Immunopharmacolooy

ELSEVIER Immunopharmacology 28 (1994) 137-143

Improvement of the T-cell response to a non-immunogenic peptide

by its tandem association with a highly efficient T-helper peptide
Franck Rouaix a, H~l~ne Gras-Masse b, Christine Mazingue a, Pierre-Richard Ridel a,
Eric Diesis b, Monique Marguerite a, J6r6me Estaquier a, Andr~ Capron a, Andr~ Tartar b,
Claude Auriault a,,
aCentre d'Immunologie des Maladies Transmissibles et Allergiques, Unit6 mixte I N S E R M UI67-CNRS 624, B.P. 245, lnstitut Pasteur de
Lille, 1 rue du Pr. Calmette, 59019 Lille C6dex, France
b Laboratoire de chimie des Biomol6cules, URA-CNRS 1309, Institut Pasteur de Lille. Lille, France

(Received 18 October 1993; accepted 28 March 1994)

Abstract

The 45-69 peptide, an helper T-cell epitope derived from HIV nef protein, is strongly immunogenic. A T-cell prolif-
erative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and
without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein
of the parasite Schistosoma mansoni, requires the presence of a carrier or the use of peptidic constructs. We demonstrate
here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic con-
structs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent
association of both peptides is necessary, since the co-injection of 45-69 and 115-131 peptides is not sufficient to induce
a detectable anti-115-131 T-cell response. The mutual orientation between the respective tandem peptides (45-115 and
115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and
immunogenicity but both allowed to reveal the existence of a 115-131 specific T-cell response normally undetectable using
115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction
of vaccines.

Key words." Efficient helper T-cell epitope; Tandem synthesis; Adjuvant; Antigenicity; Immunogenicity

I. Introduction epitopes of known protective antigen provides the

The possibility to produce recombinant or syn-
thetic peptide antigens which mimic B- and T-cell
* Corresponding author.
Abbreviations: IFA, incomplete Freund's adjuvant; APC, antigen
presenting cells; CFA, complete Freund's adjuvant; MAP, mul-
tiple antigenic peptide; PBS, phosphate buffer saline; SI, stimu-
lation index; Sm, Schistosoma mansoni; rSm28-GST, recombi-
nant Schistosoma mansoni 28-gluthatione S-transferase.
potential of a new generation of subunit vaccine
agents. However, as these immunogens become
more defined at the molecular level, they also be-
come less immunogenic, thus increasing the need for
new strategies to augment immunogenicity, such as
introduction of new immunoadjuvant, or new for-
mulation.
One immunopharmacological approach in vac-
cine was recently proposed by association of a

0162-3109/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved

SSDI 0 1 6 2 - 3 1 0 9 ( 9 4 ) 0 0 0 2 4 - A
 138 F. Rouaix et al. / lmmunopharmacoh~gy 28 (1994~ 137-143
peptide to a defined high efficient T h cell epitope
using either constructs of T- and B-characterized
epitopes of the same pathogen ( S u e t al., 1992) or a
foreign helper T-cell determinant associated to the
epitope (Good et al., 1987; Francis et al., 1987).
The purpose of this study was to analyze the im-
munogenicity of a high efficient helper T-cell peptide
in the absence of any adjuvant and then to ascertain
whether it is possible to enhance T-cell response to
a poorly immunogenic peptide by tandem associa-
tion with this strongly immunogenic peptide.
To do so, we used the 45-69 peptide derived from
HIV-1 nef protein previously described as a helper
T-cell epitope (Allan et al., 1985; Estaquier et al.,
1992a) (Fig. 1), This peptide was tandemly synthe-
tized with a poorly immunogenic peptide, the 115-
131 peptide, that represents a sequence derived from
a 28-kDa protective antigen of Schistosoma mansoni
(rSm28-GST) (Balloul et al., 1987). This peptide was
previously described as containing T and B cell
epitopes inducing the proliferation of T lymphocytes
from experimentally infected rats, mice and baboons.
However, this peptide was poorly immunogenic by
itself and required to be linked with carrier proteins
(Auriault et al., 1988).
2. Materials and methods
2.1. Animals
6-8-week-old Syngeneic male Lou M rats (Iffa
Credo) were used throughout the experiments.
45 69
(a) SSNTAATNAACAWI.EAQEEEEV(;FP
115 Igl
(b) KPQEEKEKITKEILNGK
45 6 t) l I)
(C) S S N T A A T N A A C A W L E A Q E E E E V G FPKPQEFKEKITKEILNK
I 15 IM 45
(d)KPQEEKEK rl'K EII.NG KSSNTAATN AACA W LEAQEEEEV(;I
Fig. 1. Amino-acid sequences of the ( a ) 4 5 - 6 9 , (b) 115-131, (c)
45-115 and (d) 115-45 peptides.
2.2. Peptide synthesis
115-131, 45-69 peptides and l 15-45 and 45-115
tandem peptides (Fig. l) were synthetized using the
conventional solid-phase "Boc-benzyl strategy" on
a paramethyl-benzhydrylamine resin (Applied Bio-
systems, Foster City, CA, USA) in an automated
Applied Biosystem 430A synthetizer. Protected
amino acids were purchased from Propeptide. Side
chain protections were as follows: Arg (tosyl), Asp
(cyclohexyl), Glu (cyclohexyl), Lys (2-chloro-benzy-
loxycarbonyl), Ser (benzyl), Thr (benzyl), Trp
(formyl), Tyr (2-bromo-benzyloxycarbonyl). A 4-
fold excess of amino acid was activated by DCC'
HOBt in NMP. A double coupling step was f'ol-
lowed by capping with acetic anhydride. Hydrogen
fluoride final deprotection and cleavage from the
resin was performed in a Teflon-KelF apparatus
(Asti), using a "low-high" HF procedure: an initial
treatment was performed with: HF/p-cresol/p-
thiocresol/dimethylsulfide (10:3:1:26) for 3 h at 0 ° C.
The resin was washed with ether and vacuum dried
and treated with: HF/p-cresol (10:1) for 90 rain at
0 ° C. Crude peptides were washed in ether and solu-
bilized in a small volume of TFA and precipited in
ether. Peptides were characterized by amino-acid
analysis after acidic hydrolysis (6 M HC1, l l0°C,
24 h in the presence of phenol under reduced pres-
sure) on an Beckman 7300 amino-acid analyser.
2.3. Immunization procedures
Peptides (1 /xg or 0.1 tLg) in PBS (500/~1) or in
aluminium hydroxide ( 1:20, w/w; Serva, Heidelberg,
Germany) were injected subcutaneously at the base
of the tail of animals on day 0, boosted on day 7 and
animals were killed on day 14. Inguinal lymph nodes
were aseptically removed.
2.4. Lymphocyte culture medium
RPMI-1640 (Gibco, Courbevoie, France) was
I~1 supplemented with 5 x 10 5 M 2-mercaptoethanol
(Merck), 2 mM L-glutamine (Merck), 1 mM sodium
n9 pyruvate (Gibco), antibiotics (100 IU/ml penicillin,
•
100 mg/ml streptomycin; Specia, Paris, France),
20 mM Hepes (Sigma) and 10% heat-inactivated
fetal calf serum (FCS) (Gibco).
 F. Rouaix et al. / Immunopharmacology 28 (1994) 137-143 139

2.5. Lymphocyte proliferation assay ~" 60000. [] nopeV '

[] 45-6.c
50000-
Inguinal lymph nodes were prepared aseptically.
~ 0000-
T lymphocytes were separated by passage through a
r~ 30000
nylon wool column (Julius et al., 1973) and main-
tained at 37 °C in a 5 ~o CO2 atmosphere in lympho- 20000

cyte culture medium. For the assay, 5 × 105 T cells 10000

were cultured with 10.6 irradiated syngeneic thymic
cells as antigen presenting cells (30 Grey, Phillips without adjuvant with adjuvant
RT, filter 1.7 A1, 100 Ku, 8 mA) and 15 /ag/ml of
30000 1
peptide in a total volume of 0.2 rnl in flat-bottom F" no peptide
(b)
microtiter tissue culture plates (Nunclon, Roskilde, i 115-131 peptide
Denmark). The cells were then exposed to 18.5 kBq [] 45-69 peptide I
-.-~-~20000"
of tritiated deoxythymidine ([3H]TdR) for the last
18 h of a 5-day culture period. Finally, the cells were
harvested by filtration through fiberglass discs using
~--~I0000-
a multiharvester (Skatron, Lierbyen, Norway) and
the amount of [3H]TdR uptake was measured using
a liquid scintillation counter (LKB, Wallac, Turku,
Finland). Data were expressed as mean cpm of trip-
licates samples ( + standard deviation (S.D.)). Prob- 0.1 [.tg l~ag long
Dose of immunization
ability values based on Student's test for replicates
in the presence of peptide compared to control with- 30000 • (c)
out antigens < 0.05 were taken as significant. [] no peptide

~ 20000" [] 45-69 peptide

3. Results

Three independent experiments were performed m~ 10000"

in triplicate.

3.1. Immunogenicity of the 45-69 without adjuvant

Day 3 Day 7 Day 14

Groups of three Lou M rats were subcutaneously
immunized with 10 #g of 45-69 peptide in PBS or
in the presence of aluminium hydroxide at day 0, at
day 7 and killed 7 days later. We analyzed the pro-
liferative response of T-cell-enriched lymph node
and spleen cells. The 45-69 peptide (15/~g/ml) was
able in vitro to induce a significant proliferation of
lymph node cells even when the immunization was
done in the absence of any adjuvant (Fig. 2a). In
contrast, the spleen cell response was low even for
animals receiving peptide in the presence of adjuvant
(data not shown).
We further evaluated the minimal dose of 45-69
peptide required for immunization in order to obtain
a significant lymph node cell proliferative response
Fig. 2. Immunogenicity of the 45-69 peptide. (a) In vitro prolif-
erative response of lymph node T lymphocytes in the absence of
any peptide or with 15/~g/ml of 45-69 peptide after immuniza-
tion with 10 #g of 45-69 peptide in PBS or in the presence of
aluminium hydroxide. (b) In vitro proliferative response of T
lymphocytes from rats immunized with 0.1, l or 10 #g of 45-69
peptide in PB S and cultured in the absence of any peptide or with
15 /~g/ml of 45-69 peptide or 115-131 peptide as control (c)
In vitro proliferative response o f T lymphocytes at days 3, 7 and
14 after injection of 1 #g of 45-69 peptide.
in vitro. Lou M rats received two injections of 0.1,
1 or 10 # g of 45-69 peptide. Our results indicate that
1/tg is the minimal dose required to obtain a signif-
icant T-cell level of restimulation. The immunization
with 10 #g of 45-69 peptide did not improve the
140 F. Rouaix el al. / lnmnmophamlacolog:v 28 (1994) 137-143
level of restimulation (Fig. 2b). Then, we evaluated
the minimal period of time necessary after priming
to obtain a proliferative response. Animals were in-
jected with 1 #g of 4 5 - 6 9 peptide and killed 3, 7 or
14 days later; in the latter case, rats were injected
twice. A delay of 7 days after priming with 1 #g of
peptide is necessary to obtain a significant level of
T-cell proliferation (Fig. 2c).
3.2. The 4 5 - 6 9 peptide does not enhance the anti-
genici O' or immunogenicity o f the l 15-131 peptide
In order to evaluate if the 4 5 - 6 9 peptide induces
a non-specific effect on cells, we measured the
[ 3 H ] T d R uptake of T cells from naive rats cultured
in the presence of 4 5 - 6 9 peptide. Enriched T cells
from naive Lou M rats did not significantly prolif-
erate in vitro in the presence of the 4 5 - 6 9 peptide,
demonstrating the absence of a mitogenic effect on
cells (Fig. 3). We then appreciate if the 4 5 - 6 9 pep-
tide was able to increase the antigenicity of the 115-
131 peptide already known to be poorly antigenic
and immunogenic. T cells from Lou M rats immu-
nized with 10/~g of 115-131 peptide were cultured
in vitro in the presence of a mixture of 115-131 and
4 5 - 6 9 peptides. The 4 5 - 6 9 peptide did not increase
the antigenicity of the 115-131 peptide but slightly
enhanced the background of T-cell proliferation
(Fig. 3).
20000 -
[] no peptide
[] 115-131 l~plidc
[] 45-69 peptidc
[] 45-69+115-131 pcptides
10000 "
× both tandem peptides and T cells from animals in-
z jected with the 115-45 peptide construct were stim-
naive 115-1~1 45-69 + 115-1~1
T cells primed T cells primed T cells primed T cells
Fig 3 The 45-69 peptide does not enhance the antigenicity and
immunogenicity of the uncoupled 115-131 peptide. Groups of
rats were injected with 1 #g of 115-131 or 45-69 peptides, or a
mixture of 1 #g 45-69 and l #g of 115-131 peptides in PBS. The
T-lymphocyte proliferative response was measured in cultures
done in the absence of any peptide (control) or with 45-69 and
115-131 peptides ( 15/~g/ml). Values give [ 3H ]TdR uptake (cpm)
+ S.D. ND, not done; N.S., not significant.
In order to evaluate if the 4 5 - 6 9 peptide can en-
hance the immunogenicity of the 115-131 peptide,
Lou M rats were immunized by coinjection of 4 5 - 6 9
(1 #g) and 115-131 (1, 10 or 100 #g) peptides and
lymph node proliferation was investigated in vitro in
the presence of these peptides. T cells did not pro-
liferate in vitro with the 115-131 peptide when ani-
mals were coinjected with the 4 5 - 6 9 and the 115-
131 peptides (Fig. 3). Surprisingly, while anti-45-69
primed T cells proliferated in vitro with 4 5 - 6 9 pep-
tide, T cells from animals receiving both peptides
weakly responded in vitro to 4 5 - 6 9 peptide. We
obtained the same results using different peptide
ratios (1:1, 1:10, 1:100; data not shown).
3.3. The non-responsiveness to the 115-131 peptide
overcome by tandem association to the 4 5 - 6 9 peptide
We then ascertained whether it is possible to in-
crease the imnmnogenicity of the 115-131 peptide
by association with the 4 5 - 6 9 peptide tandemly syn-
thesized at the N-terminal or C-terminal end of the
115-131 peptide. These constructs were named 4 5 -
115 and 115-45 peptides, respectively. Lou M rats
were subcutaneously injected with 1 /~g of 4 5 - 6 9 or
115-131 peptides or with 1 #g o f 4 5 - 1 1 5 or 115-45
tandem peptides and T cells were tested in vitro
toward these peptides and tandem peptides. T cells
from animals injected with the 115-131 peptide were
stimulated by the 45-115 peptide construct and
weakly by the 115-45 construct. T cells from ani-
mals injected with the 4 5 - 6 9 peptide were stimu-
lated by the 4 5 - 6 9 peptide and, to a lesser extent by'
both peptidic constructs (Fig. 4a).
Moreover, T cells from animals injected with the
4 5 - 1 1 5 peptidic construct were only stimulated by
ulated by all peptides: 115-45, 45-115 tandem pep-
45-69
tides and, to a lesser extent 115-131 and 4 5 - 6 9
peptides. Our results first indicate that T cells from
animals immunized with either 45-115 or 115-45
peptidic constructs were stimulated in vitro by both
these constructs (Fig 4b). More interestingly, the
115-45 peptide appears to be the best construct for
immunization since T cells from those animals re-
spond to both tandem peptides and also to 115-131
and 4 5 - 6 9 peptides alone. By contrast, in vitro, the
 F. Rouaix et al. / Immunopharmacology 28 (1994) 137-143 141

20000 [] no peptide (a) 4. Discussion

[] 115-131
[] 115-45 The present report supports the idea that a T h
[] 45-115 peptide, highly immunogenic in certain conditions,
[] 45-69 ~
v
could enhance by association the weak immuno-
genicity of an other epitope. We first analyzed the
IOOO0 immunogenicity of a peptide derived from the nef
protein of HIV (Allan et al., 1985), i.e., the 45-69
peptide already described as a helper T-cell epitope
in our laboratory (Estaquier et al., 1992). In this re-
port, we demonstrated that the 45-69 peptide is
highly immunogenic in Lou M rats and this in the
absence of any adjuvant. Moreover, this peptide can
115-131 peptide 45-69 peptide be administered in one low dose (1/~g in one dose
Immunizationwith injection) and this immunogenicity is unrelated to a
mitogenic effect.
50000 [] no peptide Marguerite et al. (1992) demonstrated that the
(b)
[] 115-131 O4
115-131 peptide is poorly antigenic and immuno-
[] 115-45 genic and then, the immunization with MAP-8 or
~- 4O0OO [] 45-115 MAP-2 constructs of the 115-131 peptide was nec-
v
[] 45-69 essary to obtain specific antibody and T-cell
£
30000- responses. However, one of the limitations is that
# v

these MAP were administered with CFA and IFA,

~, 20000- to
I4~//.4gl ;,
10000-
i
115-45 peptide 45-115 peptide
Immunization with
Fig. 4. Non-responsiveness to the 115-131 peptide overcome by
tandem association to the 45-69 peptide. (a) Lou M rats were
immunized with peptides alone (i.e., 45-69 or 115-131) or (b)
tandem peptides (i.e., 115-45 or 45-115). Lou M rats were im-
munized with peptides and lymph node T cells were cultured in
vitro in the presence of peptides. Results are expressed as
[3H]TdR uptake (cpm) + S.D.N.S., not significant.
45-115 peptide was more efficient for inducing the
proliferation of T cells whatever the peptide or tan-
dem peptides used for the immunization. So, 115-45
and 45-115 tandem peptides possess distinct prop-
erties of antigenicity and immunogenicity but both
allow to reveal the existence of a 115-131 specific
T-cell response normally undetectable using 115-
131 peptide alone.
which are unsuitable in man.
One promising approach in vaccine, was recently
proposed by the association of a peptide to a defined
high efficient T h cell epitope using either constructs
of T- and B-characterized epitopes of the same
pathogen of Chlamydia trachomatis (Su et al., 1992)
or a foreign helper T-cell determinant associated to
epitopes of circumsporozoite protein of Plasmodium
falciparum or foot-and-mooth disease virus (Good
etal., 1987; Francis etal., 1987). We previously
demonstrated that the 45-69 peptide was a highly
efficient T-helper peptide and we decided to evalu-
ate if it could overcome, by a tandem association, the
non-responsiveness toward the 115-131 peptide
(rSm28-GST) of Schistosoma mansoni, in the ab-
sence of any adjuvant.
We first demonstrated that 115-131 primed T cells
did not respond in vitro to a mixture of 115-131 and
45-69 peptides, suggesting that the presence of
45-69 peptide does not enhance the antigenicity of
115-131 peptide. In addition, the 45-69 mixed with
the 115-131 peptide for injection into rats does not
act as a potent adjuvant since it did not improve the
immunogenicity of the 115-131 peptide, regardless
of the dose used.
142 F. Rouaix et al. / hwnunopharmacology 28 f1994) 137-143
Using peptidic constructs (i.e., 115-45 and 45-
115 peptides) obtained by tandem synthesis of these
peptides, we observed that T cells from animals im-
munized with the 115-45 tandem peptide are able to
proliferate, not only with the 115-45 and 45-115
tandem peptides but also with peptides alone. The
orientation of peptide inside the construct seems to
be of importance since T cells from animals injected
with the 45-115 peptide construct were only able to
proliferate in vitro in the presence of the 115-45 or
the 45-115 peptide constructs and not with free pep-
tides. Surprisingly, 115-131 peptide which was not
antigenic with 115-131 or MAP-8 primed T cells
appears to be antigenic with 115-45 peptide primed
T cells. One possible explanation is that the immu-
nization with 115-45 peptide generated specific T
cells with TcR of higher affinity for the 115-131
peptide than those generated when the 115-131 pep-
tide or MAP-8 were injected. Wan et al. (1986) dem-
onstrated that a peptide, unable to stimulate T cells
when the immunization was done with the peptide
alone, was able to induce antigen specific prolifera-
tion of primed T cells when the immunization was
performed only with certain peptide-protein conju-
gates. Thus, it could be argued that the type of as-
sociation of a peptide to a protein or a peptide is a
critical factor in conferring activity to this peptide.
Of interest is that the 115-131 peptide appears to
be immunogenic in certain conditions, since the 45-
115 peptide is able to induce in vitro the prolifera-
tion of 115-131 primed T cells. The tandem asso-
ciation could provide appropriate additional amino
acids facilitating epitope binding to the T-cell recep-
tor or, although less probably, to the MHC mol-
ecules of APCs. It could also be speculated that such
a construct can protect 115-131 T-cell sites from
enzymatic degradation during the antigen process-
ing by providing additional enzymatic cleavage tar-
gets. It has also been suggested by others that this
could be attributed to different proteolytic cleavages
during the antigen processing in vivo and in the cul-
ture medium in vitro (Golvano et al., 1990).
It has been reported that copolymerisation of a
B-cell determinant with a T-cell determinant con-
ferred immunogenicity to the B-cell epitope (Leclerc
et al., 1987). However, while extensive studies have
demonstrated the influence on antibody response of
the mutual orientation of synthetic peptide dimers
(Dyrberg et al., 1986; Cox et al., 1988), few studies
concerned T-lymphocyte response. Our results in-
dicate that the mutual orientation of tandem pep-
tides is critical for the antigenicity and the immuno-
genicity of the 115-131 peptide and also reveal the
existence of a specific anti- 115-131 T-cell response
normally undetectable using the 115-131 peptide
alone or other constructs (MAP-8 or 115-131-
OVA).
Moreover, it is very obvious from our results that
peptides, injected in combination (45-69 + 115-131 )
or tandemly associated (i.e., 45-115 or 115-45), ob-
viated the generation ofT-cell response to the 45-69
peptide. This reduction of T-cell proliferation after
immunization and in vitro restimulation with antigen
evokes an anergy mechanism. It has been described
that aqueous antigens induce in vivo tolerance
(Burstein et al., 1992; 1993). These authors demon-
strated that aqueous antigens or hapten-carrier con-
jugates induce tolerance in Th-1 but not Th-2 helper
cells. Such findings have a number of significant
implications. A strategy for inducing a selective tol-
erance may be applicable to the vaccination for
avoiding deleterious T- and B-cell reactions against
conventional carriers. Thus, such tandem peptides
may be very useful in order to obtain an immune
response to a poorly immunogenic epitope together
with a dramatical decrease in the response to the
carrier.
Antigen-specific Th-cell responses are MHC class
II restricted (Berzofsky et al., 1987). In the context
of vaccine design, immunogens possessing defined
Th cell epitopes should be recognized by multiple
H-2 alleles to be effective in a genetically diverse
population. The 45-69 peptide has been shown to be
highly immunogenic in different mouse and rat
strains (Estaquier et al., 1992b). It should be inter-
esting to evaluate if such tandems are able to acti-
vate T cells from a wide range of haplotypes, con-
firming the interest of using such constructs in the
context of genetic polymorphism.
In summary, we demonstrated that the 45-69
peptide is highly immunogenic and acts as a potent
stimulatory foreign helper that reveals by tandem
association, the existence of a 115-131 specific T-cell
response normally undetectable using 115-131 pep-
tide of (rSm28-GST) of Schistosoma mansoni. Of
interest is that we did not use a foreign helper T-cell
 F. Rouaix et al. / Immunopharmacology 28 (1994) 137-143
e p i t o p e t h a t c o u l d c r o s s - r e a c t in m a n ( s u c h as o v a l -
b u m i n o r s p e r m w h a l e m y o g l o b i n ; F r a n c i s et al.,
1987), a n d t h a t t h e s e c o n s t r u c t s d o n o t n e c e s s i t a t e
the presence of adjuvant. Such colinear peptide
s y n t h e s i s c o u l d a l s o give h e l p to a m p l i f y a n t i b o d y
r e s p o n s e to a p o o r l y i m m u n o g e n i c e p i t o p e . T h i s
will b e o f i m p o r t a n c e to d e v e l o p n e w v a c c i n a l
approaches.
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