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Anti-polysaccharide Antibodies:

      Structure-Function
       8 paper Review
Variable Regions of human Hib mAb
Lucas et al 1994
• Mechanisms by which conjugate vaccines
   induce protection were poorly understood
• Anti-Hib antibody gene usage limited to:
     –   VH V3-23 and V3-15
     –   Several VL used
•   Different Hib PS conjugate vaccines induce
    different V region expression
     –   OMPC
          •   No k subgroup III in infants
          •   Common k subgroup III in adults
Lucas et al 1994
•   mAb, CA4, isolated from mouse-human
    heterohybridoma obtained from a 41 y.o. Male
    5 days after vaccination with 40 ug of Hib
    polysaccharide
•   CA4
     –    VH region 96% identical to germ line V3-21
     –    VL k subgroup III 94% identical to A27
•   CA4 utilizes VH III not previously known to
    encode anti Hib PS antibodies
•   CA4 VK III closely resembles that of a variety
    of autoantibodies
Structural Determinants of human
          idiotype HibId-1
Lucas et al 2001
• HibId-1 is expressed by kappa light chains
   encoded by A2 or A18 variable region genes
• Residues that interact to form idiotypic
   determinants may or may not be the same as
   those contributing to affinity of the combining
   site for the antigen
• HibId-1 does not require any contribution from
   the heavy chain
Lucas et al 2001
•   HibId-1 positive PS-specific antibodies have
    invariably been shown to encode:
     –   VkII A2 gene rearranged to Jk1 or Jk3
     –   Non-template arginine inserted at the Vk-Vj
         junction
     –   10 aa CDR3
•   Alterations of residues 30 and 40 result in loss
    of HibId-1
     –   Position 30 is important for interaction and
         changes in the spacing charges
Lucas et al 2001
•   HibId-1 idiotype can be dissociated from Hib
    PS binding
•   “Canonical” Hib PS-specific V3-23/A2 Fabs
    have been isolated that lack Lys30 and are
    HibId-1 negative
Structure-function Relationships for
    human Abs to PPS from transgenic
          mice with human Ig loci
Pirofski et al 2002
• PCV7 does not contain PPS3
       –   Causes disease primarily in adults
•     Can surrogates of Ab efficacy be determined
      using human mAbs to PPS3 in transgenic
      mice?
•     Vaccinated mice with PPS3 conjugated to
      tetanus toxoid
Pirofski 2002




•   mAbs isolated from hybridomas that produced
    the greatest C3 deposition in vitro, 7C5 and
    A7 were also the most protective in vivo
Molecular Ontogeny of human Ab
        repertoire to Hib PS
Lucas et al 2003
• Do variable regions of infant HibId-1 antibodies
  represent the same canonical configuration of
  adults?
• Which structural polymorphisms could account
  for functional heterogenicity?
• Infants were vaccinated, Hib PS binding B cells
  were isolated using biotinylated Hib PS and a
  Fab library was created
Lucas et al 2003
        • “We and others have
          shown that this method
          does not generate
          irrelevant chain
          combinations from
          promiscuous assembly
          but rather recapitulates
          the pairing of native
          sites.”
        • Fabs using the Jk1 gene
          conferred high affinity
          than Jk3
Correlation of antigenic epitope and
 Ab gene usage against PPS23F
Zhou et al 2003
• Human Ab response to 23F is predominated by
  Ab expressing VkL6 or VkA23
  • Reference?
• The antigenic epitopes recognized by VkL6 and
  VkA23 are different based on specificities for L-
  rhamnose, a constituent sugar of the bacterial
  capsule polymer.
Zhou et al 2003
• As the length of the L chain CDR3 decreases,
  the degree to which rhamnose contributes to
  overall affinity of interactions might also
  decrease
  • VkL6 has a longer CDR3 than VkA23
  • VkL6 Fabs are 100-fold more sensitive to inhibition
    with L-rhamnose than Vk23 Fabs
IGH V3-23*01 and its allele V23-03
    differ in capaciry to form canonical
       human Ab binding site for Hib
Lucas et al 2003
• *01 and *03 differ by 9 bases, 8 of which are
  located in the CDR2
  • These eight differences encode 5 amino acid
    substitutions
• Does the V3023*03 polymorphism affect the
  binding to Hib PS?
• Fab fragment isolated from peripheral blood of a
  vaccinated infant by combinatorial library
  cloning
Lucas et al 2003
• RABA revealed 20 fold more effective binding
  by *03 than *01
  • Fab binding was 4 fold
• “The V3-23*03 allele has not been observed in
  Hib PS antibodies.”
  • Allele more common in Asians
Codon insertion and deletion as a
 somatic diversification mechanism
Zhou et al 2006
• Codon insertions and deletions were observed
  most frequently in CDR
• Deletions were found in CDR while insertions
  were found in both CDR and frameworks 1 and
  2
• Location of I/Ds were highly correlated with
  RGYW/WRCY motifs
• In all cases codons duplicate those immediately
  5’ or 3’ to the insertion
Zhou et al 2006
• Peripheral blood was collected 7 days post adult
  vaccination with Prevnar or Pneumovax
• Expression libraries generated from individuals
• 12 of 124 independent H and L rearrangements
  contained I/D events
• I/D events were associated with somatic
  hypermutation and did not occur during V/J and
  V/D/J rearrangements
Domain specificity of the human Ab
  response to Bacillus anthracis
Zhou et al 2008
• Domain specificity of isolated mAb was biased
  toward the amino-terminal 20 kDa fragment of
  PA (protective antigen).
• However PA83 is primary immunogenic
  component of Biothrax vaccine.
• Why is there a biased antibody response to
  PA20 which is not involved in anthrax
  intoxication?
Zhou et al 2008
• Serum Ab response following vaccination is also
  biased toward determinants associated with PA20
• Selected Fab clones were expressed as IgG1
• Isolated Ab fail to neutralize PA-mediated
  cytotoxicity
• Ab may bind to PA20 so that it interferes with one
  of the obligatory funcitons
  • Receptor binding
• Shift epitope bias toward the functionally relevant
  PA63 portion
Conclusions
• Premature assumptions were made about anti-
  PPS Ab gene usage
• Data from Hib studies was used as example
• However gene usage for anti Hib antibodies has
  been well defined while in vivo anti PPS
  antibody gene usage had not been explored.
Lucas et al. Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b
capsular polysaccharide. Infection and Immunity (1994) vol. 62 (9) pp. 3873-80

Reason et al. Structural determinants of the human idiotype HibId-1. J Mol Recognit (2001) vol. 14 (6) pp. 393-400

Chang et al. Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice
with human immunoglobulin Loci. Infect Immun (2002) vol. 70 (9) pp. 4977-86

Liu and Lucas. IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site
specific for the capsular polysaccharide of Haemophilus influenzae type b. Immunogenetics (2003) vol. 55 (5) pp. 336-8

Lucas et al. Molecular ontogeny of the human antibody repertoire to the Haemophilus influenzae type B polysaccharide: expression of
canonical variable regions and their variants in vaccinated infants. Clin Immunol (2003) vol. 108 (2) pp. 119-27

Reason and Zhou. Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus
pneumoniae type 23F capsular polysaccharide. Clin Immunol (2004) vol. 111 (1) pp. 132-6

Reason and Zhou. Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires. Biol
Direct (2006) vol. 1 pp. 24

Reason et al. Domain specificity of the human antibody response to Bacillus anthracis protective antigen. Vaccine (2008) vol. 26 (32)
pp. 4041-7

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Structure function review

  • 1. Anti-polysaccharide Antibodies: Structure-Function 8 paper Review
  • 2. Variable Regions of human Hib mAb Lucas et al 1994 • Mechanisms by which conjugate vaccines induce protection were poorly understood • Anti-Hib antibody gene usage limited to: – VH V3-23 and V3-15 – Several VL used • Different Hib PS conjugate vaccines induce different V region expression – OMPC • No k subgroup III in infants • Common k subgroup III in adults
  • 3. Lucas et al 1994 • mAb, CA4, isolated from mouse-human heterohybridoma obtained from a 41 y.o. Male 5 days after vaccination with 40 ug of Hib polysaccharide • CA4 – VH region 96% identical to germ line V3-21 – VL k subgroup III 94% identical to A27 • CA4 utilizes VH III not previously known to encode anti Hib PS antibodies • CA4 VK III closely resembles that of a variety of autoantibodies
  • 4. Structural Determinants of human idiotype HibId-1 Lucas et al 2001 • HibId-1 is expressed by kappa light chains encoded by A2 or A18 variable region genes • Residues that interact to form idiotypic determinants may or may not be the same as those contributing to affinity of the combining site for the antigen • HibId-1 does not require any contribution from the heavy chain
  • 5. Lucas et al 2001 • HibId-1 positive PS-specific antibodies have invariably been shown to encode: – VkII A2 gene rearranged to Jk1 or Jk3 – Non-template arginine inserted at the Vk-Vj junction – 10 aa CDR3 • Alterations of residues 30 and 40 result in loss of HibId-1 – Position 30 is important for interaction and changes in the spacing charges
  • 6. Lucas et al 2001 • HibId-1 idiotype can be dissociated from Hib PS binding • “Canonical” Hib PS-specific V3-23/A2 Fabs have been isolated that lack Lys30 and are HibId-1 negative
  • 7. Structure-function Relationships for human Abs to PPS from transgenic mice with human Ig loci Pirofski et al 2002 • PCV7 does not contain PPS3 – Causes disease primarily in adults • Can surrogates of Ab efficacy be determined using human mAbs to PPS3 in transgenic mice? • Vaccinated mice with PPS3 conjugated to tetanus toxoid
  • 8. Pirofski 2002 • mAbs isolated from hybridomas that produced the greatest C3 deposition in vitro, 7C5 and A7 were also the most protective in vivo
  • 9. Molecular Ontogeny of human Ab repertoire to Hib PS Lucas et al 2003 • Do variable regions of infant HibId-1 antibodies represent the same canonical configuration of adults? • Which structural polymorphisms could account for functional heterogenicity? • Infants were vaccinated, Hib PS binding B cells were isolated using biotinylated Hib PS and a Fab library was created
  • 10. Lucas et al 2003 • “We and others have shown that this method does not generate irrelevant chain combinations from promiscuous assembly but rather recapitulates the pairing of native sites.” • Fabs using the Jk1 gene conferred high affinity than Jk3
  • 11. Correlation of antigenic epitope and Ab gene usage against PPS23F Zhou et al 2003 • Human Ab response to 23F is predominated by Ab expressing VkL6 or VkA23 • Reference? • The antigenic epitopes recognized by VkL6 and VkA23 are different based on specificities for L- rhamnose, a constituent sugar of the bacterial capsule polymer.
  • 12. Zhou et al 2003 • As the length of the L chain CDR3 decreases, the degree to which rhamnose contributes to overall affinity of interactions might also decrease • VkL6 has a longer CDR3 than VkA23 • VkL6 Fabs are 100-fold more sensitive to inhibition with L-rhamnose than Vk23 Fabs
  • 13. IGH V3-23*01 and its allele V23-03 differ in capaciry to form canonical human Ab binding site for Hib Lucas et al 2003 • *01 and *03 differ by 9 bases, 8 of which are located in the CDR2 • These eight differences encode 5 amino acid substitutions • Does the V3023*03 polymorphism affect the binding to Hib PS? • Fab fragment isolated from peripheral blood of a vaccinated infant by combinatorial library cloning
  • 14. Lucas et al 2003 • RABA revealed 20 fold more effective binding by *03 than *01 • Fab binding was 4 fold • “The V3-23*03 allele has not been observed in Hib PS antibodies.” • Allele more common in Asians
  • 15. Codon insertion and deletion as a somatic diversification mechanism Zhou et al 2006 • Codon insertions and deletions were observed most frequently in CDR • Deletions were found in CDR while insertions were found in both CDR and frameworks 1 and 2 • Location of I/Ds were highly correlated with RGYW/WRCY motifs • In all cases codons duplicate those immediately 5’ or 3’ to the insertion
  • 16. Zhou et al 2006 • Peripheral blood was collected 7 days post adult vaccination with Prevnar or Pneumovax • Expression libraries generated from individuals • 12 of 124 independent H and L rearrangements contained I/D events • I/D events were associated with somatic hypermutation and did not occur during V/J and V/D/J rearrangements
  • 17. Domain specificity of the human Ab response to Bacillus anthracis Zhou et al 2008 • Domain specificity of isolated mAb was biased toward the amino-terminal 20 kDa fragment of PA (protective antigen). • However PA83 is primary immunogenic component of Biothrax vaccine. • Why is there a biased antibody response to PA20 which is not involved in anthrax intoxication?
  • 18. Zhou et al 2008 • Serum Ab response following vaccination is also biased toward determinants associated with PA20 • Selected Fab clones were expressed as IgG1 • Isolated Ab fail to neutralize PA-mediated cytotoxicity • Ab may bind to PA20 so that it interferes with one of the obligatory funcitons • Receptor binding • Shift epitope bias toward the functionally relevant PA63 portion
  • 19. Conclusions • Premature assumptions were made about anti- PPS Ab gene usage • Data from Hib studies was used as example • However gene usage for anti Hib antibodies has been well defined while in vivo anti PPS antibody gene usage had not been explored.
  • 20. Lucas et al. Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide. Infection and Immunity (1994) vol. 62 (9) pp. 3873-80 Reason et al. Structural determinants of the human idiotype HibId-1. J Mol Recognit (2001) vol. 14 (6) pp. 393-400 Chang et al. Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice with human immunoglobulin Loci. Infect Immun (2002) vol. 70 (9) pp. 4977-86 Liu and Lucas. IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b. Immunogenetics (2003) vol. 55 (5) pp. 336-8 Lucas et al. Molecular ontogeny of the human antibody repertoire to the Haemophilus influenzae type B polysaccharide: expression of canonical variable regions and their variants in vaccinated infants. Clin Immunol (2003) vol. 108 (2) pp. 119-27 Reason and Zhou. Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide. Clin Immunol (2004) vol. 111 (1) pp. 132-6 Reason and Zhou. Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires. Biol Direct (2006) vol. 1 pp. 24 Reason et al. Domain specificity of the human antibody response to Bacillus anthracis protective antigen. Vaccine (2008) vol. 26 (32) pp. 4041-7