Anti-polysaccharide Antibodies: Structure-Function 8 paper Review
Variable Regions of human Hib mAbLucas 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-1Lucas 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 lociPirofski 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 PSLucas 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 PPS23FZhou 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 HibLucas 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 mechanismZhou 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 anthracisZhou 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.
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