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Eur.
J.
Immunol.
1992.
22:
689-696
Malaria-specific responses in non-exposed humans
689
Yinka Zevering.,
Fiona Amante.,
Anne Smillie.,
Jeff Currier.,
Gale Smithv,
Richard A. HoughtenA and
Michael F. Good.
Tropical Health Program.,
Queensland Institute
of
Medical
Research, Brisbane. MicroGeneSys
Inc.v, Meriden, Torrey Pines
Institute
for
Molecular StudiesA.,
San Diego
High frequency of malaria-specific
T
cells in
non-exposed humans
A major goal of current candidate malaria vaccines is to stimulate the expansion
of clones of malaria-specific lymphocytes. We have examined the
in vitro
T
cell
responses of a group of malaria exposed and non-exposed adult Caucasian donors
to recombinant circumsporozoite (CS) proteins, one of which is undergoing
clinical trials, to blood-stage parasites, and to synthetic peptides copying the CS
protein and defined blood-stage proteins. In nearly all individuals tested, CD4
T
cell proliferation or lymphokine production occurred in response to whole
parasite or CS protein stimulation, and T cells from many individuals responded
to synthetic peptides.
T
cell responses were major histocompatibility complex-
restricted, and stimulation of T cells with malaria parasites or CS protein did not
appear to expand a population of Tcell receptor
$6
cells. Malaria-specific
responses were independent of prior malaria exposure, and in some cases
exceeded the magnitude of response to tetanus toxoid. SpecificT cells are present
in high frequency in the peripheral blood of many donors who have never been
exposed to malaria. Although malaria-specific CD4
T
cells play an important role
in immunity, these data question whether vaccines need to stimulate such cells,
and focus attention on other aspects of malaria immunity which may be more
critical to a successful vaccine.
1
Introduction
Tcells develop in the thymus from fetal liver- or bone
marrow-derived hematogenous progenitor cells
[
11.
Fol-
lowing positive and negative selection events in the thymus,
during which rearrangement of the TcR genes occurs, a
population of clonally distributed precursor cells is
exported from the thymic medulla to seed the peripheral
lymph organs. Classical activation of T cells occurs when
theTcR engages a processed peptide from a foreign antigen
(epitope) bound to an MHC molecule on the surface of an
antigen-presenting cell
[2].
While MHC antigen-specific
(allo-specific)
T
cells from unprimed animals can readily be
grown
in vitro
with allogeneic stimulation, onlyT cells from
specifically primed individuals respond
in vitro
to
nominal
antigens
[3].
T cells are central to malaria immunity in that they function
as helper cells for B cells or effector cells, in their own right.
Animals which lack antibody (p-suppressed mice) can, in
fact, be protectively immunized with blood-stage malaria
parasites
[4]
or sporozoites
[5].
CD4 T cells can transfer
immunity in mice to blood-stage parasites
[6],
as well as to
sporozoites
[7-81,
and in the case of sporozoite immunity,
the circumsporozoite (CS) protein is an important antigen.
[I
96471
*
This work was Suported by UNDPlWorld Bank/WHO Special
Programme for Research and Training in Tropical Diseases,
and
by
the National Health and Medical Research Council
(Australia).
Correspondence:
Michael
F.
Good,
Tropical Health Program,
Queensland Institute
of
Medical Research, Herston Road, Bris-
bane
4029, QLD,
Australia
Abbreviations:
CS:
Circumsporozoite
Thus, central to the development
of
a malaria vaccine
aimed at cellular immune defense, is the principle of
stimulating a specific population of precursor T lympho-
cytes that will expand clonally, then recognize and destroy
the parasite encountered following natural challenge.
Recent studies, however, have shown that malaria antigen-
specific and MHC-restricted
T
cell clones could be gene-
rated from humans with no history of malaria exposure
[9-111.
This suggests that malaria-specific memory
T
cells
are present in non-exposed individuals. The frequency of
such cells is not known, but a basic premise of many current
vaccine programs (clonal expansion of a population of
malaria-specific precursor T lymphocytes) is questioned
if
memory
T
cells from non-exposed individuals which can
respond to malaria antigens are present at high frequency.
Because of the potential importance of these reports, we
have investigated the human T cell immune response to
purified bacuolovirus-expressed
F1
fakiparum
recombinant
CS protein
[12, 131
currently in use in human trials, as well
as to a yeast recombinant CS protein and to blood-stage
malaria parasites. Also, a group of synthetic peptides
copying the CS protein sequence and known to be immu-
nodominant for this group
of
donors was tested
[14].
2
Materials and methods
2.1
Donors
Half of the donors were adult Caucasian residents of
Brisbane who have lived in malaria endemic regions,
mainly Papua New Guinea, for varying periods
of
time (see
Sect.
3.1,
Table
1)
and come from a previously described
group
of
subjects
[14].
These donors are described in the
Tables as
‘E’
(exposed). The other donors
(‘C,
control)
were adult Caucasian residents
of
Brisbane with no history
of
malaria exposure.
0
VCH Verlagsgesellschaft mbH,
D-6940
Weinheim,
1YY2
0014-2980/Y2/0303-0689$3.50
+
,2510
690
Y.
Zevering,
E
Amante,
A.
Smillie et al.
Eur.
J.
Immunol. 1992.
22:
689-696
2.2 ELISA
Antibodies binding the (NANP), repeat motif of the
recombinant construct, R32tet32, were detected by ELISA
as previously described
[15,
161. Titer is defined as the
greatest dilution of test serum giving an absorbance >3 SD
above the mean of a panel of normal sera at the same
dilution.
2.3 Antigens
The baculovirus-expressed recombinant CS protein, cur-
rently in clinical studies, has previously been described
[12, 131. Its characteristics and purity are described in
Fig.
1.
A yeast-expressed recombinant
CS
protein was also
used in some studies, and has previously been described
[17]. It contains residues 43-348, and was kindly provided
by Dr. Philip Barr, Chiron.The following synthetic peptides
were used: PfCS proteinl-20 (peptide
1,
MMRKLAILSVS-
SFLFVEALF);
PfCS
protein296-315 (peptide 16, QGHNM-
PDPNRNVDENANAN); PfCS protein331-350 (peptide 21,
IEQYLKKIKNSISTEWSPCS);
PfCS
protein386-405 (pep-
tide 28, MEKCSSVFNVVNSSIGLIMV); PfMSAl (YKL-
NFYFDLLRAKL); PvMSAl (HVINFHYDLL
RANV).
The peptides are described further in listed references
[14,
18, 191.
2.4 Lymphocyte proliferation assays
Human PBL were obtained by density centrifugation
of
peripheral blood over Ficoll Hypaque. PBL were added to
round-bottom microtiter plates at 2
x
lo5
cells per well
(quadruplicate) in Hepes-buffered Eagle's minimum essen-
tial medium containing
10
%
pooled normal human serum
and antigen at various concentrations. Six or seven days
later wells were pulsed with
0.5
pCi
=
18,5 kBq of
'[Hlthymidine and incorporation of label was determined
18 h later by liquid scintillation spectroscopy. Specific
0.20
-I
cs
t
Mobility
Figure
I.
Analysis of purity
of
rCS protein. A recombinant
baculovirus expression vector containing a full-lenght
Rfulciparurn
CS
gene was used to produce
CS
protein in insect cells
(Spodoptera
frugiperdu).
The
rCs
polypeptide (66000
kDa)
was purified. Ten
micrograms
of
purified
CS
antigen was electrophoresed on
an
11.5
%
polyacrylamide gel, stained with coomassie brilliant blue,
and scanned on
an
LKB
UltroScan
XL
laser densitometer. The
arrows indicate minor contaminants which are present in the
CS
preparation.
rCS
protein represents
299
%
of the purified protein.
Amino acid sequencing revealed cleavage
of
a
signal peptide
between residues
65
and
66
of
the deduced amino acid
sequence.
proliferation was calculated as a stimulation index, defined
as (cpm in presence of antigen)/(cpm in absence of
antigen), or simply by listing cpm. Assays were performed
in quadruplicate.
2.5
Generation of
T
cell clones
The method of Sinigaglia and Pink [20] was used. Briefly,
PBL (2
x
106/well, in 24-well plates) were stimulated
in
vim with
19
fakiparum
(FCR3 clone)-infected RBC (lo6/
ml) for
7
days. Blast cells were enriched
on
Ficoll-Paque
and cultured with IL 2
(50
unitslml) for
7
days and then
plated at limiting dilution (0.3,
1
cell/well) with irradiated
allogeneic PBL (4
x
lo5
cells/well, flat-bottom 96-well
plates), I1 2 and PHA at
5
pg/ml. Medium was changed
regularly and clones were obvious by 2-3 weeks. Clones
were expanded with allogeneic filler cells, but tested with
autologous APC.
2.6
Generation of short- and long-term
T
cell lines
For short-term lines, PBL (2
x
lo5
cells/well) were
stimulated in 96-well round-bottom microwells with CS
protein at
5
pg/ml for
1
week. Cells were then washed in the
wells (X3), split into replicates, and restimulated with
either CS protein
(5
pg/ml) or peptide (30 pg/ml) in the
presence
of
fresh irradiated autologous PBM as APC. After
a further week, wells were pulsed with 3[H]thymidine and
incorporation measured after
18
h. For long-term lines,
PBL were stimulated in 24-well plates (2
x
lo6
cells/well)
with antigen and after a week the cells were washed and
rested with fresh irradiated autologous filler cells and IL 2
(50
U/ml) for 2-3 weeks. Following this, blasts were
prepared over Ficoll-Paque. Cells were then tested by
adding
5
x
lo4
cells to
lo5
irradiated APC and antigen and
measuring proliferation after
4
days.
2.7
Limiting dilution assays
T cells were purified from the lymphocytes by 2-amino-
ethyl-isothiouronium bromide-treated sheep red blood cell
rosette sedimentation [lo, 21, 221. Purified T cells were
plated out in round-bottom microtiter plates at cell con-
centrations ranging from
lo2
to 2
x
lo5.
For each concen-
tration, 24 wells with antigen (parasite or peptide), and 12
or more wells without antigen were used. Irradiated
non-T
cells were added at
lo4
cells/well, as a source of APC.
Proliferation was determined a week later by liquid scintil-
lation counting of incorporated [3H]thymidine. Antigen-
specific responses were determined to be those whose cpm
exceeded the average cpm of the negative control wells by
23
SD. Precursor frequencies of peptide-specific T cells
were determined from the slope of the line of best fit (least
squares) plotting cell number
(X
axis)
and the log
of
the
fraction
of
non-responding wells (LnF,)
(Y
axis).
2.8 Cytokine assays
IFN-y (Commonwealth Serum Laboratories, Melbourne,
Australia), and I1 6 (Genzyme, Boston, MA) were assayed
by ELISA as described in the manufacturer's kits.
Eur.
J.
Immunol.
1992.
22:
689-696
Malaria-specific responses in non-exposed humans
691
fluorescinated sheep anti-mouse IgG (Silenus Labs., Haw-
thorn, Australia) for
1
h at
4
"C. Cells were then washed and
analyzed on a Becton Dickinson FACScan (Mountainview,
CA). For T cell receptor usage studies, cloned
T
cells or
PBM gated for lymphocytes by forward and side scatter,
were incubated with directly fluorescinated anti-a@ TcR
antibody (TCR-1, Becton Dickinson) or anti-6 antibody
(TCR
61,
T Cell Sciences, Boston, MA) prior to washing
and analysis.
2.9
Depletion
of
lymphocytes with magnetic beads
PBM were incubated with optimal concentrations of either
antLCD4 mAb (ATCC CRL 8002) or anti-CD8 mAb
(ATCC CRL
8014),
washed and then incubated with mag-
netic Dynabeads (M-450; Dynal A.S., Oslo) coated with
sheep anti-mouse IgG at a ratio of 10 beads per cell for CD8
and a ratio
of
5
beads per cell for CD4. The beads were
mixed with rocking for 30 min at
4
"C before removal with a
magnet.
2.10
FCM
analysis
Briefly, cells were incubated with mouse mAb against
either human CD3, CD4, or CD8 (ATCC CRL 8001,8002
and 8014, respectively) at optimal concentration for
1
h at
4°C. Cells were then washed twice and incubated with a
3
Results
3.1
CS
protein-specific responses
The donors are described
in
Table
1
and come from a group
of previously described donors [14]. Sera from seven
Table
1.
Stimulation indeces from
20
malaria-exposed and
20
non-exposed individuals to
rCS
protein (CSP), the known immunodom-
inant
T
cell epitopes in the protein
[14],
and tetanus toxoid")
Subject
El*
E2*
E3
E7*
E8*
E9*
El0
E 18
E25
E31*
E32
E40*
E42*
E43
E47*
E56"
E65
*
E66*
E67*
E68
c1
c2
c3
C8
c10
c12
C13
C15
C19
c20
c21
c22
C23
c24
C25
C26
C27
C28
C29
C30
PeDtide number
16 21
1.1
1.1
2.4 2.8
1.6 1.9
2.6 2.6
NT NT
NT NT
1.5 31.5
4.3 3.1
1.4 1.6
1.3 2.1
1.4 1.6
0.6 2.1
5.2 2.3
4.3 7.8
0.8 0.9
1.1 1.8
2.1 4.5
1.6 1.5
1.0 1.4
9.9 4.8
2.6 5.6
0.7 1.3
2.2 1.9
1.6 13.4
4.7 12.4
6.3 8.8
3.0 3.6
8.5 26.5
2.5 2.5
12.2 18.9
2.5 6.7
1.2
1.5
0.9 2.2
7.5 6.1
0.7 0.9
1.7 4.9
3.2 7.8
1.7 2.1
1.5 1.9
2.4 4.6
28
3.5
1.3
1.9
1.8
NT
NT
2.5
4.0
1.7
2.4
3.3
3.0
4.2
9.2
0.9
1.7
6.9
1.4
2.1
26.8
2.2
4.0
2.2
20.6
13.3
5.4
4.4
20.0
12.6
38.2
6.0
4.2
36.6
43.2
2.4
3.0
2.5
5.1
2.6
3.0
Stimulation index
CSP
0.5
pg/ml
0.2
0.6
0.8
3.5
0.8
1.6
1.5
8.2
0.3
0.5
6.7
1.5
1 .9
0.1
5.6
0.7
5.2
5.4
1.6
1.2
3.1
0.9
0.0
6.0
24.0
2.2
0.6
4.1
1.1
1.7
3.7
2.2
0.8
4.0
3.1
1.3
15.0
2.2
2.4
0.9
5
pg/ml
4.1
5.1
1.1
5.5
5.5
8.6
9.9
7.5
4.0
0.2
24.0
35.0
6.4
2.5
45.2
6.3
22.3
27.0
2.3
48.7
13.9
4.8
3.3
11.2
27.6
10.9
21.6
52.2
5.4
44.2
14.2
2.4
38.0
45.0
66.7
4.6
27.5
1.6
5.2
4.5
Tetanus
toxoid
4.4
13.4
3.3
10.4
NT
NT
30.8
5.4
4.8
3.5
11.7
16.6
24.5
14.6
0.5
5.7
260
1 .5
3.0
220
56.9
41.8
6.4
27.8
36.0
96.0
0.3
199.7
141.5
88.9
62.5
557
21.6
65.0
16.8
15.6
8.7
10.8
12.4
82.0
Anti-
NANP
Ab
titer
>64
21024
<16
46
<16
<16
<16
46
<16
<I6
<16
>64
<16
<16
>64
>64
>2048
46
46
>2048
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
Years
of
exposure
4
7
10
14
14
16
3
3
4
months
10
23
19
10
9
7
14
6
6
2
months
9
a)
All
'E'
subjects have had
sporozoite exposure as
listed, but only
'E*'
subjects
have had clinical malaria.
Peptides were tested at their
optimal concentration
(30
kg/ml).
Mean cpm
of
background (no antigen):
1411.
692
Y.
Zevering,
F.
Amante, A. Smillie et
al.
malaria-exposed individuals contained antibodies against
the major
B
cell epitope of the protein ([NANP],). This
suggests that their degree of exposure to sporozoites was
less than that for lifelong residents of malaria-endemic
villages in Gambia where we have previously found that
58
Yo
-66
YO
of sera contain such antibodies [15, 161. Lym-
phocytes from 40 donors (half of whom had a history
of
sporozoite exposure) were tested for proliferation to the
recombinant baculovirus-expressed CS protein, to the
three most immunogenic synthetic peptides chosen from a
panel of 29 overlapping peptides spanning the entire
protein [14], and to tetanus toxoid.The extent of lympho-
cyte proliferation to these antigens is given inTable
1
where
it can be seen that T cells from 35 of the 40 donors (17
of
the
20 malaria-exposed and
18
of the 20 non-exposed donors)
responded by proliferation (stimulation index >3) to
recombinant CS protein at the optimal concentration
(5 yg/ml), that T cells from many donors responded to one
or more of the synthetic peptides, and that
T
cells from 35
of
38
donors tested responded to tetanus toxoid.There was
no evidence for increased affinity of the
T
cells for the CS
protein from the malaria-exposed group since
T
cells from
both groups responded comparably at the lower concentra-
tion of
0.5
yglml (Table 1). Some individuals responded
poorly to the protein but, nevertheless, did respond to one
or more of the peptides (E43, C22, C28).This may indicate
that inadequate antigen processing with respect to those
peptides was occurring for this protein, as previously
suggested [13, 23].Two individuals (E3, E67) responded to
neither the
CS
protein nor the synthetic peptides. There
was no correlation between response to tetanus toxoid and
response to CS protein.The cells responsible for the prolife-
rative response to CS protein could be removed by
antibodies to CD4 and magnetic beads but not by antibod-
ies to CD8 and magnetic beads.
Eleven short-term T cell lines generated by baculovirus-
expressed CS protein stimulation were challenged with
synthetic peptides as well as (in some cases) with recombi-
nant protein. Four of these lines proliferated in response to
the synthetic peptides, indicating that the synthetic pep-
tides can represent actual epitopes within the recombinant
protein (Table 2). The response of the lines to the peptides
does not represent a primary response to the peptides from
cells carried over in culture from the initial bleed since cells
carried in culture without CS protein stimulation did not
respond to any of the peptides. It is possible, however, that
some of the peptides that are stimulatory for a given
individual's peripheral blood
T
cells do not represent actual
epitopes present in the protein for that individual, due to
difficulties
in
antigen processing (see above, for E43, C22,
C28).
Using the yeast-expressed recombinant CS protein, we have
tested whether specific T cell responses from non-exposed
individuals are genetically restricted.Three long-termT cell
lines were generated from different donors and restimu-
lated with CS protein in the presence of irradiated PBM
from different donors. As shown in Fig.
2,
proliferation was
significantly greater
in
the presence of autologous APC for
both CS protein-specific responses and for control tetanus
toxoid-specific responses. The moderate response from
both
T
cell lines from donor C15 in the presence
of
APC
from donor C21 may result from the fact that they share
MHC class
I1
alleles (DR2 and DQwl).
Eur.
J.
Immunol.
1992. 22: 689-696
Table
2.
Proliferation of short-term
CS
protein-specificT
cell
lines
to rCs protein
and
the three immunodominant peptide epitopes
present
in
the recombinant protein")
Stimulation index
Subject
16 21 28 5
pglrnl
Peptide number
CSP
E2
E7
E8
E9
E40
E47
c12
C23
C24
c28
c29
0.9 0.9
1 .0 0.9
3.9 9.3
0.5 5.7
1.2 2.0
2.1 0.5
1.9 0.4
6.0 1.4
1.6 3.3
0.5 1.4
2.0 0.6
1.5
0.4
5.2
15.8
1.1
1.6
0.5
22.0
0.7
1 .0
0.7
a)
Mean
cpm
of
background
(no
antigen):
1497
CSP-specific
lines
tested with CSP
11.9
3.5
33.6
24.5
NT
NT
NT
NT
NT
NT
6.0
TT-apecific
line
teated with
ll
C15
C24
C25
Figure
2.
Proliferation of long-term
T
cell
lines specific for
CS
protein for
CS
protein (yeast-expressed)
and
tetanus toxoid
(TT)
in
the presence
of
autologous
and
allogeneic
APC.
3.2
Precursor frequency
of
CS
peptide-specific responses
The frequency of Tcells reactive with one of the most
immunodominant peptides from the molecule, peptide 21
(residues 331-350 [14], was then determined. For six
Eur.
J.
Immunol. 1992.
22:
689-696
..
posed donors and three non-
exposed
donors
responding
to
individuals, the T cell precursor frequencies ranged from
1/11
000
to
1/850
000
(Fig. 3).This compares with precursor
frequencies to tetanus toxoid in two individuals of
1/11
000
and
1/3500
as we previously defined using the same
methodologies as here
1241.
Due to limitations of material,
it was not possible to determine the precursor frequencies
to recombinant CS protein. However, we compared the
precursor frequencies of
T
cells specific for all the major
epitopes of the protein for a large group of malaria-exposed
and non-exposed individuals. At the same time as setting
up the limiting dilution assay for each indivudual, we also
set up a standard bulk culture. We were thus able to
compare directly the precursor frequency for each do-
nodpeptide combination with the stimulation index for
that same combination. This data (comparing
31
separate
donorlpeptide combinations) demonstrated that there was
a close correlation between the two measurements, and
that for the precursor frequencies measured, there was
no
obvious difference between the exposed and non-exposed
groups as a whole.The highest frequency we recorded from
a non-exposed individual was
286
T
cells responding to
peptide
#1
per
lo6
peripheral blood
T
cells, and the highest
response from exposed individual was
74
T
cells respond-
ing to peptide
#21
per
lo6
T
cells. Since the stimulation
indeces of T cells from both groups of donors reactive to the
recombinant protein were similar (Tables
1
and
3),
we
predict that malaria-exposed and non-exposed donors
T
CELLS
PER
WELL
(x
loa)
0
50
100
150
200
I
Donor:
El0
Frequency:
92
x
rod
Y
4
I
Donor:
€32
Frequency: 2.4
x
lod
100
30
Donor:
€43
Frequency: 1.2
x
1
0'
7
0
Malaria-specific responses in non-exposed humans
6Y3
contain comparable numbers of
T
cells specific for the CS
protein.
3.3
Cytokine production following CS protein
stimulation
T
cell cultures from malaria-exposed and non-exposed
individuals produced IFN-y and IL
6
(cytokines specifically
involved in sporozoite immunity
[25, 261)
following
CS
protein stimulation (Table
3).
T cells from two individuals
tested in this study did not proliferate to rCS protein
(C31,
C33);
however, significant quantities of lymphokines were
produced by
T
cells from both. There was
no
significant
difference in lymphokine production in response
to
CS
protein between the malaria-exposed and non-exposed
individuals, and
T
cells from two individuals tested who
had high antibody titers
(E2, E65)
produced less lympho-
kines than
T
cells from some non-exposed donors.
3.4
Blood-stage-specific responses
We have also been able to stimulate blood-stage-specific
T cells and readily generate clones from non-exposed
humans (Table
4).
In
vitro,
T
cell responses peaked at
6-7
days, and precursor frequencies ranged from
1/500-1/2000
50
100
150
100
I
I
I
I
100
I
I
I
1-
Donor:
c10
Frequency: 2.2
x
lod
50
Donor:
c10
Frequency: 2.2
x
lod
+
50
Figure
3.
Limiting dilution
analysis
of
peripheral
blood
T
cells from three malaria ex-
30
Donor:
C12
Frequency:
4.8
x
10'
?
3.
Limiting dilution
;is
of
peripheral
blood
j
from three malaria ex-
694
Eur.
J.
Immunol.
1992.
22:
689-696
Table
3.
Stimulation indeces and lymphokine production
on
following stimulation
of T
cells from malaria-exposed and non-exposed
individuals with recombinant CS protein
Y.
Zevering,
F.
Amante,
A.
Smillie et al.
Subjcct
E2
E9
E65
Eh9
E70
c3
C4
c10
C3 1
C32
c33
Stimulation index Lymphokine production following CS
protein stimulation
CSP”)
Day
1
Day
4
Tetanus
(5
pg/ml) IFN-yb)
IL
6c)
IFN-yb)
IL
64
toxoid
53.8 7.6 0.0 4.2 0.6
32.8 10.8 0.0 2.7 0.44
176 44.4 0.74 8.3 0.8
36.7 5.2 0.0 6.0 0.86
60.0 6.1 0.0 2.6 0.0
7.5 3.6 0.0 3.9 0.78
39.5 3.2 0.0 3.5 0.0
41.3 13.8 0.6 0.7
0.0
5.8 1.2
0.0
2.7 0.68
8.0 3.5 0.78 12.8 1.3
39.6 1.4 0.0 4.4 0.54
peripheral blood
T
cells. All of
10
T
cell clones examined
have stained positive for CD3, CD4 and TcR
a@,
and
negative for CD8 and TcR
y/6.
The responses of four clones
are listed in Table 4. Certain clones were specific in that
they could discriminate
P
vivax
from
l?
falciparum
(clones
C4 and C5,Table 4),whereas some other clones responded
equally well to both parasites (not shown), presumably
because they recognized shared antigens. Proliferation of
these clones was genetically restricted by APC expressing
MHC class
I1
genes in common with the clone, as has
previously been demonstrated for malaria gamete-specific
T
clones
[lo].
Following antigenic stimulation, clones
secreted
IFN-y
a lymphokine known to mediate malaria
parasite killing
[27,
281.
Clones could also be generated
from non-exposed individuals to synthetic peptides from
the MSAl proteins of
I?
falciparum
and
P
vivax
(see Sect.
2.3,
data not shown).
1.7
0.9
7.2
3.4
0.7
1.3
1.9
0.0
0.7
8.1
1.1
b) I.U./ml.
a)
CS
protein tested at
5
and
1
pg/ml(5 pglml data
only given as this was optimal in all cases). Mean
cpm of backgraound
(no
antigen):
1515.
c) ng/ml.
3.5
TcR usage
of
malaria-specific T cells
The type of
T
cell receptors used in the response to the
recombinant CS protein and whole parasite was deter-
mined. Lymphocytes from five individuals (one exposed,
four non-exposed) were analyzed by FCM to determine the
fraction of cells expressing TcR
alp
and TcR
y/6.
Lympho-
cytes were then stimulated with the yeast-expressed CS
protein or whole parasite and proliferated; thereafter the
cells were re-examined.
As
shown in Table
5,
there was no
evidence that TcR
y/6
cells were preferentially expanded in
these experiments, although it cannot be excluded that for
other individuals or for certain malaria antigens TcR
y/6
cells can be activated. There may have been some stimula-
tion of TcR
y/6
cells in our experiments, which was not
detected since we were not studying purified populations of
such cells.
Table
4.
Proliferation and specificity
of
peripheral blood lymphocytes and
T
cells clones to
R
falciparum-infected
RBC
(Pf. pRBC).”)
Subject
E42
E7 1
E72
C13
c14
c15
C16
C17
c24
C31
c34
c35
Clone
C1
Clone
C3
CloneC4
Clone
C5
PHA
60
620
34 213
34 701
70 106
32 654
13 290
36 489
25 815
53 444
64 848
77 207
59 272
Blank
204
571
284
368
102
118
264
108
95
104
272
463
CPm
Pf.
pRBC (FCR-3)lml
106 105
104
55 462
5753
3319
30 577
8363
15 877
25 455
5 078
21 214
37 209
33 303
11 515
-
44 330
2552
17 870
14 457
1412
9634
5259
654
9088
27 581
7214
277 1
8730
9424
13 192
2491
(58)
(131)
34 330
2748
11 069
745
370
1699
745
944
1903
9198
2815
2436
402
1
2176
263
308
(68)
(41)
Blank
(Day
7)
3193
2969
5398
1880
1883
943
2241
1697
33
1
1974
1569
3002
817
129
160
148
a) “E” subjects
in
this table have experienced
clinical
R
falciparum
infections;
“C”
subjects
have neither had clinical malaria nor been
exposed to sporozoites. Proliferations in re-
sponse to
PHA
was measured after
48
h, and
proliferation in response to
l?
falciparum
was
measured after
6
days. The proliferative re-
sponses of the clones
C4
and
C5
to
R
vivax
parasites is given in parentheses:
().
None
of
the
clones proliferated in the presence
of
uninfected
RBC.
Eur. J. Immunol.
1992.
22:
689-696
Table
5.
TcR analysis
of
lymphocytes before and after exposure to
CS protein or malaria parasites
Donor
El4
C4
C15
C24
C31
Percentage
of
lymphocytes
Antigen TcR
al$
rCS
protein
Tetanus toxoid
Nil
rCS
protein
Tetanus toxoid
Nil
rCS
protein
Tetanus toxoid
Nil
pRBC
PMC
Pre
70.7
70.7
70.7
NT
NT
NT
NT
NT
NT
73.1
67.6
post
85.9
86.1
88.9
NT
NT
NT
NT
NT
NT
92.2
88.8
TcR
yl6
Pre
5.1
5.1
5.1
4.6
4.6
4.6
2.5
2.5
2.5
15.6
9.6
post
1.6
1.7
2.2
2.3
1.9
2.6
2.3
3.0
1.9
10.3
7.8
4
Discussion
Malaria-specific CD4 T cells bearing TcR
alp
chains and
capable of proliferation and lymphokine production are
present
in
non-exposed non-vaccinated individuals at high
frequency. They are genetically restricted, apparently by
MHC genes. It is likely that all individuals have Tcells
which will either proliferate or secrete parasiticidal lym-
phokines (or both) in response to CS protein or whole
parasite stimulation. While previous malaria exposure does
not appear to expand this population of cells (at least to the
extent that the donors of this study were exposed), our
results would not preclude the possibility that immuniza-
tion with a sporozoite or blood-stage ‘vaccine’could expand
further the already existent population of specific
T
cells.
However, the proliferative response of some non-exposed
individuals to the CS protein was comparable to or even
greater than their response to tetanus toxoid (Tables
1
and
3).
These responses may arise through cross-reactivity [29]
since a peptide 21-specific T cell clone from a BCG-
vaccinated individual reacted strongly with
PPD
[30].
Peptide 21-specific clones from other individuals, however,
have not responded to PPD.
The antigens recognized by the blood-stage-specific T cells
have not been identified but different clones clearly
recognize different antigens based on different patterns of
reactivities to a panel of different
f?
fulcipurum
clones.
Although T cells from approximately
90
%
of individuals
responded to the CS protein by proliferation, we do not
believe that this response rcpresents a mitogenic response
since: (a) the peak of the response to both CS protein and
tetanus toxoid occurred on days
5
and
6
compared to a peak
of days 1-3 for PHA and Con
A
stimulation;
(b)
the
response
of
CS protein-specific T cell lines to synthetic
peptides from the protein suggests that the induction of the
response is not due to a mitogen or a superantigen; (c) the
response was not associated with CDS T cells and (d) the
responses of three T cell lines specific to the CS protein
were MHC restricted.
Malaria-specific
responses
in non-exposed humans
695
Trial human vaccine candidates have been designed to
expand a population
of
specific lymphocyte precursors.
While our data in no way denies an important role for CD4
T cells in malaria immunity, in pre-vaccinated adult
humans, at least, many malaria-specific precursor lympho-
cytes appear to have been already stimulated and many
apparent memory cells are already present in the circula-
tion. It is also possible that actual malaria-specific precur-
sor cells are present in the periphery at very high frequency
(comparable to the frequency of tetanus toxoid-specific
memory Tcells) and that
in
vitro
these are capable of
responding to malaria peptides or parasites. Although
non-exposed humans are not immune to malaria, it cannot
be argued that their malaria-specific T cells could play no
role in anti-parasite immunity. Other factors acting in
concert with these T cells, but not present in non-exposed
individuals, may be critical for full expression
of
cellular
immunity to malaria. One such factor could be a suitably
modified spleen
[31].
Furthermore, malaria-specific T cells
in non-exposed individuals could partially contribute to the
varying degrees of innate resistance of adult humans to
malaria parasites [32].
We
sincerely thank the donors for making this study possible, Dr.
Phil Burr, Chiron, for supplying the yeast-expressed recombinant
CS
protein and Dr. Mitch Gross,
SKB,
for supplying R32tet32.
Received June
19, 1991;
in revised form October
29, 1991.
5
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