Immunology of carbohydrates

1
Immunology of carbohydrates
2
Immunology

• Different immune systems
• Cell-mediated
• T-cells
• Humoral
• Antibodies
• Role in protection
• Interaction of polysaccharides with the immune
  system
• Modification of polysaccharides to improve
  protective response
• Endotoxin - immune system
• Example of some unique bacterial polysaccharides

3
IMMUNOLOGYOverview of the immune System
Immune System
Innate (Nonspecific)
Adaptive (Specific)
Cellular Components
Humoral Components
Cell-Mediated
Humoral (Ab)
4
Definitions

• Immunogen
• Compound that give rise to an immune response
• Antigen (Ag)
• Compound that the elicited immunity reacts with
• Hapten
• Part of an immunogen (can not elicit immune
  response itself)
• Epitope or Antigenic Determinant
• Part of an antigen
• Antibody (Ab)
• Proteins produced by the immune system upon
  stimulation with an immunogen

5
Factors Influencing ImmunogenicityContribution
of the Immunogen

• Foreignness
• Size
• Chemical Composition
• Physical Form
• Degradability

6
Factors Influencing ImmunogenicityContribution
of the Biological System

• Genetics
• Species
• Individual
• Responders vs Non-responders
• Age

7
Factors Influencing ImmunogenicityMethod of
Administration

• Dose
• Route
• Subcutaneous, dermal gt Intravenous gt Intragastric
• For mucosal immunity - intranasal
• Adjuvant
• Substances that enhance an immune response to an
  Ag

8
Chemical Nature of Immunogens

• Proteins
• Polysaccharides
• Nucleic Acids
• Lipids
• Some glycolipids and phosopholipids can be
  immunogenic for T cells and illicit a cell
  mediated immune response

9
Types of AntigensT-dependent

• Proteins

• Structure

• Examples
• Microbial proteins
• Non-self or Altered-self proteins

10
T-cell dependent antigens

• Proteins and peptides
• Presented to T cells by
• Major Histocompatibility Complex (MHC)
• Macrophages
• B cells
• Dendritic cells
• Long lasting immune response
• Formation of memory B and T cells
• High affinity antibodies
• IgA, IgM, IgG1, IgG2a, IgG2b, IgG3

11
Th cells are primed by antigen-presenting cell
B-T cell cooperation B cells receive signals from
T cells
B cells divide
B-memory cell
12
T-Independent Antigens

• Activate B cells at high concentrations
• Large polymeric molecules with repeating
  determinants
• Poorly degraded
• Some activate both immature and mature B cells
  some only mature cells
• Responses dominated by CD5 B cells

13
Types of Antigens T-independent

• Properties
• Polymeric structure
• Polyclonal B cell activation
• Yes -Type 1 (TI-1)
• No - Type 2 (TI-2)
• Resistance to degradation
• Examples
• Pneumococcal polysaccharide, lipopolysaccharide
• Flagella

14

15
Antigenic DeterminantsRecognized by B cells and
Ab

• Composition
• Proteins, polysaccharides, nucleic acids
• Sequence (linear) determinants
• Conformational determinants

16
ImmunoglobulinsStructure and Function
17
Basic immunoglobulin structure

• Immunoglobulins - heterogeneous
• Myeloma proteins - homogeneous immunoglobulins

18
Immunoglobulin Fragments Structure/Function
Relationships
Ag Binding
Complement Binding Site
Binding to Fc Receptors
19
IgG

• Structure
• Monomer (7S)
• Properties
• Major serum Ig
• Major Ig in extravascular spaces
• Placental transfer Does not require Ag binding
• Fixes complement
• Binds to Fc receptors
• Phagocytes - opsonization

20
IgM

• Structure
• Pentamer (19S)
• Properties
• 3rd highest serum Ig
• Fixes complement
• Agglutinating Ig
• Binds to Fc receptors
• B cell surface Ig

21
IgA

• Structure
• Serum - monomer
• Secretions (sIgA)
• Dimer (11S)
• Secretory component
• Properties
• 2nd highest serum Ig
• Major secretory Ig (Mucosal or Local Immunity)
• Tears, saliva, gastric and pulmonary secretions
• Does not fix complement (unless aggregated)
• Binds to Fc receptors on some cells

22
Antigen-Antibody ReactionsTests for Ag-Ab
reactions
23
Nature of Ag/Ab Reactions

• Lock and Key Concept
• Non-covalent Bonds
• Hydrogen bonds
• Electrostatic bonds
• Van der Waal forces
• Hydrophobic bonds
• Multiple Bonds
• Reversible

24
Tests Based on Ag/Ab Reactions

• All tests based on Ag/Ab reactions can be used to
  detect either Ag or Ab

25
Agglutination Tests
26
Agglutination/Hemagglutination

• Definition - tests that have as their endpoint
  the agglutination of a particulate antigen

• Qualitative agglutination test
• Ag or Ab

Bacteria, RBC, Particle with antigen
Y

Y
?
Y
27
Agglutination/Hemagglutination

• Applications
• Blood typing
• Bacterial infections
• Fourfold rise in titer
• Bacterial identification

28
Enzyme-Linked Immunoassay (EIA)
29
Solid Phase Non-Competitive EIA

• Ab detection
• Immobilize Ag
• Incubate with sample
• Add labeled anti-Ig
• Amount of labeled Ab bound is proportional to
  amount of Ab in the sample
• Quantitative

30
Solid Phase Non-Competitive EIA

• Ag detection
• Immobilize Ab
• Incubate with sample
• Add labeled antibody
• Amount of labeled Ab bound is proportional to the
  amount of Ag in the sample
• Quantitative

Labeled Ab
Y
Ag
Y
Immobilized
Solid Phase
31
Immunology of bacterial polysaccharides

• Carbohydrate antigens exhibit a large degree of
  antigenic variation
• Between and within the same species
• Basis of serogrouping or serotyping systems

32
Immunology of bacterial polysaccharides

• gt 10 different serogroups of Neisseria
  meningitidis
• gt 90 different serotypes of Streptococcus
  pneumoniae based on the CPSs
• The number of LPS O-antigens for several species
  gt 100
• Anti-polysaccharide antibodies are usually,
  serotype/serogroup-specific

33
Diversity in the carbohydrate antigens in some
clinically important bacteria
34
Immunology of bacterial polysaccharides

• T-cell independent (TI)
• Anti-polysaccharide immune response is
  characterised by
• Lack of immunological memory
• Isotype restriction of antibodies
• Children lt2 years of age and elderly respond
  poorly to polysaccharide antigens.

35
T-cell independent antigens

• Type 1
• Induce proliferation and differentiation of both
  naïve and mature B cells
• May induce immune responses in neonates and
  adults
• Example bacterial lipopolysaccharides (LPS)
• Type 2
• High molecular mass repetitive polysaccharide
  structures
• Poor in vivo degradability
• Activate mature B-cells
• Antigen-specific antibodies
• Poor response in neonates and elderly
• Examples CPS from S. pneumoniae, N. meningitidis
  and H. influenzae

36
Bacterial polysaccharides - vaccine

• Streptococcus pneumoniae
• Neisseria meningitidis
• Haemophilus influenzae

37
Polysaccharide based vaccines

• Streptococcus pneumoniae
• Gram-positive
• Adults - lower respiratory tract infections
• Children - otitis media

38
Streptococcus pneumoniae

• 83 different type specificities
• First vaccine - 14-valent
• 14 polysaccharides - 70-80 of all infections

39
Streptococcus pneumoniae

• Today a 23-valent vaccine - 90 protection
• 23 polysaccharides - most common in US and Europe
• Distribution of the CPS-types differ
  geographically

40
Streptococcus pneumoniae

• Age-related differences in distribution and
  immune response
• Infants
• Development of polysaccharide-protein conjugate
  vaccines

41
Neisseria meningitidis

• Worldwide problem
• Endemic and epidemic form
• Epidemic disease in all parts of the world
• 500/100.000

42
Neisseria meningitidis

• Mortality rates
• Not treated - 85
• Treated - 10
• Cured patients may suffer of permanent
  neurological deficiencies

43
Neisseria meningitidis

• Gram-negative
• Serological classification into groups
• A, B, C, 29e, W135, X, Y and Z
• Based on the structures of capsular
  polysaccharides

44
Neisseria meningitidis

• Groups A, B, C responsible for 90 of cases
• Vaccine available for groups A and C
• No vaccine against group B

45
Haemophilus influenzae

• Type b H. influenzae - major cause of
  meningitidis in children up to 5 years
• Mortality 5-10

46
Haemophilus influenzae

• Gram-negative
• Six CPS types (a-f)
• Type b the most common

47
Haemophilus influenzae

• Type b PS - long-lived complement mediated
  bactericidal antibodies in adults
• Age-dependence
• Only children older than 18 months - protected

48
Structural mimicry of polysaccharides

• E. coli K5 is identical to the first polymeric
  intermediate in the biosynthesis of Heparin
• E. coli K4 similar structure as chondroitin
• N. meningitidis B homopolymer of -8)aD-NeuNAc(2-
• Identical to E. coli K1 capsule

49
N. meningitidis serogroup B

• Homology between carbohydrate structures on
  bacterial surface and those of host cell
  membranes
• N. meningitidis serogroup B CPS and E. coli K1
  antigen are antigenically similar to structures
  expressed on human foetal neuronal cells
• poor immunogens in humans
• N. meningitidis serogroup B CPS in a vaccine not
  possible
• potential risk of inducing antibodies against
  self antigens

50
How to circumvent the T-cell independent property
of polysaccharides

• Protein - polysaccharide conjugates

51
Hapten-carrier conjugates

• Definition
• Structure
• native determinants
• haptenic determinants

52
T Cell-B Cell Interactions(hapten-carrier effect)

• T-helper cells recognize carrier, B cells
  recognize hapten
• T-helper and B cells cooperate by interacting

53
Mechanism of Hapten-Carrier

• Hapten recognized by Ig receptor on B cell
• Hapten-carrier endocytosed
• Carrier processed and presented on class II MHC
  to T-helper cell
• Activated T-helper cell produces cytokines
• Cytokines enable B cell to be activated to
  produce anti-hapten antibodies

54
Protein - polysaccharide conjugates

• Haemophilus influenzae b (Hib) conjugate vaccine
• Efficacy
• lt1 year - 99
• 1-2 years - 97
• 2-3 old children - 94.
• Reduction of carriage of Hib,
• lower transmission rates

55
Protein - polysaccharide conjugates - Neisseria
meningitidis

• The type A and C neoglycoconjugate vaccines
• safe and well tolerated in infants and young
  children
• specific anti-A and anti-C polysaccharide
  antibody titers.
• Meningitidis serogroup B
• homopolymer of ?-2?8-linked sialic acid residues
• poor immunogen in man.
• The poor immunogenicity due to immunologic
  tolerance induced by foetal exposure to
  cross-reactive polysialated glycoproteins
  expressed in a variety of host tissues, such as
  neuronal cell adhesion molecules.

56
Protein - PS conjugates

• Tetanus toxoid
• Diphteria toxoid
• CRM197 - mutant diphteria toxin

57
How to circumvent the T-cell independent property
of polysaccharides

• Peptides mimicking polysaccharides
• Example for V. cholerae

58
Peptides mimicking polysaccharides

• Brucella abortus LPS
• S. flexneri 5a LPS
• C. neoformans polysaccharide
• Meningococcol polysaccharide
• Streptococcal polysaccharide
• V. cholerae polysaccharide
• Blood-group antigen
• Tumor associated carbohydrate
• Carbohydrates on adeno-carcinoma cells

59
Possible candidates for V. cholerae O139 vaccine

• Whole bacteria - killed
• Attenuated live bacteria
• CPS
• Poor immunogen
• T-cell independent immune response
• LPS
• endotoxic

60
Aims

• To select peptides that mimic the capsular
  polysaccharide in V. cholerae O139
• To evaluate the specificity and protective
  efficacy of antibodies against selected peptides
  that mimic the capsular polysaccharide in
  V. cholerae O139

61
Polysaccharide capsule
HO O
P
4 6

• ?-Colp-(12)-?-D-Galp
• 3
• 6)-?-D-GlcpNAc-(14)-?-D-GalpA-(13)-?-D-QuipNAc
  -(1
• 4
• ?-Colp

62
Identification of mimotops
Peptide library
63
Phage display
64
Suckling mouse assay
65
Suckling mouse assay - dose response
66
Suckling mouse assay - specificity
67
Conclusions

• 8 peptides that mimic O139 CPS - selected
• Immune response in mice specific against O139 CPS
• Anti-peptide antibodies are protective in the
  suckling mouse assay
• Protective efficacy both specific and
  dose-dependent
• Peptides are possible candidates for development
  of a novel, safe and efficient vaccine against V.
  cholerae O139

68
Conclusions

• First example that peptides mimicking a
  bacterial polysaccharide possess protective
  efficacy against the disease

69
Advantages with polysaccharide-based vaccines

• Capsular polysaccharide
• Elicit antibodies similar to those in natural
  response
• Safe and efficacious
• Microbial products
• Can be easily purified
• Capsular polysaccharide-protein conjugate
• Elicit antibodies similar to those in natural
  response
• Convert T-cell independent to T-cell dependent
  responses in infants and children
• Increased immunogenicity in infants and young
  children
• Safe and efficacious
• Peptide mimotope of polysaccharide antigen
• Biochemically defined
• T-cell dependent antigens
• can elicit immunological memory, affinity
  maturation and isotype switching

70
Disadvantages with polysaccharide-based vaccines

• Capsular polysaccharide
• Poorly immunogenic T-cell independent-type 2
  antigens
• Do not elicit immunological memory or isotype
  switching
• Capsular polysaccharides are heterogeneous
• Capsular polysaccharide-protein conjugate
• Do not elicit classical T-cell dependent
  responses in the elderly
• Most opsonic IgG subclasses might not be produced
• Protective and non-protective antibodies can be
  produced
• Conjugates from CPS preparations are
  heterogeneous
• Possible undesired immunity against the carrier
  protein
• Peptide mimotope of polysaccharide antigen
• Poorly immunogenic if not conjugated to a carrier
  molecule
• No experience for efficacy in humans

71
Lipopolysaccharide endotoxin

• 1892 - Heat inactivated lysates of Vibrio
  cholerae induced pathophysiological reaction in
  guinea pigs (Pfeiffer)

72

• Mammals - permanent contact with Gram-negative
  bacteria and LPS

73
Endotoxin - Biological activity
74
Lipid A

• Carbohydrate
• GlcN-?-(1?6)-GlcN
• 6-7 fatty acids (all saturated)

75
Escherichia coli lipid A
76
Host cells stimulated by LPS

• Monocytes/Macrophages
• Endothelial cells
• Neutrophils
• Smooth muscle cells

77
Mechanism for host response against endotoxin
78
(No Transcript)
79
Granulocyte
Direct action Stimulation of additional
cells Recruitment of additional
mediators (complement factors, clotting cascade)
Priming for O2 release Adhesion molecules
-
Monocyte/ Macrophage
TNF-? IL-1 IL-6 PAF Oxygen radicals
Endotoxin
Fever Hypotension Tachycardia Tachypnoe Neutropeni
a DIC MOF Death
IL-1 IL-6 Adhesion molecules
Endothelial cell
80
T-cell dependent polysaccharide - example

• Bacteroides fragilis
• Anaerobe
• Member of normal flora
• Gram-negative
• Outer membrane contain LPS and CPS
• Most B. fragilis strains produce two different
  CPS
• PS A
• PS B
• Negatively and positively charged - zwitter ions

81
B. fragilis NCTC 9343 capsular polysaccharides

• Polysaccharide A (PS A)
• Polysaccharide B (PS B)
• Both contain free aminogroup and a carboxyl group

82
Capsular polysaccharide as virulence factor

• Anti-CPS antibodies do not protect against
  abscess formation
• T cells from animals immunized with B. fragilis
  protect against abscess formation
• Onderdonk et al 1982

83
B. fragilis capsular polysaccharide A
A

O
H
N
3
H
C
3
HO
O
NHAc
O
OH
O
HO
NHAc
O
O
O
HO
OH
O
OH
-
COO
O
O
O
CH
3
n
84
B. fragilis capsular polysaccharide B
B
HO
CH
3
O
O
AcHN
OH
O
O
O

-
H
N
O
OH
3
O
P
OH
OH
O
O
-
O
COO
NHAc
O
O
H
C
3
HO
HO
OH
O
O
NHAc
OH
OH
O
CH
3
n
85
Other charged bacterial polysaccharides in
abscess induction
O
OH
O
O
NHAc
O
HO
O
O
O
HO
O
OH
O
HO
NHAc
O
Streptococcus pneumoniae type 1 C-substance YES
86
Other charged bacterial polysaccharides in
abscess induction
O
O
NHAc
O
OH
O
O
O
O
HO
OH
OH
Streptococcus pneumoniae type 1 capsular
polysaccharide YES
87
Other charged bacterial polysaccharides in
abscess induction
OH
O
O
HO
O
O
O
HO
OH
OH
Streptococcus pneumoniae type 3 capsular
polysaccharide NO
88
Other charged bacterial polysaccharides in
abscess induction
O
O
HO
NHAc
O
Salmonella typhi Vi polysaccharide NO YES if
N-deAcetylated
89
Other charged bacterial polysaccharides in
abscess induction

• Both the positive and negative charge required
• Other charged polysaccharides induce
  cross-protection against abscess formation
• Tzianabos et al 1995