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Cutting Edge |
* Michael Heidelberger Division of Immunology, Department of Pathology, and
Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunization of volunteers in Africa and the USA with the CS-based RTS,S vaccine protected 50–70% of the vaccinees against infection for a very short period of time (3, 4). Although in all CS vaccine trials there was a correlation between the degree of protection and Ab titers to CS repeats (3, 5, 6), a role for cell-mediated immunity could not be excluded.
Unfortunately, there is no simple laboratory method to evaluate the neutralizing capacity of Abs to P. falciparum sporozoites. These parasites do not infect common laboratory animals, and do not develop in hepatoma cell lines in vitro. Taking advantage of the easily quantifiable infectivity of rodent malaria Plasmodium berghei sporozoites, we constructed a recombinant P. berghei parasite expressing a hybrid P. berghei/P. falciparum CS, and used it to study the efficacy of a human pre-erythrocytic P. falciparum CS vaccine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The integration plasmid contains the P. berghei
NK65 CS coding sequence and sufficient DNA 5' and 3'
of the gene and, as a selectable marker, a variant form of human
dihydrofolate reductase gene (hDHFR) that confers resistance
to the antifolate drug WR99210 (7). Two unique restriction
sites (SpeI and XhoI) were introduced by overlap
PCR to flank the sequence of the CS repeat region (Fig. 1
A). The following
oligonucleotides were used: PbXbaI,
5'-CCTCTAGATTAATAACTTGGGTTTTTGC-3'; PbSpeI,
5'-CTCCCGAAGGAACTAGTAAAAAAAATGAG-3';
PbSpeIR,
5'-CTCATTTTTTTTACTAGTTCCTTCGGGAG-3'; PbXhoI,
5'-CAAAAATAATAATAATCTCGAGGACGATTCTTATATC-3';
PbXhoIR,
5'-GATATAAGAATCGTCCTCGAGATTATTATTATTTTG-3';
PbEcoRIR,
5'-TTTTAGAATTCCCTTAAATAATATTGTAAAATAG-3'. The resulting
fragment was used to exchange wild-type (wt) CS in the integration
plasmid to give plasmid pCS(sx). The P. falciparum CS
repeat region was amplified from genomic DNA from the 7G8 isolate
(8) using primers that generated SpeI and
XhoI sites: PfSpeI,
5'-GAGATGGAACTAGTAATAACGAAGACAACGAG-3'; PfXhoI,
5'-CTTGGTTCTTCCTCGAGGTTATTATTATTTTTTAC-3'. The CS repeat
region within pCS(sx) was exchanged with the P. falciparum
CS repeat region using the SpeI and XhoI
restriction sites to give plasmid pCS(Pf). Both plasmids were confirmed
by sequencing. Parasite transfection was performed as described
previously (7, 9). Hybrid parasites were selected
in Sprague Dawley rats (The Jackson Laboratory, Bar Harbor, ME)
by i.p. injections with 5 mg kg-1 body weight
WR99210 (Jacobus Pharmaceuticals, Princeton, NJ) on days 1–3. Southern
blots of genomic DNA digested with HindIII/XhoI
or PacI were probed with internal CS
SpeI-XhoI fragments from pCS(sx) and pCS(Pf), and a
BamHI internal fragment of hDHFR. Probes were
labeled using a digoxigenin-ddUTP random priming kit and ECL was
detected as described by manufacturer (Roche, Indianapolis,
IN).
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Parasite cycles were maintained by allowing Anopheles stephensi mosquitoes to feed on SW mice (The Jackson Laboratory) infected by sporozoites or blood stages of Pb(sx) or CS(Pf) parasites. The qualitative and quantitative development of wt and hybrid parasites were analyzed as previously described (7).
CS expression on salivary gland sporozoites dissected 18–20 days post blood meal was assayed by immunofluorescence (IF) and Western Blot using mAb 2A10, specific for P. falciparum CS repeats (10) or mAb 3D11, specific for P. berghei repeats (11). Sporozoite motility was determined by gliding assay (12). Sporozoite in vitro infectivity was assayed in HepG2 hepatoma cells (American Type Culture Collection (ATCC), Manassas, VA) by counting exoerythrocytic form (EEF) at 20–24 h postinvasion in wells stained for IF using mAb 4C9, specific for malaria heat shock protein 70 (13). Sporozoite infectivity in vivo was assayed using real-time PCR to measure liver stage parasite 18S rRNA at 40–42 h after exposure of mice to infected mosquitoes (14).
Sporozoite neutralization assays
In vitro neutralization assays were conducted by adding 10-fold dilutions of mAb 2A10 or mAb 3D11 to HepG2 cultures 1 h before addition of sporozoites. Percentage of inhibition was calculated by comparing the number of EEF in cultures with or without Ab at 20–24 h postinfection. Ab-mediated neutralization in vivo was determined by i.v. injection of 100 µg of mAb 3D11 or mAb 2A10 into groups of three C57BL/6 mice. After 30 min, mice were challenged by exposure to 8–18 infective bites of CS(Pf)- or CS(sx)-infected mosquitoes. The presence of parasite rRNA in the liver was determined by real-time PCR.
(T1B)4 multiple Ag peptide (MAP)-immunized mice
A (T1B)4 MAP vaccine, recently tested in phase I trials (15), was used to immunize C57BL/10 (The Jackson Laboratory). Mice were challenged 2 wk after the third s.c. injection of 50 µg MAP formulated with 10 µg QS21, a purified saponin adjuvant. Anti-repeat ELISA titers at the time of challenge were 6 x 105 geometric mean titer.
In passive transfer experiments, 250 µl of immune sera obtained from MAP-immunized mice was injected i.v. 1 h before challenge by infected mosquitoes. Anti-repeat Ab levels in the recipients was 81,920 geometric mean titer. For CD4+ T cell depletion, 250 µg of anti-CD4 Ab (mAb GK 1.5; ATCC) was injected i.p. on days -4, -3, and -2 before challenge on day 0 by infected mosquitoes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The P. berghei CS knockout
(CS-) locus (9) was
targeted with the two plasmids containing P. berghei CS with
either wt (CS(sx)) or P. falciparum (CS(Pf)) repeat region
sequences (Fig. 1
, A and B). Southern blot
analysis of DNA from blood of CS(Pf)- or CS(sx)-infected rats confirmed
that in each transgenic clone a single copy of the construct had
integrated on the chromosome (Fig. 1C).
The development of the CS- parasite
is arrested in the mosquito at the oocyst stage (9). In
contrast, CS(sx) and CS(Pf) parasites were found in oocysts and
salivary glands in A. stephensi mosquitoes (Table I). Therefore, the introduction of the
recombinant P. berghei CS complemented the
CS- phenotype. There were no
significant differences in infectivity of recombinant or wt parasites,
as determined by percentage of midgut infections and mean number of
oocysts per mosquito. However, we noted a reduction in CS(Pf) salivary
gland infectivity and in the number of sporozoites per salivary
gland.
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A).
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When cross-linked by anti-repeat Abs, CS protein is shed as a
long precipitate at the posterior end of the parasite, termed the
circumsporozoite precipitin (CSP) reaction (16). The
CS(Pf) sporozoites gave positive CSP reactions when incubated with mAb
2A10, but not mAb 3D11 (Fig. 2B). Conversely, the CS(sx) and
wt parasites developed CSP reactions only when incubated with mAb
3D11.
Strong CSP reactions were also obtained when CS(Pf) sporozoites were
incubated with sera from human volunteers immunized with a P.
falciparum repeat peptide vaccine, (T1B)4
MAP (Fig. 2C). Reactivity of human immune sera with CS(Pf)
sporozoites was comparable to that observed with viable P.
falciparum sporozoites (15). The CSP reactions were
specific, as the immune serum did not react with wt parasites (Fig. 2C) or CS(sx) sporozoites (data not shown). Serum of a
seronegative vaccinee (volunteer 4) and normal human serum did not
react with CS(Pf) sporozoites.
The CS(Pf) and CS(sx) sporozoites were found to invade and develop
within HepG2 cells comparable to wt sporozoites (Table II). The addition of mAb 2A10 inhibited
CS(Pf) invasion of hepatoma cells in a dose-dependent manner, but had
no effect on infectivity of CS(sx) or wt sporozoites. mAb 3D11
specifically inhibited CS(sx) and wt sporozoite infectivity, but not
CS(Pf).
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A). Similarly, mAb 3D11
totally inhibited development of CS(sx) parasites, but not CS(Pf),
in vivo.
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We also examined the potential usefulness of hybrid CS(Pf)
parasites for measuring protective efficacy of P. falciparum
CS vaccines. In recent Phase I trials, the (T1B)4
MAP vaccine elicited the highest peptide-induced anti-P.
falciparum repeat Ab titers obtained to date (15). To
assess the efficacy of high levels of MAP-induced anti-repeat Abs in
vivo, C57BL mice were immunized with this vaccine and challenged by
exposure to CS(Pf)- or CS(sx)-infected mosquitoes. No liver stage
parasites were detected in the MAP-immunized mice following CS(Pf)
challenge (Fig. 3B). Protection was specific for P.
falciparum repeats as the immunized mice remained fully
susceptible to CS(sx) parasites. Sterile immunity was also demonstrated
by the total absence of blood stage parasites following CS(Pf)
challenge of a second group of MAP-immunized mice (data not
shown).
C57BL mice can develop both repeat specific CD4+
T cells (17) as well as high Ab titers. To determine the
mechanism of protection, we passively transferred sera from the
MAP-immunized mice to naive recipients and challenged them with
CS(Pf)-infected mosquitoes. All of the recipient mice were protected
(Fig. 3C). In contrast, depletion of
CD4+ T cells before CS(Pf) challenge did not
abrogate immune resistance of MAP-immunized mice (Fig. 3D).
CD4+ T cell-depleted, as well as intact,
immunized mice remained fully susceptible to challenge by
CS(sx)-infected mosquitoes (data not shown). This is the first
demonstration that vaccine-induced polyclonal anti-P.
falciparum CS repeat Abs alone are sufficient to protect against
viable sporozoite challenge in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To address this problem, we constructed a recombinant P. berghei parasite bearing P. falciparum CS repeats to provide a relatively simple tool to measure neutralizing power of anti-repeat Abs specific for P. falciparum CS. To construct this hybrid, we used a CS- parasite (9), since in the CS knockout functional sporozoites would form only if the hybrids complemented the mutant phenotype. It was predicted that hybrid parasites containing P. falciparum CS repeats would exhibit a wt P. berghei phenotype in the mosquito and rodent host, since the presence of distinct CS repeats in other malaria species does not affect sporozoite function or host specificity. Indeed, the infectivity in vitro and in vivo of CS(sx) and CS(Pf) hybrids was very similar to that of wt sporozoites. The surface expression of wt and hybrid CS, as measured by CSP reaction and gliding motility, were also indistinguishable. However, following multiple mosquito passages, CS(Pf) salivary gland infections were lower compared with wt and CS(sx). This may reflect an intrinsic property of the single CS(Pf) clone we obtained.
Our studies in the (T1B)4 MAP-immunized mice suggest that CS(Pf) parasites can be used to screen the potential efficacy of P. falciparum vaccine candidates before clinical trials. Significantly, the P. falciparum MAP-immunized mice were totally protected against challenge with CS(Pf) sporozoites and passive transfer of immune sera from these mice protected naive recipients. Although peptide-induced CD4+ T cells specific for CS can protect against rodent malaria (20), depletion of CD4+ T cells from the MAP-immunized mice did not affect immune resistance. CD8+ CTL epitopes have not been identified in CS repeats. Thus, protection elicited by the P. falciparum MAP vaccine was predominantly Ab mediated. Similar analysis of falciparum vaccines using hybrid parasites and HLA transgenic mice, which have proved useful as humanized models of disease and immunity (21) should also be informative.
The CS(Pf) parasite may have other useful applications. For example, the presence of "nonrepeat" Abs in sera of vaccinees and naturally infected individuals may be revealed by large discrepancies between IF titers obtained using CS(Pf) as compared with wt P. falciparum sporozoites. These Abs and their function have been difficult to demonstrate using peptide or recombinant protein ELISA (6, 22), possibly due to the inability of these Ags to reproduce the structural conformations of nonrepeat regions in the native protein.
The construction of additional hybrids expressing specific regions of the C terminus of P. falciparum CS, which contains the majority of T cell epitopes (20), would facilitate analysis of cellular immunity. The protective role of P. falciparum CD4+ T cell epitopes or CD8+ CTL epitopes, for example, could then be determined by challenge of vaccinated mice. The rodent hybrid parasite model is particularly advantageous for investigations of cellular immune mechanisms in liver and secondary lymphoid organs, studies which are not feasible in human or simian hosts.
Importantly, the CS(Pf) hybrids are antigenically P. falciparum, but they are functionally a rodent malaria, and thus are not a biohazard. CS(Pf) parasites provide a safe, readily available tool for analysis of immune responses to P. falciparum elicited by vaccination or natural infections.
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Acknowledgments |
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Footnotes |
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2 C.P. and G.A.O. contributed equally to this study.
3 Current address: Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden.
4 Current address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115.
5 Address correspondence and reprint requests to Dr. Elizabeth Nardin, Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010. E-mail address: nardie01{at}popmail.med.nyu.edu
6 Abbreviations used in this paper: CS, Circumsporozoite protein; hDHFR, human dihydrofolate reductase gene; CS-, CS knockout; wt, wild type; CSP, Circumsporozoite precipitin; EEF, exoerythrocytic form; IF, immunofluorescence; MAP, multiple Ag peptide.
Received for publication September 20, 2002. Accepted for publication October 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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