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.
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