (121) (silver carbonate as
insoluble catalyst) can be substituted at this step. Whichever
method is used, after chromatographic purification of the product
of the first glycosidation reaction, it can be useful to
perbenzylate to avoid any risk of intermolecular acetyl migration
during the subsequent fucosylation step.
[0244] Starting compounds, chromatography, deprotection, and
structure confirmation. All the starting materials are accessible
from L-fucose, D-galactose, and 2-acetamido-2-deoxy-D-glucose, via
procedures described in FIG. 2. Their preparation and the coupling
reactions are be followed by TLC to monitor the purification of the
intermediates. The products of coupling reactions will be purified
by silica gel column and preparative layer chromatography. GlcNAc
acceptors are shown as benzyl glycosides for simplicity, though
2-bromoethyl glycosides or 8-methoxycarbonyloctyl glycosides, for
example, may be substituted if necessary for protein conjugation.
The synthesis of 2'-fucosyl-N-acetyllactosamine (110) has been
described, providing experimental and spectroscopic data for
preparing intermediates and characterizing products. Purity of
final products is determined by TLC and HPLC, and a final
purification by passage through a column of Bio-gel P-2 and/or a
coupled column of cation and anion-exchange resins is performed
when necessary. Structures of key intermediates and final products
is confirmed by permethylation analysis and mass-spectrometry.
[0245] Analysis of Oligonucleotides
[0246] Oligosaccharides produced by any of the methods described
herein can be analyzed to assess composition and structure using
standard techniques. For example, GC analysis can be used to
analyze sugar ratios. Briefly, a sample is transferred into a
capillary tube (1 mm i.d..times.35 mm) in aqueous methanol (50%).
The solvent is removed during centrifugation under vacuum. The
sample is dried in a vacuum desiccator over P.sub.2O.sub.5. Dry
methanolic HCl (0.75 mol/L; 25 mL) and methyl acetate (5 mL) are
added before the tops of the tubes are resealed in a flame. The
tubes are incubated at 80.degree. C. for 2 h and allowed to cool to
ambient temperature, whereupon the top of the tube is scored and
cracked open. The tube is placed under vacuum with centrifugation
to remove the methanolic HCl. Internal standard (methyl
heptadecanoate, 2 nmol in 5 ml methanol) is added, and the solvent
is removed by vacuum centrifugation. Freshly made 50% acetic
anhydride in dry pyridine (5 mL) is added, the tubes are resealed,
and the acetylation allowed to proceed for 14 h at ambient
temperature (the reaction is complete after 2 h). The top of the
tube is scored and cracked open, whereupon an aliquot (1 .mu.L) of
the contents are injected into a gas chromatograph fitted with a
30-m DB-1 column. Peaks are detected by flame ionization. After
injection into the GC, the temperature is held at 150.degree. C.
for 15 min and then raised by 4.degree. C. per min to a maximum
temperature of 300.degree. C. Peak areas are calculated with an HP
integrator. This method gives results that are suitable for
determining both sugar ratios of a pure compound and absolute
quantitation of sugars in a sample. This method yields consistently
good results with approximately 1 (1 nmol) of oligosaccharide.
[0247] Products can be analyzed by mass spectrometry to assess
purity and confirm structures. The number of components in a sample
and their molecular weights are determined by matrix-assisted laser
desorption ionization mass spectrometry. MS/MS of peracetylated
sample is used to obtain compositional information on the
individual components of a mixed sample. The fragmentation pattern
in the fast atom bombardment mass spectrum gives some insight into
the structure of a pure sample. MS/MS of derivatives can be used to
obtain complete structural information even for a sample that
contains a major component in the presence of appreciable
impurities. Linkage of pure compounds is established by GC/MS
analysis of partially O-methylated hexitols and hexosaminitol
acetate (PMAAs) (123).
[0248] Polyvalent Glycoproteins
[0249] In many cases it is desirable to administer a mixture of two
or more different oligosaccharides in a polyvalent form in which
two or more different oligosaccarides are covalently attached to
the same backbone, e.g., a protein backbone. In addition it can be
desirable to administer even a single oligosaccharide in a
polyvalent form, i.e., a form in which multiple copies of the same
oligosaccaride are attached to a single backbone. Any suitable
backbone can be used, for example, a glycan, a glycolipid, a
glycoprotein, A glycosaminoglycan, a mucin or a polypeptide.
Suitable backbone polypeptides have: multiple glycosylation sites
(multiple Asn, Ser and or Thr residues) and low allergenic
potential. In some cases it is desirable to use a polypeptide that
is considered acceptable for feeding to humans. Useful backbones
include: human milk proteins such as: .kappa.-casein,
.alpha.-lactalbumin, lactoferrin, bile salt-stimulated lipase,
lysozyme, serum albumin, folate-binding protein, haptocorrin,
lipoprotein lipase, glycosaminoglycan, mucin, lactoperoxidase,
amylase, bovine milk proteins and proteins of other common
foodstuffs.
[0250] Oligosaccharides can be attached to proteins using standard
methods. For example, oligosaccharides are converted into
p-aminophenyl glycosides, followed by diazotization and conjugation
to BSA or another polypeptide using standard procedures (124,125).
The p-aminophenyl glycosides can be prepared via peracetyl
p-nitrophenyl glycosides (126-128). The p-aminophenyl glycosides
are carefully O-deacetylated (to avoid alkaline hydrolysis) and
reduced in the presence of Adams catalyst. As an alternative to the
relatively unstable diazonium salts, the stable, usually
crystalline isothiocyanates can be prepared and coupled to BSA or
another polypeptide (e.g., milk proteins) as described (124).
Alternatively, oligosaccharides with a free reducing end can be
coupled to free amino groups on a protien; as found in lysine
residues, or the amino terminus to form a Schiff base, which is
converted into a covalent bond by reductive amination by sodium
cyanoborohydride.
[0251] The attachment of the oligosaccharide to the polypeptide can
be directly without spacers, or through spacers of various chain
lengths. As a variety of different chemistries of spacers are
available. The core N-linked glycan of a natural glycoprotein can
serve as a spacer on proteins expressed in yeast.
[0252] Yeast engineered to produce proteins of choice with core
N-linked glycans attached to their natural glycosylation sites (see
Hamilton et al. Science 301:1244, 2003) can be used to create a
neoglycoprotein as a starting point for the creation of
polyfucosylated neoglycoconjugate. The following types of glycans
are representative of those that could be used as a spacer (R
represents the backbone, e.g., protein) and as the reducing end of
the desired glycans.
[0253] Asparagine (N-linked) Core
TABLE-US-00009 GlcNAc Man GlcNAc Fuc Glc.fwdarw.Man.fwdarw.GlnNAc
GlcNAc--R GlcNAc Man GlcNAc
[0254] Serine/Threonine (O-linked) Core
TABLE-US-00010 Gal GlcNAc--R 20 GlcNAc
[0255] Neolacto Core
TABLE-US-00011 GlcNAc Gal Glc---R
[0256] N-Linked Polylactosamine
TABLE-US-00012 Gal GlcNAc GlcNAc Gal GlcNAc Gal Man Gal GlcNAc Fuc
Man GlcNAc GlcNAc--R Gal GlcNAc Man Gal GlcNAc
[0257] O-Linked Polylactosamine
TABLE-US-00013 5 Gal GlcNAc Gal GlcNAc GalNAc GalNAc--R Gal (GlcNAc
Gal).sub.n GlcNAc Gal
[0258] Ceramide-Linked Polylactosamine [0259] Gal (GlcNAc
Gal).sub.n Glc-R wherein n is 1, 2, 3, 4, 5, 6, 7 or more
[0260] To create the fucosylated forms, the neoglycoprotein is used
as a substrate instead of lactose in any of the chemical,
chemienzymatic, or molecular biological approaches described above.
These proteins, when expressed in yeast that are engineered to
express GMD, GFS, and a cell wall expressed FUT-2 will produce
polyvalent forms of the H-2 epitope. An alternative approach toward
the production of these polyvalent H-2 molecules would be to
transfect the yeast engineered to express GMD, GFS, and a cell wall
expressed FUT-2 with a plasmid-carrying gene for the human milk
protein of choice and the fucosyltransferases needed for the core
and glycan structure. Such a construct could produce polyvalent H-2
neoglycoproteins, which could then be isolated by Ulex lectin
affinity chromatography. To create the fucosylated forms, the
neoglycoprotein is used as a substrate instead of lactose in any of
the chemical, chemienzymatic, or molecular biological approaches
described above. These proteins, when exposed to yeast that are
engineered to express GMD, GFS, and a cell wall expressed FUT-1 or
FUT-2 will produce polyvalent forms of the H-2 epitope; those
expressing FUT3-7 or FUT-9 on the cell wall, or a combination of
FUT-1 or 2 and FUT3-9 will produce the other fucosylated epitopes.
An alternative approach toward the production of these polyvalent
H-2 molecules would be to transfect the yeast engineered to express
GMD, GFS, and a cell wall expressed fucosyltransferases with a
plasmid-carrying gene for the human milk protein of choice and the
fucosyltransferases needed for the core and glycan structure. Such
a construct could produce polyvalent H-2 neoglycoproteins, which
could then be isolated by Ulex lectin affinity chromatography. A
third approach would be to transfect the yeast with the gene for
the protein, the glycosyltransferases needed to produce the glycan
core, antiporters of transport of sugar nucleotides, enzymes for
synthesis of the needed sugar nucleotides and the
fucosyltransferases and/or sialyltransferases needed to produce
each of the fucosylated and/or sialylated structures, or the
nonfucosylated, nonsialylated olifosaccharides.
[0261] Compositions
[0262] Oligosaccharides whether or not linked to a backbone can be
administered as a pharmaceutical composition containing the
oligosaccharides (free or linked to a backbone) and a
pharmaceutically acceptable carrier, e.g., phosphate buffered
saline solution, mixtures of ethanol in water, water and emulsions
such as an oil/water or water/oil emulsion, as well as various
wetting agents or excipients. The oligosaccharide agents can be
combined with materials that do not produce an adverse, allergic or
otherwise unwanted reaction when administered to a patient. The
carriers or mediums used can include solvents, dispersants,
coatings, absorption promoting agents, controlled release agents,
and one or more inert excipients (which include starches, polyols,
granulating agents, microcrystalline cellulose, diluents,
lubricants, binders, disintegrating agents, and the like), etc. If
desired, tablet dosages of the disclosed compositions may be coated
by standard aqueous or nonaqueous techniques.
[0263] The oligosaccharide agents can be administered orally, e.g.,
as a tablet containing a predetermined amount of the active
ingredient, pellet, gel, paste, syrup, bolus, electuary, slurry,
capsule, powder, granules, as a solution or a suspension in an
aqueous liquid or a non-aqueous liquid; as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion, or in some other form.
Orally administered compositions can include binders, lubricants,
inert diluents, lubricating, surface active or dispersing agents,
flavoring agents, and humectants. Orally administered formulations
such as tablets may optionally be coated or scored and may be
formulated so as to provide sustained, delayed or controlled
release of the active ingredient therein. The agents of the
invention can also be administered by rectal suppository, aerosol
tube, naso-gastric tube, direct infusion into the GI tract or
stomach or parenterally.
[0264] Pharmaceutical compositions containing oligosaccharide
agents can also include therapeutic agents such as antiviral
agents, antibiotics, probiotics, analgesics, and anti-inflammatory
agents.
[0265] The proper dosage is determined by one of ordinary skill in
the art and depends upon such factors as, for example, the
patient's immune status, body weight and age. In some cases, the
dosage will be at a concentration similar to that found for similar
oligosaccharides present in human breast milk.
[0266] The oligosaccharides agents can also be added to other
compositions. For example, they can be added to an infant formula,
a nutritional composition, a rehydration solution, a dietary
maintenance or supplement for elderly individuals or
immunocompromised individuals.
[0267] The oligosaccharides agents can be included in compositions
that include macronutrients such as edible fats, carbohydrates and
proteins. Edible fats include, for example, coconut oil, soy oil
and monoglycerides and diglycerides. Carbohydrates include, for
example, glucose, edible lactose and hydrolyzed cornstarch. Protein
sources include, for example, protein source may be, for example,
soy protein, whey, and skim milk.
[0268] Compositions, including nutritional compositions, containing
the oligosaccharide agents can also include vitamins and minerals
(e.g., calcium, phosphorus, potassium, sodium, chloride, magnesium,
manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E,
D, C, and B complex).
[0269] Screening and Analysis of Oligosaccharides
[0270] Oligosaccharides can be tested for their ability to bind
infectious agents using the agents themselves. For example,
prototype invasive C. jejuni strains 166-IP and 287-IP from
children with inflammatory diarrhea; C. jejuni strain 50-SP, from a
healthy child; and two V. cholerae strains, El Tor and Classic can
be used to study the effect of oligosaccharides on campylobacter
and V. cholerae.
[0271] To assess the ability of campylobacter and V. cholerae to
bind to histo-blood group antigens, bacterial binding Western blot
assays are performed with DIG-labeled bacteria (23,24).
Neoglycoproteins of blood group antigens are applied to lanes for
SDS-PAGE at 6.3.times.10.sup.-10 M oligosaccharide per lane.
Membranes are washed in TBS, immersed in a DIG-labeled bacterial
suspension of 0.2 OD600 and incubated 4 h at room temperature with
gentle stirring. Membranes are then washed and incubated for 1 h
with the alkaline phosphatase-conjugated anti-DIG antibody, washed
and stained with X-Phosphate (5-bromo-4-chloro-3-indolyl phosphate)
and Tris-buffered nitroblue tetrazolium in saline (pH 9.5)
substrate (Boehringer Mannheim).
[0272] .alpha.1,2-fucosyltransferase-transfected CHO cells
(CHO-FUT1), .alpha.1,3/4-fucoslytransferase-transfected CHO cells
(CHO-FUT3), and .alpha.1,3-fucosyltransferase-transfected CHO cells
(CHO-FUT4), and parental CHO cells transfected with the vector
pCDM.sub.7lacking the .alpha.1,2 FUT gene (CHO-WT) can be used to
test bacterial binding and bacterial/host cell agglutination.
Parental CHO cells with the vectors are used as controls.
[0273] The binding of bacteria to CHO cells transfected with the
human gene for .alpha.1,2-fucosyltransferase (FUT1), can be
assessed by bacterial-cell association assay. Briefly, transfected
CHO cells expressing the FUT1 fucosyltransferase needed for the
synthesis of human H-type antigen (.alpha.1,2-fucosyl residues) are
grown to confluency (28). Controls are wild type CHO cells,
parental CHO cells carrying only the plasmid vector, and a clone
that expresses the murine UDP
Gal:Gal.alpha.1,14GlcNAc.alpha.1,3-transferase. Monolayers are
harvested and seeded into each well of an 8-chamber slide and
incubated for 18 h, washed and incubated with a suspension of
9.times.10.sup.8 bacteria/mL. Wells are rinsed, fixed with 10%
formalin for 1 h, stained by the Warthin-Starry method, and
examined under oil immersion with light microscopy, or confocal
microscopy for mutant strains with the fluorescent plasmid.
Identical preparations grown on round cover slips are examined by
scanning electron microscopy after fixing in 2% glutaraldehyde,
dehydration through a graded series of solvents, and surface gold
deposition.
[0274] Ligands, such as .alpha.1,2-fucosyl ligands and homologs are
tested for their ability to inhibit binding of campylobacter and V.
cholerae strains to CHO-FUT1 cells. For molecules that bind to H-2
ligands, including anti-H-2 monoclonal antibodies (anti-H-2 MAbs)
and the lectins Ulex europaeus (UEA I) and Lotus tetragonolobus
(Lotus), inhibition is measured on monolayers of CHO-FUT1 cells
incubated in 8-well chamber slides for 1 h with each of the
.alpha.1,2-fucosyl ligands before adding 100 .mu.L of the bacterial
suspension containing 1.times.10.sup.8 bacteria/mL. For inhibition
using homologs to cell surface receptors, including human milk
neutral oligosaccharides (Neutral-OS), milk from secretor and
non-secretor mothers, neoglycoprotein BSA-H-2 (IsoSep AB,
Tullingen, Sweden), and 2'-fucosyllactose, 100 .mu.L of the
bacterial suspension are incubated with each of the homologs before
being added to the cell monolayer. In both assays, after a 3 h
incubation at 37.degree. C., wells are rinsed, lysed with 1% Triton
X100, and CFU (colony forming units) of bacteria per well are
determined. Data are interpreted as percent inhibition of bacteria
association to cells relative to positive controls to which no
.alpha.1,2-fucosyl ligands or homologs are added.
[0275] The effect of oligonucleotides and neoglycoproteins on
campylobacter and V. cholerae colonization in vivo will be
determined in BALB/c mice (weighing 10-20 g).
[0276] Three-week old BALB/c mice will be fed orally either BID
(twice daily) or TID (thrice daily) with escalating dose of
neoglycoprotein starting at 2 mg/100 .mu.L per intake up to 200
mg/.mu.L. Animals will be followed for 2 weeks after the last dose
to evaluate for tolerance, weight, presence of diarrhea, and
abnormal behavior.
[0277] Two different assays can be used to test the inhibition of
campylobacter and cholera colonization in vivo using the inbred
strain of mus musculus Balb/c. (1) Prophylaxis studies. In
experiments designed to study the ability of neoglycoproteins to
inhibit colonization prophylactically, three-week-old female mice
are randomly distributed into 2 experimental plus a positive and a
negative control group. The two experimental groups are challenged
with 10.sup.8 CFU per animal. Two of the four challenge groups are
treated with either the neoglycoprotein or trisaccharide 2 days
before challenge, on the day of challenge, and 2 hours after
challenge, either twice or three times per day. A negative control
group is used to ensure that the animals are initially free from
pathogens. The positive control group receives only saline. Each
experimental group is compared with each other and with the saline
control group.
[0278] Oligosaccharides and neoglycoproteins can be tested for
their ability to clear colonization of animals who are already
infected. Again, 3-week-old Balb/c mice are first infected with
10.sup.8 CFU of campylobacter, and after 7 days when the animals
exhibit persistent colonization the animals will be treated with
the neoglycoprotein or trisaccharide either twice or three times
per day at two doses established in the tolerance and safety
study.
[0279] 2-Linked Fucosylated Oligosaccharides Reduce Diarrhea Due to
Campylobacter, Caliciviruses, and Diarrhea of All Causes in
Breastfed Infants
[0280] Described below is a study demonstrating that a high ratio
of 2-linked to non-2-linked fucosylated oligosaccharides in human
milk reduces the occurrence of Campylobacter diarrhea and
calicivirus diarrhea in breastfed infants.
[0281] A cohort of 316 mother-infant pairs was enrolled and
monitored from birth to two years postpartum in San Pedro Martir, a
transitional neighborhood of Mexico City. Enrollment was restricted
to term, normal birthweight infants. This research was approved by
institutional review boards in Mexico and Cincinnati. Written
informed consent was obtained from mothers who participated. Infant
illness and feeding history were collected by trained field workers
who made weekly home visits. Milk samples were collected from
mothers weekly in the first month, and monthly thereafter. Samples
were collected in the morning by an experienced study nurse using
an Egnell electric breast pump to obtain the complete content of
one breast. Samples were transported on ice from the study
household to the laboratory, where they were stored at -70.degree.
C. Infant stool samples were collected weekly with additional
samples obtained whenever diarrhea occurred. Diarrhea samples were
routinely tested for Campylobacter jejuni, diarrheagenic E. coli,
Shigella, Salmonella, Aeromonas, and rotavirus, as detailed in
previous publications (129, 136-138). Calicivirus testing of stool
samples was later performed by enzyme immune assay (EIA) and
reverse transcription-PCR; a positive result by either test was
considered calicivirus positive (139-141). Diarrhea episodes were
defined throughout the study as three or more watery stools within
a 24-hour period or loose-to-watery bowel movements that exceeded
the child's usual daily stool frequency by two or more stools as
determined by a study physician. Using the severity scoring system
of Ruuska and Vesikari, an episode of diarrhea was classified as
moderate-to-severe if the score was .gtoreq.10 (136, 142).
Classification of disease severity was based on the standardized
history of each diarrhea episode recorded by a study physician, and
was blind to and independent of milk oligosaccharide analysis.
Diarrhea was attributed to campylobacter or calicivirus if the
pathogen was detected in a stool sample collected during or within
seven days of an episode of diarrhea. Diarrhea episodes associated
with two or more pathogens were excluded from pathogen-specific
analyses. Upon completion of the initial cohort study, mothers were
requested to participate in a blood draw to determine maternal
blood group type.
[0282] A mother-infant pair was included in the present study if
they were followed in the cohort and breastfed for at least 2
weeks; the mother consented to participate in blood collection for
blood group typing; and they had at least one vial of milk in
storage that contained 2 mL or more of milk collected between 1-5
weeks postpartum. Reasons for exclusion were that 40 did not
breastfeed and remain in the study for at least 2 weeks; 91 mothers
did not consent to blood collection; and 92 had insufficient volume
of milk sample in storage. A total of 93 mother-infant pairs met
all three criteria and were included for study. If more than one
sample was available per mother, the one closest to 30 days
postpartum with at least 2 mL volume was selected.
[0283] Milk samples were transported to Boston and analyzed as
described previously (143). Milk oligosaccharides were isolated,
perbenzoylated, and resolved by reversed-phase HPLC (C-8) with an
acetonitrile/water gradient and detected at 229 nm. This
chromatography system produces eight major peaks in human milk
samples, which correspond to the most common oligosaccharides of
human milk: four 2-linked fucosylated oligosaccharides
(lacto-N-fuco-pentaose I [LNF-I], 2'-FL, lacto-N-difucohexaose
[LDFH-I] and lactodifucotetraose [LDFT]); two fucosylated
oligosaccharides that are not 2-linked (LNF-II and 3-fucosyllactose
[3-FL]); and their two precursors (lacto-N-tetraose [LNT] and
lacto-N-neotetraose [LNneoT]). These eight oligosaccharides are
homologs of Lewis histo-blood group antigens, respectively: H-1,
H-2, Lc.sup.b, Lc.sup.y, Lc.sup.a, Lc.sup.x, and types 1 and 2
precursors. Detection of oligosaccharides in human milk samples was
not adversely affected by storage or freeze-thaw.
[0284] Statistical Analysis
[0285] The primary analysis focused on the incidence of diarrhea
during breastfeeding defined as the total number of cases of
diarrhea that occurred during breastfeeding per 100 child-months of
breast-feeding. Child-months of breastfeeding was calculated as the
sum of all months spent breastfeeding, from birth to the end of
breastfeeding (or termination from study, whichever occurred
sooner). A secondary analysis was conducted of the incidence of
diarrhea during post-breastfeeding child-months, i.e., from the end
of breastfeeding to termination from study. Time during diarrheal
illness was not included in the denominator used to calculate
incidence rates. Study outcomes were defined as the rates of
diarrhea associated with C. jejuni, calicivirus, all causes of
diarrhea, and all causes of moderate-to-severe diarrhea. The major
independent variables were the specific and total 2-linked
fucosylated oligosaccharides characterized in terms of
concentration in milk (mmol/L) and percent of milk oligosaccharide
(the quantity of specific or total 2-linked fucosylated
oligosaccharide divided by the sum of the eight oligosaccharides
measured). The percent of milk oligosaccharide measure was used to
correct for variability in concentrations due to lactation
physiology, sampling, collection, storage, and testing.
[0286] Correlations were analyzed among oligosaccharide measures.
The associations between milk oligosaccharide measures and rates of
diarrhea outcomes were examined using a generalized linear model
with a Poisson link function. This model was selected as optimal
for analysis of incidence rates with one or more outcomes per
person, accounting for variable lengths of follow-up time.
Potential interactions or confounding by factors shown in table 1
were analyzed in relation to milk oligosaccharide measures and
rates of infant diarrhea. Significant (P<0.05) risk factors
associated with the rate of infant diarrhea in univariate and/or
multivariate models were the percent of infant feedings that were
breast milk (calculated from weekly follow-up data for the duration
of breastfeeding as the number of breast milk feedings divided by
the total number of feedings in the past 24 hours), infant birth
order, maternal age, and maternal ABO blood group type. These
factors were included in multiple regression models but they were
not associated with milk oligosaccharide values and did not
confound the associations between oligosaccharide measures and
diarrhea outcomes. Thus, final regression models included only
specific or total 2-linked fucosylated oligosaccharide expressed as
a percent of milk oligosaccharide in relation to diarrhea outcomes.
In addition to analysis of milk oligosaccharides as continuous
variables, the dose-response pattern of the data was further
examined by classifying mother-infant pairs into low, intermediate,
and high tertiles (n=31 per group) of milk oligosaccharide values,
with rates of infant diarrhea calculated by group.
[0287] Study Population
[0288] The 93 mother-infant pairs in this study were monitored for
857 breastfeeding infant-months and 765 post-breastfeeding
infant-months between birth and 2 years of age. These 93
mother-infant pairs were compared to the 183 mothers in the cohort
who were not included in this study but had breastfed their infants
for at least 2 weeks; they were comparable regarding the incidence
of infant diarrhea during breastfeeding and all sociodemographic,
hygiene, and infant factors (table 1), except that mothers included
in this study breastfed longer (median duration 9 vs 5 months,
P<0.01) and were more likely to complete a secondary education
or higher (P<0.01) than those not included. Analysis of these
factors found that they were not associated with milk
oligosaccharide levels and were not confounding. These differences
were thus unlikely to affect the internal validity of this study.
Among the 93 study pairs, the mean percent of feedings that were
breast milk was 49% during the breastfeeding period; none practiced
exclusive breastfeeding. In addition to their own mother's milk,
study infants were given differing amounts of formula, juice, tea,
water, solid foods, and gruel. Two-thirds of mothers were 0 blood
type; nearly three-quarters were Lewis positive secretors (Le a-b+)
and one-quarter were Lewis negative secretors (Le a-b-). The
serologic classification for two mothers was Le a+b-, which is
considered to indicate obligate non-secretors. However, since the
milk from these two mothers contained 2-linked fucosylated
oligosaccharide, inconsistent with being a non-secretor, the
discrepancy between milk and blood group phenotypes was resolved by
classifying the blood group as indeterminate (Table 1).
TABLE-US-00014 TABLE 1 Characteristics of the 93 breastfeeding
mother-infant pairs Characteristic Measure Value Number of people
in the household Median (range) 5 (3-11) Kept animals in the
household No. (%) 66 (71%) Age of mother (years) Median (range) 23
(15-41) Maternal education: None/Elementary No. (%) 42 (45%) Middle
school 31 (33%) High school and 20 (22%) beyond Primiparous mother
No. (%) 31 (33%) Duration of breast-feeding (months) Median (range)
9 (0.7-24) Percent of infant feedings that were Median (range) 49
(4-82%)* breast milk Maternal Lewis blood a-b+ No. (%) 67 (72%)
group: a-b- 24 (26%) indeterminate 2 (2%) Maternal ABO blood O No.
(%) 62 (67%) group: A 18 (19%) B 12 (13%) AB 1 (1%) Male infant No.
(%) 43 (46%) Age in months at diarrhea outcomes during
breastfeeding: Median (range) All diarrhea (234 cases): 6.9 (0.1,
23.4) Moderate-to-severe (77 cases): 6.7 (0.1, 20.3) Campylobacter
(31 cases): 9.2 (1.6, 15.6) Calicivirus (16 cases): 9.1 (1.2,
14.4)**
[0289] Milk Analysis
[0290] A single milk sample was analyzed for each mother; all
analyzed samples were collected 1-5 weeks (median, 3 weeks)
postpartum in a standardized manner to avoid sampling variation.
The representativeness of this sample for the course of lactation
was analyzed using longitudinal data from 11 Mexican secretor
mothers. The 2-linked fucosylated oligosaccharide measured in the
milk sample collected at 3 weeks postpartum was highly correlated
(r=0.73) with the average of the same measure in milk collected
from each mother at 3, 6, 9, and 12 months of lactation (144).
[0291] Milk oligosaccharide concentrations ranged from 1.0 to 36.1
mmol/L. Total 2-linked fucosylated oligosaccharide concentrations
ranged from 0.8 to 20.8 mmol/L (50 to 92 percent of milk
oligosaccharide) (Table 2). The most commonly occurring specific
2-linked fucosylated oligosaccharides were 2'-FL (34 percent of
milk oligosaccharide) and LNF-I (25 percent of milk
oligosaccharide). The milk of the 24 Le a-b- mothers had
significantly (P<0.05) higher percent of total 2-linked
fucosylated oligosaccharide compared to the 67 Le a-b+ mothers (80
vs 71 percent of total, respectively). The 2-linked fucosylated
oligosaccharides, whether analyzed as milk concentrations or
percent of milk oligosaccharide, were not associated with maternal
sociodemographic factors or ABO blood group. Correlations between
specific oligosaccharides expressed as milk concentrations ranged
from r=-0.1 to +0.8, and as percent of milk oligosaccharide ranged
from r=-0.5 to +0.6.
TABLE-US-00015 TABLE 2 Characterization of the concentration (mean
.+-. SD) and the percent of specific and total .alpha.1,2-linked
fucosylated oligosaccharides in maternal milk. Concentration
Percent of Milk (mmol/L) milk oligosaccharide Oligosaccharide Mean
.+-. SD Range Mean .+-. SD Range lacto-N-fucopentaose I (LNF-I)
3.21 .+-. 1.75 0, 7.0 25.1 .+-. 9.9 0, 43.7 2'-fucosyllactose
(2'-FL) 3.85 .+-. 1.04 0.5, 6.2 33.7 .+-. 10.4 0.05, 66.7
lacto-N-difucohexaose I (LDFH-I) 1.26 .+-. 1.00 0, 5.2 9.5 .+-. 5.7
0, 25.4 lactodifucotetraose (LDFT) 0.70 .+-. 0.72 0.03, 5.0 5.1
.+-. 3.4 0.01, 16.9 Total 2-linked fucosyl 9.02 .+-. 3.23 0.78,
20.8 73.3 .+-. 8.8 50.5, 92.3 oligosaccharide Total milk
oligosaccharide+ 12.44 .+-. 4.8 1.0, 36.1 100 *Percent of milk
oligosaccharide is the quantity of each specific oligosaccharide
divided by the total quantity of the milk oligosaccharides measured
in this study. +Total milk oligosaccharide includes eight
oligosaccharides: the four 2-linked fucosylated oligosaccharides,
two non-2-linked fucosylated oligosaccharides (3-FL and LNF II) and
their two precursors.
[0292] Association with Diarrhea
[0293] During breastfeeding, a total of 234 diarrhea episodes were
identified (median, 2 diarrhea episodes per child; range, 0-12
episodes per child), of which a total of 77 (33%) diarrhea episodes
were moderate-to-severe. The incidence of diarrhea was 28.8 cases
per 100 child-months of breastfeeding and of moderate-to-severe
diarrhea was 9.5 cases per 100 child-months of breastfeeding. Among
all diarrhea episodes, 40 were associated with C. jejuni, 25 with
calicivirus, 10 with enteropathogenic E. coli, 9 with rotavirus, 5
with shigella, and 4 with stable toxin-associated E. coli.
Excluding diarrhea episodes with detected co-infections, 31
diarrhea episodes were associated with C. jejuni in 22 children,
and 16 episodes of diarrhea were associated with calicivirus in 13
children.
[0294] Rates of C. jejuni diarrhea during breastfeeding were
inversely associated (P=0.004) with 2'-FL as a percent of milk
oligosaccharide (Table 3), and directly associated (P=0.047) with
LDFH-I as a percent of milk oligosaccharide, but association with
LDFH-I did not persist after controlling for 2'-FL in regression
models. 2'-FL as a percent of milk oligosaccharide, however,
remained significantly (P<0.05) inversely associated with rates
of C. jejuni diarrhea, whether in univariate or multivariate
models. Analyzed categorically, the group with low 2'-FL as a
percent of milk oligosaccharide had significantly (P<0.01)
higher rates of Campylobacter diarrhea during breastfeeding than
each of the medium and high 2'-FL groups (FIG. 6A). For calicivirus
diarrhea during breastfeeding, several milk oligosaccharides tended
towards protective associations (Table 3), but only LDFH-I was
significant (P=0.012). The inverse association between LDFH-I as a
percent of milk oligosaccharide and calicivirus diarrhea had a
visible dose-dependent relationship (FIG. 6B).
TABLE-US-00016 TABLE 3 Univariate analyses of specific
.alpha.1,2-linked fucosylated oligosaccharides as a percent of milk
oligosaccharide and protection against C. jejuni-associated
diarrhea and calicivirus- associated diarrhea in study children, by
Poisson regression* Milk Campylobacter.sup..dagger.
Caliciviruses.sup..dagger. Oligosaccharide .beta. (SE) P .beta.
(SE) P LNF-I -0.51 (1.75) 0.772 3.30 (2.66) 0.215 2'-FL -5.60
(1.93) 0.004 3.77 (2.14) 0.078 LDFH-I 5.87 (2.95) 0.047 -13.32
(5.33) 0.012 LDFT 3.09 (4.74) 0.514 -16.82 (11.00) 0.126
[0295] *Each model included only one independent variable, a
specific 2-linked fucosylated oligosaccharide as percent of milk
oligosaccharide, and pathogen-specific diarrhea as the dependent
variable. Negative beta coefficients indicate protection.
Significant protective associations are in bold type.
.sup..dagger.22 subjects had 31 cases of campylobacter diarrhea, 13
subjects had 16 cases of calicivirus diarrhea.
[0296] A significant inverse association (.beta.=-3.9.+-.1.2
(SE[.beta.], P=0.001) was found by Poisson regression between total
2-linked fucosylated oligosaccharide as a percent of milk
oligosaccharide and rates of all moderate-to-severe diarrhea during
breastfeeding, but no association was found with all diarrhea.
Whether analyzed as a continuous or categorical variable, total
2-linked fucosylated oligosaccharide in milk had an inverse,
dose-dependent relationship with moderate-to-severe diarrhea (FIG.
6C). The inverse associations between 2-linked fucosylated
oligosaccharides of milk and diarrhea outcomes persisted for the
duration of the breastfeeding, analyzed by stratifying on age while
breastfeeding (0-5, 6-12, 13-18, and 19-24 months). Exclusion of
the 6 study children with <1 month duration of breastfeeding did
not alter the observed associations between milk oligosaccharides
and diarrhea outcomes.
[0297] In the post-breastfeeding period, a total of 89 children
continued in the study and experienced 188 diarrhea episodes, 60
moderate-to-severe diarrhea episodes, and 36 campylobacter diarrhea
episodes (calicivirus testing was not conducted for the
post-breastfeeding period). No associations were observed between
milk oligosaccharides and post-breastfeeding diarrhea outcomes.
[0298] In this study of breastfed Mexican infants, we found that
low levels of specific 2-linked fucosylated oligosaccharides in
human milk were significantly associated with increased rates of
pathogen-specific infant diarrhea. C. jejuni and caliciviruses,
excluding co-infections, together accounted for 20% of all diarrhea
episodes in our study population. A low level of 2'-FL as a percent
of milk oligosaccharide was associated with a high rate of C.
jejuni diarrhea in breastfed infants. Similarly, lower levels of
LDFH-I as a percent of milk oligosaccharide had a dose-dependent
association with higher rates of calicivirus diarrhea. We also
found that lower levels of total 2-linked fucosylated
oligosaccharide as a percent of milk oligosaccharide had a
dose-dependent association with higher rates of moderate-to-severe
diarrhea of all causes. The association between milk
oligosaccharide measured during the first month postpartum and
diarrhea in breastfed infants persisted through the course of
breastfeeding, but not after termination of breastfeeding. This
observation was consistent with our proposed mechanism of
protection: the presence of milk oligosaccharide in the infant
gastrointestinal tract to inhibit pathogen binding. In our study
population, all maternal milk contained some 2-linked fucosylated
oligosaccharide, but there was a wide range of expression, with
specific and total 2-linked oligosaccharide measured on a
continuous scale. The association between milk oligosaccharide and
diarrhea in breastfed infants was observed only for percent of milk
oligosaccharide, suggesting the importance of a denominator to
correct for variability in recovery of oligosaccharide that
occurred despite standardized milk sample collection and laboratory
methods.
[0299] The human milk oligosaccharides measured in this study are
Lewis epitopes, products of the same genes that control maternal
Lewis histo-blood group type. Blood group types are the result of
genetic polymorphisms that determine oligosaccharide-containing
glycoconjugate expression on host cell surfaces. Associations have
been previously reported between histo-blood group type and
differing susceptibility to bacterial and viral diseases. Glass et
al. showed that O blood group individuals have increased
susceptibility to cholera (145). P blood group type has been
associated with susceptibility to hemolytic uremic syndrome (146).
Ikehara et al. found an association between Lewis and secretor
histo-blood group genotypes and risk of infection with Helicobacter
pylori (147). Hutson et al. reported that O blood group individuals
have increased susceptibility to Norwalk virus (148). Influenza
virus binding has been shown to vary in relation to host blood
group antigens (149)..sup.3 Further, Raza et al reported that
secretor children have increased risk of hospitalization for
respiratory infections due to influenza viruses A and B,
rhinoviruses, respiratory syncytial virus, and echoviruses
(150).
[0300] This study is unique in examining the mother-infant dyad.
Phenotypic variation in the relative quantities of 2-linked
fucosylated oligosaccharides in mothers' milk determines the
protection offered to breastfed infants. Because the same genotype
that produces milk oligosaccharides in the mother is expected to
produce cell surface receptors that increase risk in the infant,
the lack of control for infant susceptibility is likely to have
biased our results such that the true association between milk
oligosaccharides and protection against disease is stronger than
observed. Further, we note that certain major endemic pathogens not
included in this study, e.g., ST-associated E. coli (130, 134, 135)
are also inhibited by 2-linked fucosylated oligosaccharides, while
others, e.g., rotavirus, are inhibited by human milk
glycoconjugates encoded by products of genes other than the
secretor and Lewis genes (129). Thus, the association we have
described provides only a glimpse into the potential protective
role of the innate immune system of human milk.
[0301] A growing body of research suggests that common mechanisms
of pathogenesis may exist between some bacterial and viral
pathogens..sup.17-20,33-35,37 We have found that both C. jejuni, a
bacterium, and caliciviruses bind to 2-linked fucosylated
oligosaccharides..sup.17-19 Fucosylated oligosaccharide milk
fractions inhibit C. jejuni adherence to human epithelial cells in
vitro and colonization in experimental mice, and 2'-FL inhibits C.
jejuni binding to human intestinal mucosa ex vivo (131). Further,
we also found that Chinese hamster ovary cells transfected with a
human fucosyltransferase gene bind C. jejuni, and that this binding
is inhibited by Lewis epitopes containing 2'-FL. Our studies with
caliciviruses have shown that Norwalk virus-like particles bind to
tissue sections of the gastro-duodenal junction from secretors but
not from nonsecretors (132), and that binding is blocked by milk
from a secretor (133). Volunteers challenged with Norwalk virus
become symptomatically infected only if they are secretors.
Consistent with our finding that LDFH-I oligosaccharide (an
Le.sup.b oligosaccharide homolog) is associated with protection
against calicivirus diarrhea, our laboratory data suggest that
Le.sup.b epitopes and other 2-linked fucosylated oligosaccharide
structures inhibit binding by common strains of caliciviruses.
[0302] Potential limitations of this study should be considered. A
single milk sample collected in a standardized manner from each
mother was analyzed in relation to infant diarrhea for the duration
of breastfeeding. Our data indicate, however, that the expression
of 2-linked fucosylated milk oligosaccharide in the first month is
highly correlated with its expression across lactation. In this
study, we found oligosaccharide associated with protection against
moderate-to-severe but not mild diarrhea. With a larger sample size
and analysis of multiple samples across lactation, future studies
might be better able to determine whether milk oligosaccharides
protect against mild diarrhea. Inclusion of mothers in this study
required sufficient milk volume in cryogenic storage and
participation in an additional blood draw. As a result, mothers in
our study had significantly longer duration of breastfeeding and a
higher educational level than others in the original cohort, but
these factors were not associated with the milk oligosaccharides
under study.
[0303] Our findings suggest that heterogeneous expression of
oligosaccharides in human milk provides infants with varying
degrees of protection against specific pathogens, consistent with
the concept that heterogeneous expression of oligosaccharide
epitopes in infants underlies their individual susceptibility to
different pathogens. Because many of the oligosaccharides found in
human milk are unique, this study supports the importance of
breastfeeding. In summary, the associations observed from this
study provide initial clinical evidence that human milk
oligosaccharides may offer clinically relevant protection against
diarrhea, and suggests the potential for oligosaccharides to form
the basis of oral agents with potent antibacterial and antiviral
activity.
[0304] 2-Linked Fucosylated Oligosaccharides Reduce Campylobacter
and Cholera Adherence to Cells
[0305] The specific binding of campylobacter in HEp2 cells is
inhibited by fucosylated carbohydrate moieties containing the H(O)
blood group epitope (FucI1,2Gal.theta.1,4GlcNAc). Studies of
campylobacter binding to histo-blood group antigens as
neoglycoproteins immobilized in nitro-cellulose membranes
demonstrated a high avidity for the H-2 antigen as confirmed by
specific inhibition with monoclonal antibodies. In studies on the
mechanism of adherence, C. jejuni, which normally does not bind to
Chinese hamster ovary (CHO) cells, bound avidly when the cells were
transfected with a human I1,2-fucosyltransferase gene that caused
over-expression of H-2 antigen. Similarly, V. cholerae adheres to
transfected but not to parental cells. This binding was
specifically inhibited by H-2 ligands (Ulex europaeus lectin, Lotus
tetragonolobus lectin, and H-2 monoclonal antibody), H-2 mimetics,
and human milk oligosaccharides (FIG. 7). Invasive campylobacter
287-IP binds to FUT1, but not FUT3 or FUT4-transfected CHO cells
(FIG. 8). In experimental models, human milk oligosaccharides
inhibited campylobacter colonization in mice in vivo and in human
intestinal mucosa ex vivo. (FIGS. 9A and 9B).
[0306] The role of milk .alpha.1,2 glycoconjugates in passive
protection against campylobacter infection was evaluated in litters
of B6-SJL transgenic female mice carrying the human
.alpha.1,2-fucosyltransferase gene (FUT2) with a whey promoter that
induces the expression of histo-blood group antigens primarily in
mammary gland during lactation, and thus, in milk. As a control,
non-transgenic parental mice were used. Suckling mice were
challenged with 10.sup.4, 10.sup.6 and 10.sup.8 CFU of C. jejuni
and were returned to the dams. Gut colonization was monitored for
15 days. Up to 90% of non-transgenic litters remained colonized
during follow-up. Colonization of transgenic mice was transient and
the time of colonization was directly related to the inoculum
(FIGS. 10A and 10B). These experiments strongly support the role of
.alpha.1,2-linked fucosylated glycoconjugates of milk in protection
against campylobacter infection, and suggest that the main
intestinal ligands for campylobacter are the H-2 histo-blood group
antigens. Milk fucosyloligosaccharides and specific fucosyl
.alpha.1,2-linked molecules inhibit this binding. Preliminary
experiments of cholera infection in suckling pups from pWAP FUT1
transgenic dams expressing H(O) experiments of cholera infection in
suckling pups from pWAP FUT1 transgenic dams expressing H(O)
antigen in mammary gland, demonstrated, as with campylobacter, a
significant reduction in colonization with an inoculum of 10.sup.8
CFU and a significant reduction in mortality with an inoculum of
10.sup.10 CFU when compared with non-transgenic controls. Our data
strongly support the role of .alpha.1,2-fucosyl glycoconjugates in
the protection against campylobacter infection and suggest that the
main intestinal ligands for campylobacter are the H-2 histo-blood
group antigens.
REFERENCES
[0307] 1. Newburg D S. Human milk glycoconjugates that inhibit
pathogens. Curr Med Chem 1999; 6: 117-127. [0308] 2. Ruiz-Palacios
G, Cervantes L E, Ramos P, Prieto P A, Newburg D S. Campylobacter
jejuni binds intestinal H(O) antigen (Fuc.alpha.1,2Galb1,4GlcNAc),
and fucosyloligosaccharides of human milk inhibit its binding and
infection. J Biol Chem 2003; 278: 14112-14120. [0309] 3. Huang P,
Farkas T, Marionneau S, et al. Noroviruses bind to human ABO, Lewis
and secretor histo-blood group antigens: Identification of four
distinct strain-specific patterns. J Infect Dis 2003 [0310] 4.
Morrow A L, Ruiz-Palacios G M, Altaye M, et al. Human milk
oligosaccharide blood group epitopes and innate immune protection
against diarrhea in breast-fed infants. Under revision for J
Pediatrics. [0311] 5. Marionneau S, Ruvoen N, Le Moullac-Vaidye B,
et al. Norwalk virus binds to histo-blood group antigens present on
gastroduodenal epithelial cells of secretor individuals.
Gastroenterology 2002; 122: 1967-1977. [0312] 6. Newburg D S,
Pickering L K, McCluer R H, Cleary T G. Fucosylated
oligosaccharides of human milk protect suckling mice from
heat-stabile enterotoxin of Escherichia coli. J Infect Dis 1990;
162: 1075-1080. [0313] 7. Newburg D S. Bioactive components of
human milk: Evolution, efficiency, and protection. Adv Exp Med Biol
2001; 501: 3-10. [0314] 8. American Academy of Pediatrics.
Breastfeeding and the use of human milk. American Academy of
Pediatrics. Work Group on Breastfeeding. Pediatrics 1997; 100:
1035-1039. [0315] 9. WHO Collaborative Study Team. Effect of
breastfeeding on infant and child mortality due to infectious
diseases in less developed countries: a pooled analysis. WHO
Collaborative Study Team on the Role of Breastfeeding on the
Prevention of Infant Mortality. Lancet 2000; 355: 451-455. [0316]
10. Kramer M S, Chalmers B, Hodnett E D, et al. Promotion of
Breastfeeding Intervention Trial (PROBIT): a randomized trial in
the Republic of Belarus. JAMA 2001; 285: 413-420. [0317] 11. Morrow
A L, Guerrero M L, Shults J, et al. Efficacy of home-based peer
counselling to promote exclusive breastfeeding: a randomised
controlled trial. Lancet 1999; 353: 1226-1231. [0318] 12. Arifeen
S, Black R E, Antelman G, Baqui A, Caulfield L, Becker S. Exclusive
breastfeeding reduces acute respiratory infection and diarrhea
deaths among infants in Dhaka slums. Pediatrics 2001; 108: E67.
[0319] 13. Hanson L A, Ceafalau L, Mattsby-Baltzer I, et al. The
mammary gland-infant intestine immunologic dyad. Adv Exp Med Biol
2000; 478: 65-76. [0320] 14. Pickering L K, Granoff D M, Erickson J
R, et al. Modulation of the immune system by human milk and infant
formula containing nucleotides. Pediatrics 1998; 101: 242-249.
[0321] 15. Horton S, Sanghvi T, Phillips M, et al. Breastfeeding
promotion and priority setting in health. Health Policy Plan 1996;
11: 156-168. [0322] 16. Morrow A L, Pickering L K. Human milk
protection against diarrheal disease. Semin Pediatric Infectious
Diseases 1994; 5: 236-242. [0323] 17. Hamosh M. Bioactive factors
in human milk. Pediatr Clin North Am 2001; 48: 69-86. [0324] 18.
Garofalo R P, Goldman A S. Expression of functional
immunomodulatory and anti-inflammatory factors in human milk. Clin
Perinatol 1999; 26: 361-377. [0325] 19. Noguera-Obenza M, Cleary T
G. The role of human milk secretory IgA in protecting infants from
bacterial enteritis. Adv Nutr Res 2001; 10: 213-229. [0326] 20.
Morrow A L, Pickering L K. Human milk and infectious diseases. In:
Long S S, Pickering L K, Prober C G, eds. Principles and Practice
of Infectious Diseases. Second ed. New York: Churchill Livingstone,
2002. [0327] 21. Viverge D, Grimmonprez L, Cassanas G, Bardet L,
Solere M. Discriminant carbohydrate components of human milk
according to donor secretor types. J Pediatr Gastroenterol Nutr
1990; 11: 365-370. [0328] 22. Thurl S, Henker J, Siegel M, Tovar K,
Sawatzki G. Detection of four human milk groups with respect to
Lewis blood group dependent oligosaccharides. Glycoconj J 1997; 14:
795-799. [0329] 23. Zopf D, Roth S. Oligosaccharide anti-infective
agents. Lancet 1996; 347: 1017-1021. [0330] 24. Newburg D S.
Oligosaccharides in human milk and bacterial colonization. J
Pediatr Gastroenterol Nutr 2000; 30 Suppl 2: S8-17. [0331] 25. Moro
G, Minoli I, Mosca M, et al. Dosage-related bifidogenic effects of
galacto- and fructooligosaccharides in formula-fed term infants. J
Pediatr Gastroenterol Nutr 2002; 34: 291-295. [0332] 26. Boehm G,
Lidestri M, Casetta P, et al. Supplementation of a bovine milk
formula with an oligosaccharide mixture increases counts of faecal
bifidobacteria in preterm infants. Arch Dis Child Fetal Neonatal Ed
2002; 86: F178-181. [0333] 27. Crane J K, Azar S S, Stam A, Newburg
D S. Oligosaccharides from human milk block binding and activity of
the Escherichia coli heat-stable enterotoxin (STa) in T84
intestinal cells. J Nutr 1994; 124: 2358-2364. [0334] 28. Cervantes
L E, Newburg D S, Ruiz-Palacios G M. a1-2 Fucosylated chains (H-2
and Lewisb) are the main human milk receptor analogs for
Campylobacter. Pediatr Res 1995; 37: 171A. [0335] 29. Shen Z,
Warren C D, Newburg D S. High-performance capillary electrophoresis
of sialylated oligosaccharides of human milk. Anal Biochem 2000;
279: 37-45. [0336] 30. Newburg D, Peterson J, Ruiz-Palacios G, et
al. Role of human-milk lactadherin in protection against
symptomatic rotavirus infection. Lancet 1998; 351: 1160-1164.
[0337] 31. Shen Z, Warren C D, Newburg D S. Resolution of
structural isomers of sialylated oligosaccharides by capillary
electrophoresis. J Chromatogr A 2001; 921: 315-321. [0338] 32. Dai
D, Nanthkumar N N, Newburg D S, Walker W A. Role of
oligosaccharides and glycoconjugates in intestinal host defense. J
Pediatr Gastroenterol Nutr 2000; 30: S23-33. [0339] 33. Yolken R H,
Peterson J A, Vonderfecht S L, Fouts E T, Midthun K, Newburg D S.
Human milk mucin inhibits rotavirus replication and prevents
experimental gastroenteritis. J Clin Invest 1992; 90: 1984-1991.
[0340] 34. Newburg D S, Peterson J A, Ruiz-Palacios G M, et al.
Role of human-milk lactadherin in protection against symptomatic
rotavirus infection. Lancet 1998; 351: 1160-1164. [0341] 35.
Chaturvedi P, Warren C D, Altaye M, et al. Fucosylated human milk
oligosaccharides vary between individuals and over the course of
lactation. Glycobiology 2001; 11: 365-372. [0342] 36. Henry S,
Oriol R, Samuelsson B. Lewis histo-blood group system and
associated secretory phenotypes. Vox Sang 1995; 69: 166-182. [0343]
37. Koda Y, Tachida H, Pang H, et al. Contrasting patterns of
polymorphisms at the ABO-secretor gene (FUT2) and plasma
alpha(1,3)fucosyltransferase gene (FUT6) in human populations.
Genetics 2001; 158: 747-756. [0344] 38. Henry S, Mollicone R,
Fernandez P, Samuelsson B, Oriol R, G L. Homozygous expression of a
missense mutation at nucleotide 385 in the FUT2 gene associates
with the Le(a+b+) partial-secretor phenotype in an Indonesian
family. Biochem Biophys Res Commun 1996; 219: 675-678. [0345] 39.
Newburg D S. Are all human milks created equal? Variation in human
milk oligosaccharides. J Pediatr Gastroenterol Nutr 2000; 30:
131-133. [0346] 40. Blackwell C C, Jonsdottir K, Hanson M F, Weir D
M. Non-secretion of ABO blood group antigens predisposing to
infection by Haemophilus influenzae. Lancet 1986; 2: 687. [0347]
41. Kallenius G, Mollby R, Svenson S B, Winberg J, Hultberg H.
Identification of a carbohydrate receptor recognized by
uropathogenic Escherichia coli. Infection 1980; 8: 288-293. [0348]
42. Ikehara Y, Nishihara S, Yasutomi H, et al. Polymorphisms of two
fucosyltransferase genes (Lewis and Secretor genes) involving type
I Lewis antigens are associated with the presence of
anti-Helicobacter pylori IgG antibody. Cancer Epidemiol Biomarkers
Prey 2001; 10: 971-977. [0349] 43. Newburg D S, Chaturvedi P, Lopez
E L, Devoto S, Gayad A, Cleary T G. Susceptibility to
hemolytic-uremic syndrome relates to erythrocyte glycosphingolipid
patterns. J Infect Dis 1993; 168: 476-479. [0350] 44. Glass R I,
Holmgren J, Haley C E, et al. Predisposition for cholera of
individuals with O blood group. Possible evolutionary significance.
Am J Epidemiol 1985; 121: 791-796. [0351] 45. Erney R M, Malone W
T, Skelding M B, et al. Variability of human milk neutral
oligosaccharides in a diverse population. J Pediatr Gastroenterol
Nutr 2000; 30: 181-192. [0352] 46. Warren C D, Chaturvedi P,
Newburg A R, Oftedal O T, Tilden C D, Newburg D S. Comparison of
oligosaccharides in milk specimens from humans and twelve other
species. Adv Exp Med Biol 2001; 501: 325-332. [0353] 47. Tauxe R V.
Epidemiology of Campylobacter jejuni infections in the United
States and other industrialized nations. In: Nachamkin I, Blaser M
J, Tomkins L S, eds. Campylobacter jejuni Current Status and Future
Trends. Washington, D.C.: American Society for Microbiology, 1992:
9-19. [0354] 48. Allos B M. Campylobacter jejuni Infections: update
on emerging issues and trends. Clin Infect Dis 2001; 32: 1201-1206.
[0355] 49. Mead P S, Slutsker L, Dietz V, et al. Food-related
illness and death in the United States. Emerg Infect Dis 1999; 5:
607-625. [0356] 50. Wheeler J G, Sethi D, Cowden J M, et al. Study
of infectious intestinal disease in England: rates in the
community, presenting to general practice, and reported to national
surveillance. The Infectious Intestinal Disease Study Executive. Br
J Med 1999; 318: 1046-1050. [0357] 51. de Wit M A S, Koopmans M P
G, Kortbeek L M, van Leeuwen N J, Vinje J, van Duynhoveri Y T H P.
Etiology of gastroenteritis in sentinel general practices in The
Netherlands. Clin Infect Dis 2001; 33: 280-288. [0358] 52. Shallow
R K, Samuel S, McNees A, al. e. Preliminary Food Net data on the
incidence of foodborne illnesses. Selected sites, United States
2000. MMWR CDC Surveill Summ 2001; 50: 241-246. [0359] 53. C D SC.
Trends in selected gastrointestinal infections-2000. Commun Dis Rep
C DR Weekly 2001; 10: 8. [0360] 54. Havelaar A H, de Wit M A, van
Koningsveld R, van Kempen E. Health burden in the Netherlands due
to infection with thermophilic Campylobacter spp. Epidemiol Infect
2000; 125: 505-522. [0361] 55. Calva J J, Ruiz-Palacios G M,
Lopez-Vidal A B, Ramos A, Bojalil R. Cohort study of intestinal
infection with campylobacter in Mexican children. Lancet 1988; 1:
503-506. [0362] 56. Albert M J, Farouque A S G, Farouque S M, Sack
R B, Mahalanabis D. Case-control study of enteropathogens
associated with childhood diarrhea in Dhaka, Bangladesh. J Clin
Microbiol 1999; 37: 3458-3464. [0363] 57. Smith K E, Besser J M,
Hedberg C W, et al. Quinolone-resistant Campylobacter jejuni
infections in Minnesota, 1992-1998. Investigation Team. N Engl J
Med 1999; 340: 1525-1532. [0364] 58. Kallenius G, Mollby R,
Svensson S B, et al. The Pk antigen as receptor for the
haemagglutinin of pyelonephritogenic Escherichia coli. FEMS
Microbiol Lett 1980; 7: 297. [0365] 59. Lomberg H, Leffler H,
Svanborg-Eden C. Influence of secretor status on the availability
of receptors for attaching Escherichia coli on human uroepithelial
cells. In: Lark DL, ed. Protein-Carbohydrate Interactions in
Biological Systems. London: Academic Press, 1986: 235-238. [0366]
60. Blackwell C C, May S J, Brettle R P, MacCallum C J, Weir D M.
Host-parasite interactions underlying non-secretion of blood group
antigens and susceptibility to recurrent urinary tract infections.
In: Lark D L, ed. Protein-Carbohydrate Interactions in Biological
Systems. London: Academic Press, 1986: 229-230. [0367] 61. Howie P
W, Forsyth J S, Ogston S A, Clark A, du V Florey C. Protective
effect of breast feeding against infection. Br Med J 1990; 300:
11-16. [0368] 62. Teele D W, Klein J O, Rosner B, Group GBOMS.
Epidemiology of otitis media during the first seven years of life
in children in Greater Boston: A prospective, cohort study. J
Infect Dis 1989; 160: 83-94. [0369] 63. Newburg D S.
Oligosaccharides and glycoconjugates in human milk: Their role in
host defense. J Mammary Gland Biol Neoplasia 1996; 1: 271-283.
[0370] 64. Ruiz-Palacios G M, Cervantes L E, Newburg D S,
Lopez-Vidal Y, Calva J J. In vitro models for studying
Campylobacter jejuni infections. In: Nachamkin I, Blaser M J,
Tomkins L S, eds. Campylobacter jejuni Current Status and Future
Trends. Washington, D.C.: American Society for Microbiology, 1992:
176-183. [0371] 65. Reguigne-Arnould I, Couillin P, Mollicone R, et
al. Relative positions of two clusters of human
a-L-fucosyltransferases in 19q (FUT1-FUT2) and 19p (FUT6-FUT3-FUT5)
within the microsatellite genetic map of chromosome 19. Cytogenet
Cell Genet 1995; 71: 158-162. [0372] 66. Bry L, Falk P G, Gordon J
L. Genetic engineering of carbohydrate biosynthetic pathways in
transgenic mice demonstrates cell cycle-associated regulation of
glycoconjugate production in small intestinal epithelial cells.
Proc Natl Acad Sci USA 1996; 93: 1161-1166. [0373] 67. Parkhill J,
Wren B W, Mungall K, et al. The genome sequence of the food-borne
pathogen Campylobacter jejuni reveals hypervariable sequences.
Nature 2000; 403: 665-668. [0374] 68. Walker R I, Caldwell M B, Lee
E C, Guerry P, Trust T, Ruiz-Palacios G M. Pathophysiology of
Campylobacter enteritis. Microbiol Rev 1986; 50: 81-94. [0375] 69.
van Vliet A H, Ketley J M. Pathogenesis of enteric Campylobacter
infection. J Appl Microbiol 2001; 90: 45S-56S. [0376] 70. Wassenaar
T M, Blaser M J. Pathophysiology of Campylobacter jejuni infection
of humans. Microbes Infect 1999; 1: 1023-1033. [0377] 71. Fauchere
J L, Rosenau A, Veron M, Moyen E N, Richard S, Pfister A.
Association with HeLa cells of Campylobacter jejuni and
Campylobacter coli isolated from human feces. Infect Immun 1986;
54: 283-287. [0378] 72. Pei Z, Blaser M J. PEB 1, the major
cell-binding factor of Campylobacter jejuni, is a homologue of the
binding component in Gram negative nutrient transport systems. J
Biol Chem 1993; 267: 18717-18725. [0379] 73. Pei S, Doye A, Boquet
P. Mutation of specific acidic residues of the CNF1 T domain into
lysine alters cell membrane translocation of the toxin. Mol
Microbiol 2001; 41: 1237-1247. [0380] 74. Wooldridge K G, Ketley J
M. Campylobacter-host cell interactions. Trends in Microbiology
1997; 5: 96-102. [0381] 75. Marchant J, Wren B W, Ketley J M.
Exploiting genome sequence: prediction for mechanisms of
campylobacter chemotaxis. Trends Microbiol 2002; 10: 155-159.
[0382] 76. Hugdahl M B, Beery J T, Doyle M P. Chemotactic behavior
of Campylobacter jejuni. Infect Immune 1988; 56: 1560-1566. [0383]
77. Jagannathan A, Constantinidou C H, Penn C H W. Roles of rpoN,
fliA, and flgR in expression of flagella in Campylobacter jejuni. J
Bacteriol 2001; 183: 2937-2942. [0384] 78. Allen K J, Griffiths M
W. Effect of environmental and chemotactic stimuli on the activity
of the Campylobactger jejuni flaA s28 promoter. FEMS Microbiol Lett
2001; 205: 43-48. [0385] 79. Szymanski C H M, Burr D H, Guerry P.
Campylobacter protein glycosylation affects host cell interactions.
Infect Innume 2002; 70: 2242-2244.
[0386] 80. Cinco M, Banfi B, Ruaro E, et a. Evidence for L-fucose
(6 deoxy-1-galactopyrasone) mediated adherence of Campylobacter
spp. to epithelial cells. FEMS Microbiol Lett 1984; 21: 347-351.
[0387] 81. Prieto P A, Larsen R D, Cho M, et al. Expression of
human H-type a1,2-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 1997; 272: 2089-2097. [0388] 82. Prieto P A, Mukerji P, Kelder
B, et al. Remodeling of mouse milk glycoconjugates by transgenic
expression of a human glycosyltransferase. J Biol Chem 1995; 270:
29515-29519. [0389] 83. Barua D, Paguio A S. ABO blood groups and
cholera. Ann Hum Biol 1977; 4: 489-492. [0390] 84. Levine M M,
Nalin D R, Rennels M B, et al. Genetic susceptibility to cholera
Ann Hum Biol 1979; 6: 369-374. [0391] 85. Lagos R, Avendano A,
Prado V, et al. Attenuated live cholera vaccine strain CVD 103-HgR
elicits significantly higher serum vibriocidal antibody titers in
persons of blood group O. Infect Immun 1995; 63: 707-709. [0392]
86. Hanne L F, Finkelstein R A. Characterization and distribution
of the hemagglutinins produced by Vibrio cholerae. Infect Immun
1982; 36: 209-214. [0393] 87. Holgersson J, Stromberg N, Breimer M
E. Glycolipids of human large intestine: difference in glycolipid
expression related to anatomical localization,
epithelial/non-epithelial tissue and the ABO, Le and Se phenotypes
of the donors. Biochimie 1988; 70: 1565-1574. [0394] 88. Galvan E
M, Roth G A, Monferran C G. Participation of ABH glycoconjugates in
the secretory response to Escherichia coli heat-labile toxin in
rabbit intestine. J Infect Dis 1999; 180: 419-425. [0395] 89. Boat
T F, Davis J, Stern R C, Cheng P W. Effect of blood group
determinants on binding of human salivary mucous glycoproteins to
influenza virus. Biochim Biophys Acta 1978; 540: 127-133. [0396]
90. Raza M W, Blackwell C C, Molyneaux P, et al. Association
between secretor status and respiratory viral illness. Bmj 1991;
303: 815-818. [0397] 91. Huang P W, Zhong W M, Morrow A L,
Ruiz-Palacios G M, Pickering L K, Jiang X. Human milk contains
elements that block Norwalk-like viruses binding to histo-blood
group antigens in saliva. American Society of Virology Meeting, .
[0398] 92. Albermann C, Piepersberg W, Wehmeier U F. Synthesis of
the milk oligosaccharide 2'-fucosyllactose using recombinant
bacterial enzymes. Carbohydr Res 2001; 334: 97-103. [0399] 93.
Mattila P, Rabina J, Hortling S, Helin J, Renkonen R. Functional
expression of Escherichia coli enzymes synthesizing GDP-L-fucose
from inherent GDP-D-mannose in Saccharomyces cerevisiae.
Glycobiology 2000; 10: 1041-1047. [0400] 94. Priem B, Gilbert M,
Wakarchuk W W, Heyraud A, Samain E. A new fermentation process
allows large-scale production of human milk oligosaccharides by
metabolically engineered bacteria. Glycobiology 2002; 12: 235-240.
[0401] 95. Abe H, Shimma Y-i, Jigami Y. In vitro oligosaccharide
synthesis using intact yeast cells that display
glycosyltransferases at the cell surface through cell wall-anchored
protein Pir. Glycobioloogy 2003; 13: 87-95. [0402] 96. Cleary T G,
Chambers J P, Pickering L K. Protection of suckling mice from
heat-stable enterotoxin of Escherichia coli by infant formulas. J
Pediatr Gastroenterol Nutr 1985; 4: 125-127. [0403] 97. Cleary T G,
Chambers J P, Pickering L K. Protection of suckling mice from
heat-stable enterotoxin of Escherichia coli by human milk. J Infect
Dis 1983; 148: 1114-1119. [0404] 98. Newburg D S, Chaturvedi P,
Crane J K, Cleary T G, Pickering L K. Fucosylated
oligosaccharide(s) of human milk inhibits stable toxin of
Escherichia coli. In: Agrawal V P, Sharma C B, Sah A, Zingde M D,
eds. Complex Carbohydrates and Advances in Biosciences.
Muzaffarnagar, India: Society of Biosciences, 1995: 199-226. [0405]
99. Ruvoen-Clouet N, Ganiere J P, Andre-Fontaine G, Blanchard D, Le
Pendu J. Binding of rabbit hemorrhagic disease virus to antigens of
the ABH histo-blood group family. J Virol 2000; 74: 11950-11954.
[0406] 100. Huang R T C. Isolation and characterization of the
gangliosides of butter milk. Biochim Biophys Acta 1973; 306: 82-84.
[0407] 101. Lindesmith L, Moe C, LePendu J, Jiang X, Baric R.
Determinants of susceptibility and protective immunity to Norwalk
virus infection Annual Meeting of the International Congress of
Virology, Paris, France. [0408] 102. Morrow A L, Ruiz-Palacios G M,
Altaye M, et al. Human milk oligosaccharide homologs of Lewis blood
group epitopes and protection against diarrhea in breastfed
infants. Glycobiology 2002; 12: Abst 21. [0409] 103. Igarashi K.
The Koenigs-Knorr reaction. Adv Carbohydr Chem Biochem 1977; 34:
243-283. [0410] 104. Paulsen H. Advances in selective chemical
syntheses of complex oligosaccharides. Angewandte Chemie
International Edition in English 1982; 21: 155-224. [0411] 105.
Flowers H M. Chemical synthesis of oligosaccharides. Methods
Enzymol 1987; 138: 359-404. [0412] 106. Lemieux R U, Hendriks K B,
Stick R V, James K. Halide ion catalyzed glycosidation reactions.
Syntheses of a-linked disaccharides. J Am Chem Soc 1975; 97:
4056-4062. [0413] 107. Dejter-Juszynski M, Flowers H M. Studies on
the Koenigs-Knorr reaction. Part II. Synthesis of an a-L-linked
disaccharide from tri-O-benzyl-a-L-fucopyranosyl bromide. Carbohydr
Res 1971; 18: 219-226. [0414] 108. Lemieux R U, Driguez H. The
chemical synthesis of
2-acetamido-2-deoxy-4-O-(a-L-fucopyranosyl)-3-O-(b-D-galactopyranosyl)-D--
glucose. The Lewis a blood-group antigenic determinant. J Am Chem
Soc 1975; 97: 4063-4068. [0415] 109. Abbas S A, Barlow J J, Matta K
L. Synthesis of
O-a-L-fucosyranosyl-(1-2)-O-b-D-galactopyranosyl-(1-4)-D-glucosyranose
(2'-O-a-L-fucopyranosyl-lactose). Carbohydr Res 1981; 88: 51-60.
[0416] 110. Hindsgaul O, Norberg T, Pendu J L, Lemieux R U.
Synthesis of type 2 human blood-group antigenic determinants. The
H, X, and Y haptens and variations of the H type 2 determinant as
probes for the combining site of the lectin I of Ulex europaeus.
Carbohydr Res 1982; 109: 109-142. [0417] 111. Nashed M A.
Oligosaccharides from "standard intermediates". The
2-amino-2-deoxy-D-galactose analog of the blood-group O(H)
determinant, type 2, and its precursors. Carbohydr Res 1983; 114:
53-61. [0418] 112. Slife C W, Nashed M A, Anderson L. "Standardized
intermediates" for oligosaccharide synthesis. Precursors of
b-linked, interior D-galactopyranose units having chain extension
at position 4, or positions 4 and 2. Carbohydr Res 1981; 93:
219-230. [0419] 113. Nashed M A, Chowdhary M S, Anderson L.
"Standardized intermediates" for oligosaccharide synthesis.
Precursors of D-galactopyranose residues having chain extension at
position 3, or positions 3 and 2. Carbohydr Res 1982; 102: 99-110.
[0420] 114. Jain R K, Locke R D, Matta K L. A convenient synthesis
of
O-a-L-fucopyranosyl-(1-2)-O-b-D-galactopyranosyl-(1-4)-D-glucopyranose(2'-
-O-a-L-fucopyranosyllactose). Carbohydr Res 1991; 212: c1-c3.
[0421] 115. Liptak A, Jodal I, Nanasi P. Stereoselective
ring-cleavage of 3-O-benzyl- and
2,3-di-O-benzyl-4,6-O-benzylidenehexopyranoside derivatives with
the LiAlH4-AlCl3 reagent. Carbohydr Res 1975; 44: 1-11. [0422] 116.
Lemieux R U, Driguez H. The chemical synthesis of
2-O-(a-L-fucopyranosyl)-3-O-(a-D-galactopyranosyl)-D-galactose. The
terminal structure of the blood-group B antigenic determinants. J
Am Chem Soc 1975; 97: 4069-4075. [0423] 117. Hanessian S, Banoub J.
Chemistry of the glycosidic linkage. An efficient synthesis of
1,2-trans-disaccharides. Carbohydr Res 1977; 53: C13-C16. [0424]
118. Hanessian S, Banoub J. Preparation of 1,2-trans-glycosides in
the presence of silver trifluoromethanesulfonate. Methods in
Carbohydrate Chemistry 1980; 8: 247-250. [0425] 119. Banoub J,
Bundle D R. 1,2-Orthoacetate intermediates in silver
trifluoromethanesulphonate promoted Koenigs-Knorr synthesis of
disaccharde glycosides. Can J Chem 1979; 57: 2091-2097. [0426] 120.
Helferich B, Wedemeyer K-F. Ann 1949; 563: 139-145. [0427] 121.
Koenigs W, Knorr E. Ueber einige Derivate des Traubenzuckers and
der Galactose. Chemische Berichte 1901; 34: 957-981. [0428] 122.
Wiesner D A, Sweeley C C. Microscale analysis of glycospingolipids
by methanolysis, peracetylation, and gas chromatography. Anal
Biochem 1994; 217: 316-322. [0429] 123. Levery S B, Hakomori S.
Microscale methylation analysis of glycolipids using capillary gas
chromatography-chemical ionization mass fragmentography with
selected ion monitoring. In: Ginsburg V, ed. Complex Carbohydrates,
Part E. New York: Academic Press, 1987: 13-25. [0430] 124. McBroom
C R, Samanen C H, Goldstein I J. Carbohydrate antigens: Coupling of
carbohydrates to proteins by diazonium and phenylisothiocyanate
reactions. Complex Carbohydrates, Part B 1972; 28: 212-219. [0431]
125. Zopf D A, Tsai C-M, Ginsburg V. Carbohydrate antigens:
Coupling of oligosaccharide-phenethylamine derivatives to edestin
by diazotization and characterization of antibody specificity by
radioimmunoassay. Methods Enzymol 1978; 50: 204-206. [0432] 126.
Yariv J, Rapport M M, Graf L. The interaction of glycosides and
saccharides with antibody to the corresponding phenylazo
glycosides. Biochem J 1962; 85: 383-388. [0433] 127. Rosenfeld L,
Lee Y C. A practical preparation of p-nitrophenyl
b-D-mannopyranoside. Carbohydr Res 1976; 46: 155-158. [0434] 128.
Pazur J H. Affinity chromatography of macromolecular substances on
adsorbents bearing carbohydrate ligands. Adv Carbohydr Chem Biochem
1981; 39: 405-447. [0435] 129. Newburg D S, Peterson J A,
Ruiz-Palacios G M, Matson D O, Morrow A L, Schults J, et al. Role
of human-milk lactadherin in protection against symptomatic
rotavirus infection. Lancet 1998; 351: 1160-4. [0436] 130. Newburg
D S, Ruiz-Palacios G M, Altaye M, Chaturvedi P, Meinzen-Derr J,
Guerrero M L, Morrow A L. Innate protection conferred by
fucosylated oligosaccharides of human milk against diarrhea in
breastfed infants. Glycobiology 2004; 14:253-263. [0437] 131.
Ruiz-Palacios G M, Cervantes L E, Ramos P, Prieto P A, Newburg D S.
Campylobacter jejuni binds intestinal H(O) antigen
(Fuca1,2Gal.beta.1,4GlcNAc), and fucosyloligosaccharides of human
milk inhibit its binding and infection. J Biol Chem 2003;
278:14112-14120. [0438] 132. Marionneau S, Ruvoen N, Le
Moullac-Vaidye B, Clement M, Cailleau-Thomas A, Ruiz-Palacios G M,
et al. Norwalk virus binds to H types 1/3 histo-blood group
antigens present on gastro-duodenal epithelial cells of "secretor"
individuals. Gastroenterology 2002; 122: 1967-77. [0439] 133. Huang
P, Farkas T, Marionneau S, Zhong W, Ruvoen-Clouet N, Morrow A L, et
al. Noroviruses bind to human ABO, Lewis and secretor histo-blood
group antigens: Identification of four distinct strain-specific
patterns. J Infect Dis 2003; 188:19-31. [0440] 134. Newburg D S,
Pickering L K, McCluer R H, Cleary T G. Fucosylated
oligosaccharides of human milk protect suckling mice from
heat-stabile enterotoxin of Escherichia coli. J Infect Dis 1990;
162:1075-80. [0441] 135. Crane J K, Azar S S, Stam A, Newburg D S.
Oligosaccharides from human milk block binding and activity of the
Escherichia coli heat stable enterotoxin (Sta) in T84 intestinal
cells. J Nutr 1994; 124:2358-64. [0442] 136. Velazquez F R, Matson
D O, Calva J J, Guerrero M L, Morrow A L, Carter-Campell S, et al.
Rotavirus infections in infants as protection against subsequent
infections. N Engl J Med 1996; 335: 1022-8. [0443] 137. Morrow A L,
Reves R R, West M S, Guerrero M L, Ruiz-Palacios G M, Pickering L
K. Protection against infection with Giardia lamblia by
breastfeeding in a cohort of Mexican infants. J Pediatr. 1992;
121:363-70. [0444] 138. Ruiz-Palacios G M, Calva J J, Pickering L
K, Lopez-Vidal Y, Volkow P, Pezzarossi H, West M S. Protection of
breast-fed infants against Campylobacter diarrhea by antibodies in
human milk. J Pediatr 1990; 116:707-13. [0445] 139. Farkas T, Jiang
X, Guerrero M L, Zhong W, Wilton N, Berke T, et al. Prevalence and
genetic diversity of human caliciviruses (HuCVs) in Mexican
children. J Med Virol 2000; 62: 217-23. [0446] 140. Jiang X, Huang
P W, Zhong W M, Farkas T, Cubitt D W, Matson D O. Design and
evaluation of a primer pair that detects both Norwalk- and
Sapporo-like caliciviruses by RT-PCR. J Virol Methods 1999; 83:
145-54. [0447] 141. Jiang X, Wilton N, Zhong W M, Farkas T, Huang P
W, Barrett E, et al. Diagnosis of human caliciviruses by use of
enzyme immunoassays. J Infect Dis 2000; 181: S349-59. [0448] 142.
Ruuska T, Vesikari T. Rotavirus disease in Finnish children: use of
numerical scores for clinical severity of diarrhoeal episodes.
Scand J Infect Dis 1990; 22: 259-67. [0449] 143. Chaturvedi P,
Warren C D, Ruiz-Palacios G M, Pickering L K, Newburg D S. Milk
oligosaccharide profiles by reversed-phase HPLC of their
perbenzoylated derivatives. Anal Biochem 1997; 251:89-97. [0450]
144. Newburg D S, Altaye M, Morrow A L. Is the expression of human
milk fucosylated oligosaccharide during the first month of
lactation representative of the first year of lactation?
Glycobiology 2003; 13:885. [0451] 145. Glass R I, Holmgren J, Haley
C E, Khan M R, Svennerholm A M, Stoll B J, et al. Predisposition
for cholera of individuals with O blood group. Possible
evolutionary significance. Am J Epidemiol 1985; 121: 791-6.34.
[0452] 146. Newburg D S, Chaturvedi P, Lopez E L, Devoto S, Fayad
A, Cleary T G. Susceptibility to hemolytic-uremic syndrome relates
to erythrocyte glycosphingolipid patterns. J Infect Dis 1993; 168:
476-9. [0453] 147. Ikehara Y, Nishihara S, Yasutomi H, Kitamura T,
Matsuo K, Shimizu N, et al. Polymorphisms of two fucosyltransferase
genes (Lewis and Secretor genes) involving type I Lewis antigens
are associated with the presence of anti-Helicobacter pylori IgG
antibody. Cancer Epidemiol Biomarkers Prey 2001; 10: 971-7. [0454]
148. Hutson A M, Atmar R L, Graham D Y, Estes M K. Norwalk virus
infection and disease is associated with ABO histo-blood group
type. J Infect Dis 2002; 185:1335-7. [0455] 149. Boat T F, David J,
Stern R C, Cheng P W. Effect of blood group determinants on binding
of human salivary mucous glycoproteins to influenza virus. Biochim
Biophys Acta 1978; 54:127-33. [0456] 150. Raza M W, Blackwell C C,
Molyneaux P, James V S, Ogilvie M M, Inglis J M, et al. Association
between secretor status and respiratory viral illness. BMJ 1991;
303:815-8. [0457] 151. Ciarlet M, Crawford S E, Estes M K.
Differential infection of polarized epithelial cell lines by sialic
acid-dependent and sialic-acid independent rotavirus strains. J
Virol 2001; 75: 11834-50. [0458] 152. Endo T, Koizumi S.
Large-scale production of oligosaccharides using engineered
bacteria. Curr Opin Struct Biol 2000; 10:536-541. [0459] 153. Endo
T, Koizumi S, Tabata K, Kakita S, Ozaki A. Large-scale production
of the carbohydrate portion of the sialyl-Tn epitope,
alpha-Neup5Ac-(2.fwdarw.6)-D-GalpNAc, through bacterial coupling.
Carbohydr Res 2001; 330:439-443.
[0460] 154. Endo T, Koizumi S, Tabata K, Ozaki A. Large-scale
production of CM P-NeuAc and sialylated oligosaccharides through
bacterial coupling. Appl Microbiol Biotechnol 2000; 53:257-261.
[0461] 155. Chen X, Zhang J, Kowal P, Liu Z, Andreana P R, Lu Y,
Wang P G. Transferring a biosynthetic cycle into a productive
Escherichia coli strain: large-scale synthesis of galactosides. J
Am Chem Soc 2001; 123:8866-8867. [0462] 156. Chen X, Liu Z, Zhang
J, Zhang W, Kowal P, Wang P G. Reassembled biosynthetic pathway for
large-scale carbohydrate synthesis: a-Gal epitope producing
`superbug`. Chembiochem 2002; 3:47-53. [0463] 157. Dumon C, Priem
B, Martin S L, Heyraud A, Bosso C, Samain E. In vivo fucosylation
of lacto-N-neotetraose and lacto-N-neohexaose by heterologous
expression of Helicobacter pylori alpha-1,3 fucosyltransferase in
engineered Escherichia coli. Glycoconj J 2001; 18:465-474. [0464]
158. Priem B, Gilbert M, Wakarchuk W W, Heyraud A, Samain E. A new
fermentation process allows large-scale production of human milk
oligosaccharides by metabolically engineered bacteria. Glycobiology
2002; 12:235-240. [0465] 159. Samain E, Chazalet V, Geremia R A.
Production of O-acetylated and sulfated chitooligosaccharides by
recombinant Escherichia coli strains harboring different
combinations of nod genes. J Biotechnol 1999; 72:33-47. [0466] 160.
Luhn K, Wild M K, Eckhardt M, Gerardy-Schahn R, Vestweber D. The
gene defective in leukocyte adhesion deficiency II encodes a
putative GDP-fucose transporter. Nat Genet 2001; 28:69-72. [0467]
161. Ishida N, Kawakita M. Molecular physiology and pathology of
the nucleotide sugar transporter family (S LC35). Pflugers Arch
2004; 447:768-775. [0468] 162. Wu B, Zhang Y, Wang P G.
Identification and characterization of GDP-d-mannose
4,6-dehydratase and GDP-1-fucose snthetase in a GDP-1-fucose
biosynthetic gene cluster from Helicobacter pylori. Biochem Biophys
Res Commun 2001; 285:364-371. [0469] 163. Shao J, Zhang J, Kowal P,
Lu Y, Wang P G. Efficient synthesis of globoside and isogloboside
tetrasaccharides by using beta(1.fwdarw.3)
N-acetylgalactosaminyltransferase/UDP-N-acetylglucosamine C4
epimerase fusion protein. Chem Commun (Camb) 2003:1422-1423. [0470]
164. Bulik D A, van Ophem P, Manning J M, Shen Z, Newburg D S,
Jarroll E L. UDP-N-acetylglucosamine pyrophosphorylase, a key
enzyme in encysting Giardia, is allosterically regulated. J Biol
Chem 2000; 275:14722-14728. [0471] 165. Sener K, Shen Z, Newburg D,
Jarroll E. Amino sugar phosphate levels in Giardia change during
cyst wall formation. Microbiology 2004; 150:1225-1230. [0472] 166.
Hamilton S, Bobrowicz P, Bobrowicz B, Davidson R, Li H, Mitchell T,
Nett J, Rausch S, Stadheim T, Wischnewski H, Wildt S, Gerngross T.
Production of complext human glycoproteins in yeast. Science 2003;
301:1244-1246. [0473] 167. Choi B K, Bobrowicz P, Davidson R C,
Hamilton S R, Kung D H, Li H, Miele R G, Nett J H, Wildt S,
Gerngross T U. Use of combinatorial genetic libraries to humanize
N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad
Sci USA 2003; 100:5022-5027. [0474] 168. Nakayama K, Maeda Y,
Jigami Y. Interaction of
GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase with
GDP-mannose-4,6-dehydratase stabilizes the enzyme activity for
formation of GDP-fucose from GDP-mannose. Glycobiology 2003;
13:673R-680R. [0475] 169. Mattila P, Rabina J, Hortling S, Helin J,
Renkonen R. Functional expression of Escherichia coli enzymes
synthesizing GDP-L-fucose from inherent GDP-D-mannose in
[0476] Saccharomyces cerevisiae. Glycobiology 2000; 10:1041-1047.
170. Gao X D, Dean N. Distinct protein domains of the yeast Golgi
GDP-mannose transporter mediate oligomer assembly and export from
the endoplasmic reticulum. J Biol Chem 2000; 275:17718-17727.
[0477] 171. Gao X D, Nishikawa A, Dean N. Identification of a
conserved motif in the yeast golgi
[0478] GDP-mannose transporter required for binding to nucleotide
sugar. J Biol Chem 2001; 276:4424-4432.
[0479] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *