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Previous Article | Next Article 
Infection and Immunity, February 2001, p. 1202-1206, Vol. 69, No. 2
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.1202-1206.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
DNA Immunization by Plasmodium
falciparum Liver-Stage Antigen 3 Induces Protection against
Plasmodium yoelii Sporozoite Challenge
Jean-Pierre
Sauzet,
Blanca-Liliana
Perlaza,
Karima
Brahimi,
Pierre
Daubersies, and
Pierre
Druilhe*
Laboratoire de Parasitologie Médicale,
Institut Pasteur, 75724 Paris Cedex 15, France
Received 21 August 2000/Returned for modification 26 September
2000/Accepted 6 November 2000
 |
ABSTRACT |
DNA-based immunization of mice by Plasmodium falciparum
liver-stage antigen 3 (PfLSA3), a novel highly conserved P. falciparum preerythrocytic antigen, was evaluated. Animals
developed a dominant Th1 immune response (high gamma interferon T-cell
responses and predominance of immunoglobulin G2a) to each of three
recombinant proteins spanning the molecule. We have exploited the
immunological cross-reactivity of PfLSA3 with its putative homologue on
sporozoites of the rodent parasite Plasmodium yoelii, and
we show for the first time that responses induced by PfLSA3 in mice
significantly protect against a heterologous challenge by P. yoelii sporozoites. These results support a significant effect of
DNA-induced immune responses on preerythrocytic stages.
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TEXT |
To date, the only means of inducing
strong, sterile protection against preerythrocytic stages of
Plasmodium falciparum in humans has been through repeated
immunizations with nonlethally irradiated sporozoites (4).
Therefore, efforts have focused on the identification of subunit
vaccines able to induce similar protection. Two important limitations
of the vaccine candidates proposed to date are their substantial
polymorphism within immunologically important regions and their
suboptimal immunogenicity. We previously identified several vaccine
candidates which might overcome these limitations. P. falciparum liver-stage antigen 3 (PfLSA3) is the first
preerythrocytic antigen selected by screening based on differences in
the immune response between protected and nonprotected volunteers immunized with irradiated sporozoites. PfLSA3 is a 200-kDa protein, expressed in both sporozoite and liver stages, and is highly conserved among parasites from various geographical regions (70 isolates have
been tested so far). PfLSA3 displays promising antigenic, immunogenic,
and protective properties in Aotus monkeys (11) and chimpanzees (1-3). Cross-reactivity was observed at
the immunological level with Plasmodium yoelii but not with
P. berghei, which suggests the existence of an as yet
uncharacterized homologous antigen in the rodent parasite P. yoelii (K. Brahimi et al., unpublished data).
In this study, we set out to assess the immunogenicity and protective
efficacy of a DNA vaccine encoding PfLSA3 in mice. Immunization with
antigen-encoding plasmid DNAs offers a number of advantages over
classical immunization strategies and has been used to induce immune
responses to infectious diseases in several animal models (9). The fact that strong T-cell responses are often
elicited indicates that DNA constructs might be very suitable for
vaccination against malaria preerythrocytic stages (4). We
show here that PfLSA3 DNA immunization induces potent Th1 responses
with protective properties and confers protection against heterologous
P. yoelii challenge in mice. This confirms the interest
shown in PfLSA3 as a DNA vaccine candidate.
Plasmids.
The PfLSA3 plasmid pVR2555, referred to as pV AB,
was constructed by cloning a PCR fragment corresponding to the nearly
full-length LSA3 gene (spanning over 3,920 bp) from P. falciparum clone 3D7 in plasmid pVR1020, licensed by Vical.
Construct pV AD corresponds to the N-terminal half of the gene (1,930 bp spanning the nonrepeat region NR-A and the repeat regions R1 and
R2), whereas construct pV DC corresponds to the nonrepeat region NR-B
(2,145 bp) of the LSA3 gene (Fig. 1).
Supercoiled plasmids were produced in Escherichia coli and
purified with EndoFree Plasmid Giga kits (Qiagen, Hilden, Germany). The
endotoxin concentration was between 5 and 50 EU/mg of DNA, as
determined by the Limulus amebocyte lysate test
(BioWhittaker).
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FIG. 1.
Location in the lsa3 gene of the various
peptide and DNA sequences used in this study. R1, R2, and R3 represent
repeat regions, and NR-A, NR-B, and NR-C represent nonrepeat regions.
DG729, NN, and PC sequences were expressed as either GST-fused or
His-tagged recombinant proteins (see Materials and Methods).
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Antigens.
The PfLSA3 recombinant proteins NN (LSA3-NN) and PC
(LSA3-PC) were produced as glutathione S-transferase (GST)
fusion proteins in the pGEX vector as previously described
(3). The recombinant 729-H protein corresponds to the
previously reported DG729 sequence (3) produced as a
histidine (His)-tailed fusion protein using the plasmid pTcr His as
recommended by the manufacturer (BioWhittaker) (Fig. 1)
(3).
Two groups of 18 BALB/c and C3H mice (7- to 10-week-old female mice
purchased from CERJ, Le Genest St. Isle, France) were immunized three
times with 100 µg of pV AB intramuscularly (i.m.) at 3-week intervals
(each time, the dose was equally distributed between the two tibialis
anterior muscles). Sera were collected 3 weeks after each injection and
tested against each of the three recombinant proteins using a standard
enzyme-linked immunosorbent assay (ELISA) procedure (1).
Antibodies became detectable after two injections both in BALB/c mice
(Fig. 2) and in C3H mice (data not
shown). The mean ELISA titers increased after further immunizations (Fig. 2A), and the proportion of responding mice reached 100%. Antibodies (Abs) to NRI and NRII peptides derived from the nonrepeat region of LSA3-729 (2) became detectable only after the
third immunization. A high proportion of mice (94% of C3H mice and
78% of BALB/c mice) produced Abs mostly to the repeat region (i.e., LSA3-NN). Ab titers were higher to LSA3-NN and LSA3-729 than to LSA3-PC
(Fig. 2). Immunofluorescent-antibody assays performed using P. yoelii and P. falciparum sporozoites showed that Abs elicited by PfLSA3 DNA immunization specifically reacted with the
surface of both sporozoite species at similar levels (titers of 1:50 to
1:200). By contrast, preliminary experiments indicated that Abs
elicited by protein immunization recognized the native protein better
than did those obtained by DNA immunization, even after four injections
of DNA (data not shown). This would be consistent with previous
observations that, in contrast to protein immunization, increasing the
number of DNA injections does not increase Ab avidity, suggesting
little or no affinity maturation of specific Abs during the course of
DNA immunization (8).
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FIG. 2.
Ab responses in LSA3 DNA immune mice are directed to
different regions of LSA3. (A) A group of 18 BALB/c mice were immunized
i.m. with 100 µg of pV AB, and Ab responses to recombinant proteins
LSA3-729, LSA-NN, and LSA-PC and to peptides in both repeat and
nonrepeat regions of LSA3 were monitored after several injections.
Results represent the mean ELISA ratio compared to that obtained with
sera collected before immunizations. (B) 729-H-specific antibodies in
BALB/c LSA3 DNA immune mice contain predominantly IgG2a. Isotypes of
729-H-specific antibodies in the same sera were analyzed by ELISA.
Results represent the mean ELISA ratio compared to that in sera
collected before immunizations. Reported P values are those
obtained after comparative analysis of IgG1 to IgG2a at each date using
Student's test.
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Although DNA immunization induced Abs of all subclasses, the
immunoglobulin G2a (IgG2a) isotype was predominant with respect to IgG1
and IgG2b in a statistically significant manner both in BALB/c (Fig.
2B) and in C3H (data not shown) immunized mice. IgG3 (particularly in
C3H mice) and IgM were found at low levels. Over the course of the
immunization, the level of IgG2a (an isotype promoted by Th1-like
responses) increased more than that of IgG1 (an isotype facilitated by
Th2-like responses) (P < 0.05), and the differences
increased with the number of immunizations (Fig. 2B).
At the T-cell level, major differences were observed between
proliferative and gamma interferon (IFN-
) responses (Fig.
3). Low proliferative responses were
detected in DNA-immunized BALB/c mice (six of eight mice) only after in
vitro stimulation of spleen cells with recombinant protein PC (Fig.
3A). No such responses were detectable in C3H mice. Surprisingly, in
both C3H and BALB/c mice, high IFN- secretion was consistently
detected (100% of mice), both in the supernatants of stimulated cells
(Fig. 3B) and by enzyme-linked immunospot assay (Fig. 3C). IFN-
titration in supernatants from 48-h spleen cell proliferation cultures
(using a pair of capture and detection rat anti-mouse IFN-
monoclonal Abs from Becton Dickinson, San Diego, Calif.) showed high to
very high levels of the cytokine in each mouse in response to at least one of the three antigens tested. The highest levels were obtained in
C3H mice in response to LSA3-NN (up to 900 IU/ml). By contrast, responses elicited in BALB/c mice were lower (highest titer at 380 IU/ml) and directed to all regions of the protein. These results were
further confirmed using the sensitive ELISPOT assay with fresh,
unstimulated spleen cells incubated with each of the three LSA3
recombinant proteins for 40 h. Activated cells were found in all
mice tested, and a high frequency was found in C3H mice in response to
NN (Fig. 3C).
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FIG. 3.
T-lymphocyte responses in DNA LSA3-immunized mice. C3H
and BALB/c mice were injected three times i.m. with 100 µg of pV AB
DNA construct. At 1 month after the last injection, their spleens were
removed and T-cell responses to selected regions of LSA3 were studied.
Results of lymphoproliferation (A), IFN- titration in supernatants
(B), and ELISPOT assays for IFN- (C) of eight individually tested
C3H ( ) and BALB/c ( ) mice are represented. Unstimulated cells did
not produce detectable amounts of IFN-. The mean values obtained in
wells with GST alone as negative control were as follows: stimulation
index (S.I.), 0.99 ± 0.23; IFN-, 1.31 ± 1.49; and
ELISPOT, 21 ± 18.4 (BALB/c mice); and S.I., 0.6 ± 0.17;
IFN-, 13.9 ± 10.1; and ELISPOT, 5.6 ± 2.6 (C3H mice).
SFC, spot-forming cell; PBMC, peripheral blood mononuclear cells.
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The results obtained by DNA immunization contrast with those previously
obtained using LSA3 recombinant proteins with various adjuvants or by
lipopeptide immunization (reference 1 and our unpublished
observations). Indeed, C3H mice immunized with peptides or proteins
showed consistent proliferative responses, which was not the case for
those given DNA immunizations. When recombinant proteins were used, the
highest proliferative responses and IFN- were directed mainly
against LSA3-PC and infrequently against LSA3-NN or LSA3-729, whereas
DNA-induced responses were restricted to the repeat region NN.
Similarly, protein-based immunization induced Ab responses mainly to PC
rather than to NN, which was the main target of DNA-induced Ab
responses. It is noteworthy that naturally acquired Abs to LSA3 in
malaria-exposed individuals have a pattern of recognition of LSA3
similar to that of Abs in DNA-immunized mice (B. L. Perlaza, J. P. Sauzet, A. Toure-Balde, K. Brahini, P. Daubersies, G. P. Corradin, and
P. Druilhe, submitted for publication). This similarity might be the
result of a correctly folded NN region by in vivo DNA-transfected
cells. Finally, protein immunization generated a rapid and equally
distributed isotype profile, suggesting a mixed Th1-Th2 pattern of
response. In contrast, our results indicate the ability of LSA3 DNA
immunization to channel mouse responses toward epitopes preferentially
recognized by humans. The response generated is directed toward a
Th1-biased type of immune response, as supported by the fact that the
IgG2a/IgG1 ratio and the frequency of LSA3-specific peripheral
IFN--secreting T cells evolved in parallel (Fig.
4).
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FIG. 4.
Correlation between the IgG2a/IgG1 ratio and the
frequency of IFN--producing cells in LSA3 DNA-immunized mice. The
IgG2a/IgG1 ratio was found to be correlated with the frequency of
IFN--producing spleen cells specific for LSA3-729 ( ) and LSA3-PC
() determined by an ELISPOT assay in both BALB/c and C3H mice
immunized with LSA3 DNA. SFC, spot-forming cell.
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IFN- secretion in response to DNA immunization was found to be high,
and this cytokine has been proposed to be a major effector of defense
mechanisms against the liver stages of the parasite (5, 6, 10,
12).
To study the protective capacities of the PfLSA3 DNA vaccine
formulation, we exploited the interspecies B- and T-cell
cross-reactivity of LSA3-specific immune responses between P. falciparum and P. yoelii (Brahimi et al., unpublished).
Mice immunized with four injections of pV AB were challenged by
intravenous inoculation with 150 P. yoelii sporozoites
(clone 1.1, derived from the 17XNL strain), dissected from infected
salivary glands. All five C3H mice and five of seven BALB/c mice (71%)
were protected against sporozoite challenge (Table
1). Protection was defined either as the
complete absence of blood-stage parasitemia from days 4 to 14 postchallenge (two of five C3H mice [40%] and one of seven BALB/c
mice [14%]) or as a delayed onset of parasitemia compared to control
groups. In the latter case, the influence of the parasite load in the
liver on the subsequent course of blood-stage parasitemia has been
estimated in naive mice as a function of the size of the sporozoite
inoculum; hence, a 1-, 2-, or 3-day-delayed onset of parasitemia would
represent an 80, 96, and 99.2% reduction in parasite burden,
respectively (Table 2). Of seven LSA3 DNA immune BALB/c mice, four (57%) achieved such partial protection, with
three mice being delayed by 2 days and one mouse being delayed by 1 day. Similarly, three of five C3H mice (60%) were partially protected,
with one mouse even having a 3-day delay and the two others having a
1-day delay. In contrast, the onset of blood parasitemia in all eight
nonimmunized control mice, as in all eight mice immunized with control
DNA (influenza virus gene cloned in the same Vical vector), always
occurred on the same day (day 4 postchallenge) in parallel experiments
and with the same sporozoite preparation (Table 1). Taken together, our
results indicate that PfLSA3 DNA-based immunization induces a
significant, although not always complete, protective effect on
parasite preerythrocytic development.
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TABLE 2.
Influence of parasite load in the liver on the subsequent
course of blood-stage parasitemia estimated in naive mice as a function
of the size of the sporozoite inoculuma
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To try to identify the regions of PfLSA3 responsible for the induced
protection against P. yoelii, C3H mice were immunized with
DNA constructs encoding either the N-terminal (pV AD) or C-terminal (pV
DC) region of the protein. As shown in Fig. 4, pV AD induced protection
in two of three mice, with one of them being totally protected. It is
noteworthy that pV AD encodes the LSA3-729 region, which contains
several B- and T-cell epitopes with protective potential in chimpanzees
(2). The three mice immunized with pV DC were partially
protected, since parasitemia was only delayed in all three. It is
unclear whether one or both regions are important for full protection.
The results described in this report show that a naked DNA encoding the
PfLSA3 antigen is strongly immunogenic in mice, inducing both B- and
T-cell responses to different subregions of the molecule. Furthermore,
these responses translate into a biological effect. These results
compare favorably with DNA vaccination against a wide range of
infectious diseases (9), including mice immunized by a
plasmid DNA encoding the P. yoelii circumsporozoite protein (7). The fact that the highest levels of protection were
achieved in C3H mice might be related to the ability of this mouse
strain to secrete high IFN- levels in response to the repeat region of LSA3. Although total protection was not obtained in all the mice, it
must be noted that protection was obtained in a very demanding
heterologous system, where the immunogen and challenge sporozoites are
from different Plasmodium species. This differs from other
immunization experiments such as those with circumsporozoite protein,
where protection was demonstrated against homologous species and
homologous strain challenge. Finally, the variability in protection in
individual mice might be related to the surprisingly large variations
in the levels of immune responses found among inbred mice.
The selection of LSA3 as a new P. falciparum liver-stage
molecular target is based on a strong rationale; antigenicity studies in humans, immunogenicity data in mice and non-human primates, and
sequence conservation have further increased the interest in this
molecule. The present results obtained by genetic immunization lend
further support to its vaccine potential.
| |
ACKNOWLEDGMENTS |
This work was supported by European Commission Inco-DC grant
98-0387 and WHO-Tdr grant 940023.
We thank Aventis-Pasteur for assistance and encouragement and for the
gift of the control plasmid used in these experiments, Georges Snounou
for critical review of the manuscript, and Nicolas Puchot for excellent
technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Parasitologie Médicale, Institut Pasteur, 28 Rue du Docteur Roux,
75724 Paris Cedex 15, France. Phone: 33 1 45 68 85 78. Fax: 33 1 45 68 86 40. E-mail: druilhe{at}pasteur.fr.
Editor:
W. A. Petri Jr.
| |
REFERENCES |
| 1.
|
BenMohamed, L.,
H. Gras-Masse,
A. Tartar,
P. Daubersies,
K. Brahimi,
M. Bossus,
A. Thomas, and P. Druilhe.
1997.
Lipopeptide immunization without adjuvant induces potent and long-lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees.
Eur. J. Immunol.
27:1242-1253[Medline].
|
| 2.
|
BenMohamed, L.,
A. Thomas,
M. Bossus,
K. Brahimi,
J. Wubben,
H. Gras-Masse, and P. Druilhe.
2000.
High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new plasmodium falciparum pre-erythrocytic molecules.
Vaccine
18:2843-2855[CrossRef][Medline].
|
| 3.
|
Daubersies, P.,
A. W. Thomas,
P. Millet,
K. Brahimi,
J. A. M. Langermans,
B. Ollomo,
L. BenMohamed,
B. Slierendregt,
W. Eling,
C. Guérin-Marchand,
A. Van Belkum,
G. Dubreuil,
J. F. G. M. Meis,
S. Cayphas,
J. Cohen,
H. Gras-Masse, and P. Druilhe.
2000.
Protection against P. falciparum malaria in chimpanzees by immunization with a conserved pre-erythrocytic antigen, LSA3.
Nat. Med.
6:1258-1263[CrossRef][Medline].
|
| 4.
|
Druilhe, P. L.,
L. Renia, and D. A. Fidock.
1998.
Immunity to liver stages, p. 513-543.
In
I. W. Sherman (ed.), Malaria: parasite biology, pathogenesis, and protection. ASM Press, Washington, D.C.
|
| 5.
|
Ferreira, A.,
L. Schofield,
V. Enea,
H. Schellekens,
P. van der Meide,
W. E. Collins,
R. S. Nussenzweig, and V. Nussenzweig.
1986.
Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon.
Science
232:881-884[Abstract/Free Full Text].
|
| 6.
|
Hoffman, S. L.,
J. M. Crutcher,
S. K. Puri,
A. A. Ansari,
F. Villinger,
E. D. Franke,
P. P. Singh,
F. Finkelman,
M. K. Gately,
G. P. Dutta, and M. Sedegah.
1997.
Sterile protection of monkeys against malaria after administration of interleukin-12.
Nat. Med.
3:80-83[CrossRef][Medline].
|
| 7.
|
Hoffman, S. L.,
M. Sedegah, and R. C. Hedstrom.
1994.
Protection against malaria by immunization with a Plasmodium yoelii circumsporozoite protein nucleic acid vaccine.
Vaccine
12:1529-1533[CrossRef][Medline].
|
| 8.
|
Kang, Y.,
P. A. Calvo,
T. M. Daly, and C. A. Long.
1998.
Comparison of humoral immune responses elicited by DNA and protein vaccines based on merozoite surface protein-1 from Plasmodium yoelii, a rodent malaria parasite.
J. Immunol.
161:4211-4219[Abstract/Free Full Text].
|
| 9.
|
Lai, W. C., and M. Bennett.
1998.
DNA vaccines.
Crit. Rev. Immunol.
18:449-484[Medline].
|
| 10.
|
Maheshwari, R. K.,
C. W. Czarniecki,
G. P. Dutta,
S. K. Puri,
B. N. Dhawan, and R. M. Friedman.
1986.
Recombinant human gamma interferon inhibits simian malaria.
Infect. Immun.
53:628-630[Abstract/Free Full Text].
|
| 11.
|
Perlaza, B. L.,
M. Arevalo-Herrera,
K. Brahimi,
G. Quintero,
J. C. Palomino,
H. Gras-Masse,
A. Tartar,
P. Druilhe, and S. Herrera.
1998.
Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys.
Infect. Immun.
66:3423-3428[Abstract/Free Full Text].
|
| 12.
|
Sedegah, M.,
F. Finkelman, and S. L. Hoffman.
1994.
Interleukin 12 induction of interferon gamma-dependent protection against malaria.
Proc. Natl. Acad. Sci. USA
91:10700-10702[Abstract/Free Full Text].
|
Infection and Immunity, February 2001, p. 1202-1206, Vol. 69, No. 2
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.1202-1206.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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