Strategies for Landing an Oracle DBA Job as a Fresher
Ociit conference 2010 final
1. Promiscuous
Variable
Light
Chain
Recombina6on
with
Pneumococcal
Polysaccharide
Specific
Variable
Heavy
Chain
Rebecca
Thompson
OCIIT
Forum
Tuesday, June 5, 12
2. • Streptococcus
pneumoniae
is
a
significant
cause
of
morbidity
and
mortality
worldwide.
• The
main
virulence
factor
of
S.
pneumoniae
is
the
capsular
polysaccharide.
• An;bodies
elicited
against
PPS
provide
protec;on
against
invasive
disease.
Tuesday, June 5, 12
3. • The
adult
23
valent
purified
PPS
vaccine
has
an
80%
protec6ve
efficacy
in
healthy
young
adults.
• However
efficacy
in
the
elderly
is
dras6cally
reduced
despite
normal
an6body
levels.
• This
phenomenon
is
thought
to
be
due
to
an6body
structure.
Tuesday, June 5, 12
4. • Previous
data
characterizing
the
structure-‐func;on
rela;onship
of
an;-‐pneumococcal
an;bodies
has
been
gathered
primarily
from
the
use
of
combinatorial
libraries.
• Combinatorial
libraries
pool
mul;ple
B
cells
and
PCR
the
variable
immunoglobulin
regions
not
conserving
the
natural
VH/VL
pairings.
• Combinatorial
libraries
are
used
for
an;body
repertoire
analysis
and
assume
that
the
random
heavy
and
light
chain
pairs
formed
represent
the
na;ve
repertoire.
• This
is
supported
by
data
that
demonstrates
the
in
vivo
use
of
similar
variable
heavy
and
light
combina;ons.
Tuesday, June 5, 12
5. • The
consistent
pairing
of
various
VH
with
iden6cal
VL,
as
seen
in
response
to
Haemophilius
influenza
type
B
(Hib)
polysaccharide
and
pneumococcal
polysaccharide,
suggests
a
restricted
VH-‐VL
pairing
in
nature.
• In
response
to
Hib,
the
predominant
variable
light
chain,
A2
is
used
in
the
repertoire
across
different
individuals
and
within
the
same
individual.
• The
phenomenon
responsible
for
this
pairing
limita6on
has
yet
to
be
elucidated.
Tuesday, June 5, 12
6. • The
goal
of
our
study
was
to
explore
the
reported
restricted
gene
usage
in
response
to
polysaccharide
an;gens
and
to
determine
if
this
observa;on
is
the
result
of
physiological
or
structural
limita;ons
of
an;bodies
that
recognize
polysaccharide
an;gens.
• To
test
these
concepts,
one
variable
heavy
chain
specific
for
PPS23F
was
paired
with
several
alterna;ve
PPS-‐specific
variable
light
chains.
• Recombinant
human
an;bodies
were
tested
for
func;onality
and
influences
on
pneumococcal
polysaccharide
binding.
Tuesday, June 5, 12
7. • Variable
light
gene
fragments
were
obtained
from
PPS-‐
specific
B
cells
isolated
from
immunized
donors.
Fragments
were
PCR
amplified
to
add
expression
plasmid
specific
restric;on
sites.
• CBE2,
a
PPS23F
specific
variable
heavy
chain
was
obtained
from
Baxendale
et
al
and
restric;on
sites
were
inserted.
• The
variable
chains
were
each
ligated
into
the
appropriate
pHC-‐huC
plasmid
(McLean
et
al).
pHC-‐huC
containing
variable
heavy
and
light
sequences
were
then
transfected
into
HEK293
cells.
• Surface
plasmon
resonance
(SPR)
measured
the
avidity
of
the
an;body
(Reichert
3700DC).
The
sensor
chip
with
immobilized
an;-‐human
Ig
an;body
bound
to
its
surface
was
used
to
capture
recombinant
human
an;body.
PPS23F
was
injected
over
the
chip
surface
to
determine
binding
avidity.
Tuesday, June 5, 12
14. • Analysis
of
these
recombinant
an;bodies
by
ELISA
demonstrates
that
these
an;bodies
maintain
their
specificity
for
PPS23F.
However,
analysis
with
SPR
demonstrates
that
these
an;bodies
possess
unique
binding
characteris;cs.
These
findings
were
also
confirmed
by
disrup;on
ELISA.
• Although
there
are
certain
capsular
polysaccharides
where
a
specific
variable
light
chain
gene
is
required
for
epitope
binding
this
does
not
seem
to
be
the
case
with
PPS23F.
It
is
plausible
that
the
variable
light
chain
does
not
significantly
contribute
to
the
structural
requirements
of
PPS23F
binding.
• In
contrast
for
Hib
PS,
an
arginine
in
posi;on
95a
of
the
light
chain
is
essen;al
for
binding.
To
date,
we
have
not
iden;fied
an
essen;al
amino
acid
sequence
in
the
CDR3
of
VL
for
PPS.
Tuesday, June 5, 12
15. Future
Work
• Comparison
of
IgG1
to
IgG2
isotypes
o Predominate
an;body
response
to
S.
pneumoniae
o IgG1
levels
drop
with
age
• Analysis
of
an;body
structure
in
response
to
S.
pneumoniae
o Fc,
Fab
and
Fab’2
fragments
• Defining
the
binding
requirements
for
pneumococcal
polysaccharide
23F
Tuesday, June 5, 12
16. Acknowledgements
• Major
Advisor • Collaborators
– Dr.
Julie
Westerink,
MD – Dr.
Baxendale
• Lab
Members – Dr.
McLean
– Jason
Mosakowski • Commidee
Members
– Jieying
Wang – Dr.
Dignam
– Dr.
Noor
Khaskhely,
PhD – Dr.
Malhotra
• Funding – Dr.
Ruch
– NIH
RO1 – Dr.
Wooten
Tuesday, June 5, 12