Monoclonal Antibody ITD Method

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Monoclonal Antibody ITD Method
Javier Martin
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• ITD tests have been used for many years in labs
  of the Polio Network to differentiate wild
  polioviruses from Sabin-homologous strains
  excreted from vaccinees

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Intratypic differentiation (ITD) methods
recommended by WHO

• PAb-E ELISA using cross-adsorbed antisera (RIVM)
• ProHyb Probe hybridisation (CDC)
• PCR Diagnostic PCR method (CDC)
• RFLP PCR-RFLP (IP and NIID Tokyo)
• MAb-N MAb-neutralisation (IP, NIBSC) and NIBSC)

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MAb-ITD Method
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Type 3 Non Sabin-like poliovirus
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Type 3 Sabin-like poliovirus
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NIBSC MAbs
Hybridoma
Immunogen and
Fusion
Subclass
Specificity
source
partner
955
Poliovirus type 1
NSO
IgM
Reacts only with Sabin 1-
Sabin vaccine virus
homologous strains. Site 1
1233
Trypsin treated
NSO
IgM
Reacts only with Sabin 2-
poliovirus type 2
homologous strains. Site 3?
Sabin vaccine virus
889
Trypsin treated
NSO
IgM
Reacts only with Sabin 3-
poliovirus type 3
homologous strains. Site 3
Sabin vaccine virus
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Results of MAb screen (1993-2001)
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From Olen Kew, 2001
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Vaccine-derived polioviruses can replicate for
long periods of time in antibody deficient
individuals
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Reported iVDPV cases
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Vaccine-derived polioviruses, in some
circumstances, can circulate for long periods of
time and cause poliomyelitis outbreaks
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Examples of circulating VDPVs

• Confirmed
• Hispaniola 2000-2001 type 1 12 cases
• Egypt 1982-1993 type 2 32 cases
• Philippines 2001-2002 type 1 3 cases
• Madagascar 2002 type 2 5 cases

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Can current ITD methods detect VDPV strains (gt1
VP1 sequence divergence from Sabin)?
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MAb-ITD Results with VDPVs
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Properties of PV3/31043/99 virus from Belarus

• Isolate from a child with VAPP
• Non Sabin-like by MAb-ITD
• VDPV type 3/type 2/type 1 tripartite recombinant
• Type 3/type 2 capsid intertypic recombinant
• Reversion at attenuation/temperature-sensitive
  mutations

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Genomic structure of PV3/31043/99
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Intertypic capsid recombination

• First example described in field isolates
• Insertion of 120 nt coding for VP1 from type 2 in
  a type 3 capsid background
• Amino acid changes at VP1 281, 288-290, 292 and
  295
• Residues involved in antigenic site 3a and
  receptor contact
• No transfer of type 2 antigenic properties

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Protein structure of PV3/31043/99
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Long-term polio excreter in Ireland

• Healthy immunocompetent child born in Zimbabwe
• 17 type 1 poliovirus isolates taken from 16 May
  to 16 September 2002.
• All isolates were non Sabin-like by the MAb-ITD
  test.
• First isolate (16/05/02) contains 0.99
  nucleotide changes in the VP1 with respect to
  Sabin 1 vaccine strain.
• All isolates characterized are genetically
  related and non-recombinants
• Isolates contained reversions at attenuation
  mutations 5'NCR-480, VP3-225, VP1-106 and 3D-73
  and showed reversion of attenuation and
  temperature-sensitivity phenotypes.

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VP1 sequence evolution
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The MAb-ITD method could be used as a rapid
filter to select out Sabin-homologous strains
from, for example, large numbers of poliovirus
samples from environmental surveillance
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MAb-ITD Method as rapid screening filter
1. Ready MAbs that neutralise Sabin-homologous
isolates are available for each serotype from
NIBSC and IP. Method already in use
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MAb-ITD Method as rapid screening filter
2. Simple MAb neutralisation can be done at the
same time as routine polyclonal serotyping at
National Laboratory level
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MAb-ITD Method as rapid screening filter
3. Fast Isolates that are not neutralised are
either wild type or VDVP and rapidly flagged for
further ITD and sequencing