Shoulder disorders and postural stress in automobile assembly work

Abstract

A case-referent study was conducted in an automobile assembly plant to evaluate the risk of shoulder disorders associated with nonneutral postures.
The cases were workers who reported shoulder pain to the plant clinic during a 10-month period and met symptom criteria (pain frequency or duration in the past year) in an interview; more than one-half also had positive findings in a physical examination. The referents were randomly selected workers who were free of shoulder disorders according to the clinic records, the interview, and the physical examination. For each of the 79 cases and 124 referents, 1 job was analyzed for postural and biomechanical demands by an analyst blinded to the case-referent status.
Forty-one percent of the subjects flexed or abducted the right arm "severely" (above 90 degrees) during the job cycle, and 35% did so with the left arm. The peak torques at the shoulder were rather low. Shoulder disorders were associated with severe flexion or abduction of the left [odds ratio (OR) 3.2, 95% confidence interval (95% CI) 1.5-6.5] and the right (OR 2.3, 95% CI 1.2-4.8) shoulder. The risk increased as the proportion of the work cycle exposed increased. The relationships were similar for the cases with and without physical findings. Use of hand-held tools increased the risk and also modified the association with postural stress, although the joint exposure distributions limited full analysis of this finding.
The findings support the conclusion that severe shoulder flexion or abduction, especially for 10% or more of the work cycle, is predictive of chronic or recurrent shoulder disorders.

Full text

Proc.
Nati.
Acad.
Sci.
USA
Vol.
86,
pp.
9514-9518,
December
1989
Medical
Sciences
Long-term
culture
and
fine
specificity
of
human
cytotoxic
T-lymphocyte
clones
reactive
with
human
immunodeficiency
virus
type
1
(AIDS/cell-mediated
immunity/reverse
transcriptase/immunogenic
viral
epitopes)
BRUCE
D.
WALKER*§,
CHARLES
FLEXNERt,
KAREN
BIRCH-LIMBERGER*,
LAURA
FISHER*,
TIMOTHY
J.
PARADIS*,
ANNA
ALDOVINIt,
RICHARD
YOUNGt,
BERNARD
MOSSt,
AND
ROBERT
T.
SCHOOLEY*
*Infectious
Disease
Unit,
Massachusetts
General
Hospital
and
Harvard
Medical
School,
Boston,
MA
02114;
tLaboratory
of
Viral
Diseases,
National
Institute
of
Allergy
and
Infectious
Diseases,
National
Institutes
of
Health,
Bethesda,
MD
20892;
and
tWhitehead
Institute
for
Biomedical
Research,
Massachusetts
Institute
of
Technology,
Cambridge,
MA
02142
Contributed
by
Bernard
Moss,
August
7,
1989
ABSTRACT
The
definition
of
human
immunodeficiency
virus
type
1
(HIV-1)
immunogenic
epitopes
is
central
to
the
rational
design
of
AIDS
vaccine
strategies.
In
this
study,
we
have
generated
seven
HIV-1
reverse
transcriptase-specific
cytotoxic
T-lymphocyte
(CTL)
clones
from
the
peripheral
blood
of
two
seropositive
subjects.
Epitopes
recognized
by
these
CTL
clones
were
identified
by
using
target
cells
infected
with
recombinant
HIV-1-vaccinia
virus
vectors
expressing
truncated
reverse
tran-
scriptase
proteins
and
further
defined
by
using
target
cells
incubated
with
overlapping
25-amino
acid
synthetic
reverse
transcriptase
peptides.
Five
different
CTL
epitopes
were
iden-
tified,
and
in
each
case
recognition
was
restricted
by
class
I
human
leukocyte
antigens
(HLA).
Clones
maintained
specific
cytolytic
function
in
continuous
culture
for
up
to
11
months,
requiring
only
periodic
restimulation
with
a
CD3-specific
mono-
clonal
antibody.
These
results
indicate
that
HIV-1-specific,
major
histocompatibility
class
I-restricted
CTL
recognize
mul-
tiple
epitopes
of
a
single
viral
gene
product
in
conjunction
with
different
host
HLA
antigens.
In
addition,
they
demonstrate
that
human
virus-specific
CTL
can
be
grown
in
long-term
culture
without
the
need
for
reexposure
to
viral
antigen.
Human
immunodeficiency
virus
type
1
(HIV-1)
infection
is
usually
associated
with
a
persistent
viremia;
virus
is
readily
cultured
from
the
peripheral
blood
of
infected
individuals
(1).
Despite
ongoing
exposure
to
the
virus,
most
persons
remain
healthy
for
years
after
infection,
suggesting
that
a
virus-
specific
host
immune
response
plays
a
role
in
maintaining
the
asymptomatic
state.
Cytotoxic
T-lymphocytes
(CTL)
may
be
an
important
host
defense,
as
has
been
demonstrated
in
other
viral
infections,
including
those
caused
by
retroviruses
(2-7).
CTL
responses
against
the
major
HIV-1
gene
products
have
been
detected
in
infected
individuals
(8-15);
however,
little
information
is
available
on
actual
epitopes
recognized
by
these
CTL.
An
immunodominant
T-cell
epitope
has
been
identified
in
mice
immunized
with
a
recombinant
vaccinia
virus
express-
ing
the
HIV-1
envelope
gene
product
(16).
In
addition,
a
human
leukocyte
antigen
(HLA)-B27-restricted
gag
peptide
epitope
has
been
identified,
using
bulk
stimulated
peripheral
blood
mononuclear
cells
(PBMC)
from
a
seropositive
individ-
ual
as
effector
cells
(12).
Whereas
human
CTL
clones
respon-
sive
to
HIV-1
have
been
reported
(13),
further
characterization
of
epitopes
has
been
hampered,
at
least
in
part,
by
difficulties
in
propagating
human
CTL
clones
in
vitro.
Here
we
report
a
method
for
generating
and
maintaining
HIV-specific
CTL
clones
in
continuous
culture.
This,
in
turn,
has
facilitated
a
detailed
analysis
of
the
fine
specificity
apd.
major
histocompatibility
complex
(MHC)
restriction
of
these
CTL.
In
these
studies
we
have
concentrated
on
HIV-1
reverse
transcriptase
(RT)-specific
CTL
clones,
since
pol
is
the
most
highly
conserved
gene
among
sequenced
viral
isolates
and
RT
is
a
target
of
both
humoral
and
cellular
immune
responses
in
infected
individuals
(10,
17).
Ourresults
indicate
that
the
CTL
response
to
HIV-1
is
heterogeneous,
in
that
multiple
epitopes
of
a
given
viral
gene
product
-re
recognized
in
conjunction
with
different
host
HLA
antiges
and
that
a
single
HLA
antigen
can
serve
as
the
restricting
element
for
more
than
one
of
these
epitopes.
This
work.ha~s
been
presented,
in
part,
elsewhere.¶
MATERIALS
AND
METHODS
Effector
Cells.
PBMC
were
obtained
by
Ficoll/Hypaque
density
gradient
centrifugation
of
blood
from
two
asymptqm-
atic
HIV-1-seropositive
subjects
(subjects
63
and
68).
Th%'e
PBMC
were
either
used
directly
as
effector
cells
in
a
cyto-
toxicity
assay
or
cloned
at
limiting
dilution.
For
cloning,.cells
were
seeded
at
50
cells
per
well
in
96-well
plates.
Irradia4d
allogeneic
PBMC
from
HIV-1-seronegative
subjects
were
added
as
feeder
cells
at
2
x
105
cells
per
well,
in
a
final
volume
of
200
,ul
(per
well)
of
medium
[RPMI
1640
medium
contain-
ing
10%
(vol/vol)
heat-inactivated
fetal
calf
serum,
antibiot-
ics,
and
100
units
of
recombinant
interleukin-2
(DuPont)
per
ml].
The
CD3-specific
monoclonal
antibody
12F6
(8)
was
added
at
0.05-0.1
,ug/ml
as
a
stimulus
to
T-cell
proliferation.
Plates
were
incubated
at
37°C
in
a
humidified
chamber
in
S%
CO2
and
fed
1
or
2
times
per
week
with
medium
exchanges.
After
3-4
weeks,
cells
from
wells
showing
cell
proliferation
were
transferred
to
24-well
plates
and
restimulated
by
adding
2
ml
of
medium
containing
2
x
106
irradiated
allogeneic
feeder
cells
plus
12F6
at
0.05-0.1
,ug/ml.
At
no
time
were
cells
exposed
to
exogenous
viral
antigens.
Cells
were
tested
1-3
weeks
later
for
CTL
activity.
HIV-1
antigen-specific
activity
was
detected
in
4%
or
less
of
wells
showing
proliferation,
and
no
cultures
with
specificity
for
more
than
one
HIV-1
antigen
were
detected.
These
cultures
were
therefore
likely
to
rep-
resent
clonal
expansion
of
a
single
virus-specific
cell
and,
due
to
their
demonstrated
unique
specificities,
are
operationally
referred
to
as
clones.
Abbreviations:
HLA,
human
leukocyte
antigen;
MHC,
major
histo-
compatibility
complex;
RT,
reverse
transcriptase;
PBMC,
peripheral
blood
mononuclear
cells;
EBV,
Epstein-Barr
virus;
CTL,
cytotoxic
T
lymphocytes;
HIV-1,
human
immunodeficiency
virus
type
1.
§To
whom
reprint
requests
should
be
addressed.
$Fifth
International
Conference
on
AIDS,
Montreal,
PQ,
Canada,
June
4-9,
1989
(abstract
W.C.O.
43).
9514
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
9515
Target
Cells.
(i)
Epstein-Barr
virus
(EBV)
lymphoblasts
infected
with
recombinant
HIV-1-vaccinia
viruses.
EBV-
immortalized
B-cell
lines
were
established
and
maintained
as
described
(8).
These
were
infected
with
recombinant
vaccinia
viruses
containing
the
bacterial
/3-galactosidase
gene
(VAC/
lac,
a
negative
control)
or
the
env,
gag,
or
RT
genes
of
HIV-1
(VAC/env,
VAC/gag,
and
VAC/RT,
respectively)
and
were
used
as
target
cells
in
a
4-
or
6-hr
chromium
release
assay
(8,
10).
(ii)
EBV
lymphoblasts
infected
with
recombinant
vaccinia
.viruses
containing
truncated
RT
gene
inserts.
The
RT
gene
of
HTLV-IIIB
subclone
HXB.2
(18),
with
added
start
and
stop
codons,
was
excised
from
the
replicative
form
of
the
M13
bacteriophage
recombinant
mpCF19
(described
in
ref.
19)
an&
ligated
into
the
Sma
I
site
of
pUC19,
yielding
the
rceombinant
plasmid
pCF31.
The
following
six
restriction
fragments
were
then
purified
from
this
plasmid:
Xba
I-SSt
I,
Xba
I-Asp718,
Xba
I-Bsp1286,
Xba
I-Hinfl,
Xba
I-Pvu
II,
Xba
I-EcoRV.
These
were
then
blunt-ended
with
the
Klenow
,fragment
of
DNA
polymerase
I
for
ligation
into
the
Stu
I
site
ofpSCllss,
a
vaccinia
expression
vector
derived
from
pSC11
.(20)
and
containing
Stu
I
and
Sal
I
sites
in
place
of
the
Sma
Lckining
site,
as
well
as
stop
codons
in
all
three
reading
.frames
immediately
downstream
from
the
Stu
I
site
(P.
Earl,
personal
communication).
Plasmids
containing
correctly
ori-
ented
RT
fragments
were
then
used
to
transfect
vaccinia-
inficted.
cells,
and
the
resulting
recombinant
viruses
were
id.tified,
grown,
and
purified
as
described
(18).
Expression
(.t~he
truncated
RT
peptides
in
cells
infected
with
recombi-
toWt&
virus
containing
all
but
the
smallest
(amino
acids
168-
*.45)
insert
was
confirmed
by
Western
blotting
(data
not
lsbown).
Infection
of
autologous
EBV
blasts
with
these
vac-
4ia-truncation
constructs
was
performed
as
described
(8,
lp)f,;and
these
cells
were
then
used
as
targets
in
a
4-hr
cIromium
release
assay.
(iii)
EBV
lymphoblasts
incubated
with
synthetic
HIV-1
RT
p~ptides.
Synthetic
HIV-1
RT
peptides
were
synthesized
as
.inaribed
(21),
using
the
sequence
of
the
pv22
clone
of
HIV-1
(2Zy,
Peptides
overlapped
with
adjacent
peptides
by
eight
amrwo
acids.
Chromium-labeled
EBV
lymphoblasts
were
i~eubated
for
45
min
with
the
designated
peptide
at
20
,ug/ml,
aMikthen
autologous
RT-specific
CTL
clones
were
added
as
effector
cells.
The
percent
specific
chromium
release
was
*determined
after
a
4-hr
incubation.
RESULTS
Generation
of
HIV-i-Specific
CTL
Clones.
PBMC
were
obtained
from
two
asymptomatic
HIV-1
seropositive
subjects
shown
to
have
cytolytic
responses
to
autologous
EBV-
immortalized
B-cell
targets
expressing
the
HIV-1
envelope
and
RT
proteins
(Table
1).
The
PBMC
were
cloned
at
limiting
dilution,
a
CD3-specific
monoclonal
antibody
was
used
as
a
stimulus
for
T-cell
proliferation,
and
recombinant
interleukin
2
was
added
as
a
growth
factor.
Developing
clones
were
tested
for
virus-specific
lysis
by
using
a
panel
of
autologous
EBV-
immortalized
B-cell
targets
expressing
the
HIV-1
envelope,
gag,
or
RT
protein
(8-10).
After
several
weeks
in
culture,
1-4%
of
developing
clones
demonstrated
HIV-1-specific
CTL
responses,
specific
for
either
the
viral
RT
or
envelope
(Table
1).
No
HIV-1
gag-specific
CTL
were
detected
with
either
fresh
PBMC
or
cloned
cells
from
these
two
subjects.
At
no
time
were
these
cells
stimulated
with
viral
antigen
in
vitro,
indicat-
ing
that
their
viral
specificities
had
been
determined
in
vivo.
Clones
demonstrated
no
evidence
of
natural
killer
activity,
as
assayed
by
lysis
of
K562
cells
(data
not
shown).
MHC
Restriction
of
HIV-1
RT-Specific
CTL
Clones.
Clones
demonstrating
RT-specific
cytotoxicity
were
examined
in
detail
to
further
define
the
CTL/target
cell
interaction.
Rec-
ognition
of
virus-infected
cells
by
CTL
has
been
shown
to
Table
1.
HIV-1-specific
CTL
clones
from
two
seropositive
subjects
%
specific
cytotoxicity
of
target
cells
Effector
E:T
VAC/
VAC/
VAC/
VAC/
Subject
cells
ratio
lac
env
gag
RT
63
PBMC
50:1 5
39
1
25
63A3
5:1
1
2
1
41
63A7
5:1
3
0
2
72
63D35
5:1
1
0
3
48
63D45
2.5:1
2
72
2
4
63H24
5:1
1
1
2
56
6319
*
1
5
7
44
68
PBMC
50:1
4
34
7
43
68A13
2.5:1
2
1
4
82
68A33
25:1
4
29
2
0
68A62
2.5:1
6
2
4
71
Fresh
PBMC
and
cloned
cells
were
tested
for
specific
lysis
of
autologous
EBV-immortalized
B-cell
targets
expressing
the
recom-
binant env,
gag,
or
RT
protein
(VAC/env,
VAC/gag,
and
VAC/RT,
respectively)
or
a
control
target
cell
(VAC/lac).
For
the
initial
screening,
clones
were
not
counted
but
were
used
at
an
effector-
to-target
(E:T)
ratio
estimated
to
be
10:1
(*).
All
clones
except
63I9
were
subsequently
retested
against
these
four
targets
at
the
specific
effector-to-target
ratios
shown.
Lysis
of
autologous
target
cells
by
fresh
bulk
PBMC
from
each
subject
is
shown
for
comparison.
Average
percent
spontaneous
lysis
for
the
assays
shown
in
this
table
was
always
<30o,
except
for
the
assay
using
clone
63A7,
where
it
was
38%.
require
association
of
a
processed
viral
protein
product
with
a
"self'
MHC
molecule,
either
class
I
(A,
B,
or
C
locus)
or
class
II
(D
locus)
(23,
24).
To
examine
the
role
of
the
MHC
in
restricting
the
cytotoxic
activity
of
each
of
these
clones,
allogeneic
target
cells
partially
HLA-matched
with
the
donor
of
the
CTL
were
infected
with
a
recombinant
vaccinia
virus
expressing
the
full-length
HIV-1
RT
protein
(10,
19).
As
shown
in
Tables
2
and
3,
for
each
RT-specific
clone
a
single
class
I
antigen
was
identified
as
the
restriction
element.
For
subject
63,
two
clones
(63A3
and
63H24)
were
restricted
by
Table
2.
HIV-1
RT-specific
CTL
clones
from
subject
63
are
MHC
class
I-restricted
Target
cell
%
RT-specific
lysis
(shared
Ag)
63A3
63A7
63D35
63H24
6319
Autologous
100
100
100
100
100
A1,
DQw2
0
0
2
0
NT
All,
DRw52
6
83
111
6
108
Al,
All,
DRw52,
DQw2
NT
NT
NT
NT
93
B8
149
12
3
NT
NT
Al,
B8,
DR3,
DRw52
91
0
0
75
1
Bw62,
DRw52
NT
0
0
1
8
DR3
NT
NT
NT
2
NT
None
NT
NT
NT
1
NT
CTL
clones
were
tested
for
their
ability
to
lyse
allogeneic
RT-
expressing
EBV
lymphoblasts
matched
at
specific
HLA
class
I
and
class
II
loci,
as
indicated.
Results
represent
RT-specific
lysis
ex-
pressed
as
the
percentage
of
that
observed
against
autologous
RT-expressing
target
cells.
Clones
63D35
and
63H24
were
tested
at
an
effector-to-target
ratio
of
5:1,
and
clones
63A3,
63A7,
and
6319
were
tested
at
a
ratio
of
10:1
in
4-hr
chromium
release
assays.
Average
percent
specific
lysis
of
the
autologous
RT-expressing
target
cell
and
the
paired
vaccinia
control
target
cell
for
each
clone
was
65%
vs.
6%
for
63A3,
72%
vs.
3%
for
63A7,
66%
vs.
1%
for
63D35,
62%
vs.
1%
for
63H24,
and
88%
vs.
3%0
for
63I9.
Average
spontaneous
lysis
for
these
assays
was
always
<30o.
NT,
not
tested;
Ag,
antigen.
The
complete
HLA
type
of
subject
63
is
Al,
All;
B8,
Bw62;
Cw4,
-;
DR3,
-;
DQw2;
DRw52.
Medical
Sciences:
Walker
et
al.

9516
Medical
Sciences:
Walker
et
al.
Table
3.
HIV-1
RT-specific
CTL
clones
from
subject
68
are
MHC
class
I-restricted
Target
cell
%
RT-specific
lysis
(shared
Ag)
68A13
68A62
Autologous
100
100
A2,
DR4
7
96
A2,
B7
10
82
A2,
B7
0
106
A32
12
4
B7,
DR1
0
2
Bw6O
108
0
None
5
2
Clones
were
tested
as
described
in
Table
2,
except
that
an
effector-
to-target
ratio
of
2.5:1
was
used
in
4-hr
chromium
release
assays.
Average
percent
lysis
of
the
autologous
RT-expressing
target
cell
and
the
paired
vaccinia
control
target
cell
for
each
clone
was
39o
vs.
3%
for
68A13
and
59%o
vs.
2%
for
68A62.
Average
spontaneous
lysis
for
these
assays
was
always
<30%o.
The
complete
HLA
type
of
subject
68
is
A2,
A32;
B7,
Bw6O;
Cw3,
w7;
DR1,
4;
DQw1,
3;
DRw53.
the
HLA-B8
antigen,
whereas
three
others
(63A7,
63D35,
and
6319)
were
restricted
by
the
HLA-A11
antigen
(Table
2).
The
two
RT-specific
clones
derived
from
subject
68
(68A13
and
68A62)
were
restricted
by
the
HLA
class
I
antigens
Bw6O
and
A2,
respectively
(Table
3).
Phenotypic
analyses
revealed
the
clones
to
be
CD3+,
CD8+
(data
not
shown).
In
no
instance
was
class
II-restricted
CTL
activity
(25)
detected
using
the
cloning
strategy
we
employed.
CTL
Lysis
of
Target
Cells
Expressing
Truncated
RT
Gene
Products.
The
viral
epitopes
recognized
by
these
RT-specific
clones
were
then
investigated
with
a
series
of
recombinant
vaccinia
viruses
constructed
to
express
the
RT
protein
with
sequentially
greater
carboxyl-terminal
deletions,
as
shown
in
Fig.
1.
Six
different
vectors
were
used,
expressing
from
100%
(amino
acids
168-706)
to
27%
(amino
acids
168-315)
of
the
RT
protein
(18).
Clones
63A3
and
63H24,
both
restricted
by
the
HLA-B8
antigen,
were
able
to
lyse
target
cells
infected
with
each
of
the
truncated
vectors,
even
when
only
the
amino-terminal
148
residues
of
RT
were
expressed
(amino
acids
168-315).
These
results
indicate
that
the
epitope
rec-
ognized
by
these
clones
lies
somewhere
in
this
amino-
terminal
region.
Lysis
by
clones
63A7
and
63D35,
both
restricted
by
the
HLA-A11
antigen,
was
completely
abro-
gated
when
amino
acids
316-422
were
no
longer
expressed.
In
contrast,
a
third
HLA-A11-restricted
clone
(63I9)
from
this
RT
AMINO
ACIDS
EXPRESSED
subject
recognized
an
entirely
different
epitope,
in
that
CTL
activity
was
lost
when
amino
acids
481-531
were
no
longer
expressed.
Clone
68A62
from
subject
68
also
recognized
a
viral
epitope
in
this
same
region
(amino
acids
481-531),
whereas
amino
acids
lying
in
the
region
316-422
were
es-
sential
for
recognition
by
clone
68A13
from
this
subject.
CTL
Lysis
of
Target
Cells
Incubated
with
Synthetic
HIV-1
RT
Peptides.
HLA
class
I-restricted
CTL
generally
recognize
viral
antigen
that
has
been
processed
endogenously
in
infected
cells
and
presented
at
the
cell
surface
as
a
binary
complex
with
the
restricting
HLA
molecule
(26).
Target
cells
can
also
be
sen-
sitized
to
virus-specific
lysis
in
vitro
by
incubation
with
the
appropriate
synthetic
viral
peptide
(27),
presumably
through
formation
of
a
binary
complex
involving
synthetic
viral
pep-
tide
and
surface
HLA
class
I
molecule
of
the
uninfected
target
cell.
The
fine
specificity
of
the
RT-specific
clones
was
there-
fore
investigated
by
using
autologous
target
cells
incubated
with
overlapping
25-amino
acid
synthetic
peptides
derived
from
the
predicted
amino
acid
sequence
of
the
RT
protein
(21).
As
shown
in
Table
4,
target
cells
were
sensitized
to
lysis
by
either
a
single
peptide
or
two
adjacent
peptides
in
the
regions
predicted
by
the
truncation
constructs
to
contain
the
CTL
epitopes.
Clones
63D35
and
63A7
were
able
to
lyse
cells
incubated
with
either
peptide
49
or
50,
suggesting
that
the
epitope
recognized
lies
within
the
8-amino
acid
overlap
region
(amino
acids
342-349).
For
all
other
clones,
target
cells
were
sensitized
by
only
a
single
peptide.
For
clones
68A62
and
63D35,
MHC
restriction
was
also
investigated
with
peptide-
sensitized
target
cells
and
showed
the
same
MHC
restriction
specificity
as
had
been
demonstrated
using
recombinant
vac-
cinia
virus
to
express
the
full-length
RT
protein
in
autologous
cells
(data
not
shown).
Long-Term
Culture
of
HIV-1-Specific
CTL.
Two
of
these
CTL
clones
have
now
maintained
virus-specific
CTL
activity
in
continuous
culture
for
10
and
11
months,
respectively,
requiring
only
periodic
restimulation
with
the
CD3-specific
monoclonal
antibody.
For
example,
after
315
days
in
culture,
clone
68A62
demonstrated
54%
specific
lysis
of
autologous
RT-expressing
target
cells
at
an
effector-to-target
ratio
of
2.5:1,
with
only
3%
lysis
of
the
paired
control
target
cell
(Table
5).
Maintenance
of
virus-specific
killing
has
not
re-
quired
in
vitro
exposure
to
viral
antigen.
Analysis
of
super-
natant
fluid
from
the
clones
for
HIV-1
p24
antigen
(28)
as
well
as
analysis
of
cloned
cells
by
polymerase
chain
reaction
(29,
30)
for
evidence
of
HIV-1
genomic
sequences
have
both
been
%
SPECIFIC
CYTOTOXICITY
63A3
63A7
63D35
63H24
63I9
68A13
68A62
(B8)
(All)
(Ail)
(B8)
(All)
(Bw6O)
(A2)
1168-706
1
75
..........
&
r
O
=z
-:-
:.
::::::::::::::
:::
:::R-.:::::::::::::::::
r
.......:-::-:::-::-::-:-::::.:-::-
.........:::::
......_...>>.
=>.a...
.........
E-
-
75
80
69
67
50
72
72
80
93
82
67
46
74
73
69
82
79 79 45
89
75
79
91
72
0
41
0
88
86
101
62
0
47
l
70
0
7
78
2
2
l
FIG.
1.
Specific
lysis
of
target
cells
infected
with
recombinant
vaccinia
viruses
expressing
truncated
RT
gene
products.
A
panel
of
vaccinia
viruses
containing
sequential
carboxyl-terminal
truncated
HIV-1
RT
gene
inserts
were
used
to
infect
EBV-immortalized
B
cells
from
subjects
63
and
68.
Autologous
clones
were
then
tested
for
their
ability
to
lyse
these
cells.
Results
are
shown
at
an
effector-to-target
ratio
of
25:1,
except
for
clone
6319,
which
was
tested
at
a
ratio
of
10:1.
Similar
results
were
obtained
when
clones
were
used
at
an
effector-to-target
ratio
of
2.5:1.
Average
spontaneous
lysis
for
these
assays
was
always
<30%o.
The
amino
acids
expressed
by
these
constructs
are
indicated;
the
numbers
correspond
to
the
pol
gene
sequence
of
HIV-1
(17).
Amino
acids
168-706
represent
the
full-length
RT
protein.
Proc.
Natl.
Acad
Sci.
USA
86
(1989)

Proc.
Natl.
Acad.
Sci.
USA
86
(1989)
9517
Table
4.
Lysis
by
RT-specific
CTL
clones
of
autologous
lymphoblasts
incubated
with
synthetic
25-amino
acid
RT
peptides
Peptide
%
specific
cytotoxicity
no.
Residues
63A3
63A7
63D35
63H24
6319
68A13
68A62
39
155-179
4
0
0
0
-
0
40
172-1%
15
0
2
16
0
0
41
189-213
8
2 2
0
-
0
42
206-230
0
7
90
-
0
43
223-247
2
0 0
0
-
0
44
240-264
1
0
0
0
-
0
45
257-281
2
0 0
0
-
0
0
46
274-298
2
0
2
0
-
0
0
47
291-315
3
2
0
0
-
0
0
48
308-332
8
0 0
0
1
0
49
325-349
0 89
90
0
1
1
0
50
342-366
0
67
50
0
0
2
0
51
359-383
6
5
2
0
3
14
0
52
376-400
0
0
0
2
2
0
53
393-417
1
0 0
0
0
1
0
54
410-434
-
0
3
0
2
0
0
55
427-451
NA
NA
-
NA
56
444-468
-
0
0
-
0
57
461-485
0
6
3
0
4
0
31
58
478-502
-
-
-
0
0
59
495-519
-68
0
60
512-536
2
1
61
529-553
-_2-
0-
62
546-570
-
0
0
Effector-to-target
ratios
for
the
specific
cell
lines
were
2.5:1
for
clones
from
subject
68
and
5:1
for
clones
from
subject
63,
except
6319,
which
was
tested
at
10:1.
Spontaneous
lysis
was
always
<30o,
except
for
target
cells
incubated
with
peptide
55,
which
was
toxic
to
target
cells,
with
spontaneous
release
values
ranging
from
40-70o.
Boxes
indicate
the
regions
predicted
by
the
vaccinia
truncation
construct
data
to
contain
the
recognized
epitope.
-,
Not
tested;
NA,
data
not
available
due
to
spontaneous
release
>30%o.
The
amino
acid
sequences
of
RT
peptides
found
to
sensitize
target
cells
were
IETVPVKLKPGMDGPKVKQWPLTEE
(peptide
40),
AIFQSSMTKILEPFRKQNPDIVIYQ
(peptide
49),
NPDI-
VIYQYMDDLYVGSDLEIGQHR
(peptide
50),
DLEIGQHRTK-
IEELRQHLLRWGLTT
(peptide
51),
PLTEEAELELAEN-
REILKEPVHGVY
(peptide
57),
and
EIQKQGQGQWTY-
QIYQEPFKNLKTG
(peptide
59).
negative
(data
not
shown),
indicating
that
the
clones
them-
selves
are
not
infected
with
HIV-1.
DISCUSSION
These
results
indicate
that
multiple
epitopes
of
a
given
viral
gene
product
can
act
as
immunogens
for
the
cytotoxic
Table
5.
RT-specific
lysis
by
clone
68A62
%
specific
cytotoxicity
Days
in
culture
VAC/lac
VAC/RT
67
6
71
77
2
66
91
5
75
104
2
74
111
1
77
154
2
98
168
0
66
202
0
66
265
1
67
314
3
54
Target
cells
are
autologous
Iymphoblasts
infected
with
the
control
T-cell-mediated
immune
response
in
HIV-1-infected
individ-
uals.
For
each
CTL
clone
examined,
the
epitope
mapping
using
vaccinia-HIV-1
truncation
constructs
correlated
pre-
cisely
with
data
obtained
using
synthetic
RT
peptides
to
sensitize
target
cells.
None
of
the
viral
peptides
containing
CTL
epitopes
was
recognized
by
CTL
from
both
subjects
studied,
suggesting
that
the
immunogenic
viral
epitopes
are
different
in
different
individuals.
The
full
spectrum
of
epitopes
recognized
by
RT-specific
CTL
is
yet
to
be
deter-
mined,
but
it
may
be
large;
in
this
study,
CTL
restricted
by
four
different
HLA
class
I
antigens
recognized
five
different
peptide-defined
epitopes.
The
demonstration
that
more
than
one
immunogenic
viral
epitope
of
a
given
viral
gene
product
can
associate
with
the
same
HLA
class
I
antigen
(All,
in
this
case)
is
further
evidence
for
the
heterogeneity
of
the
HIV-
1-specific
CTL
response.
These
data
also
indicate
that
CTL
directed
at
the
viral
RT
should
be
at
least
partially
cross-reactive.
Peptides
demon-
strated
in
these
studies
to
contain
CTL
epitopes
were
exam-
ined
for
sequence
similarity
among
sequenced
HIV-1
iso-
lates.
In
each
case,
considerable
sequence
similarity
was
present,
indicating
that
CTL
specific
for
these
epitopes
should
cross-react
with
more
than
one
viral
isolate.
For
example,
the
predicted
amino
acid
sequence
for
peptide
40
(amino
acids
172-1%)
is
identical
for
the
HIV-1
isolates
HXB.2,
HB102,
HB5,
PV22,
BRU,
RF,
MN,
SF2,
and
ELI
and
differs
by
only
one
amino
acid
for
the
HIV-1
MAL
isolate
(31).
The
least
conserved
sequence
for
these
peptides
con-
taining
CTL
epitopes
was
that
of
peptide
51
(amino
acids
359-383),
which
differed
by
as
many
as
four
amino
acids
among
these
sequenced
isolates.
It
will
be
of
interest
to
determine
if
the
minimal
complement
of
amino
acids
neces-
sary
for
recognition
by
these
clones
will
fit
computer-assisted
predictions
of
T-cell
epitopes
(32,
33).
This
establishment
of
long-term
cultures
of
human
virus-
specific
CTL
provides
a
useful
reagent
for
the
elucidation
of
mechanisms
involved
in
cell-mediated
immunity.
Detailed
mapping
of
the
immunogenic
epitopes
of
HIV-1
using
the
system
described
here
may
identify
immunodominant
CTL
epitopes
to
direct
the
rational
design
of
an
AIDS
vaccine
that
elicits
CTL
responses.
Whether
CTL
responses
to
HIV-1
RT
or
other
HIV-1
proteins
will
protect
from
initial
infection
or
retard
disease
progression
remains
unknown.
If
CTL
re-
sponses
are
of
importance
in
HIV-1
vaccine
development,
our
data
suggest
that
a
subunit
vaccine
may
need
to
be
polyvalent
to
be
immunogenic
in
individuals
of
many