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Department of Microbiology and Immunology, Allegheny University of the Health Sciences, Philadelphia, PA 19129
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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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With the widespread development of drug resistance in the parasite and insecticide resistance in the mosquito vectors, malaria remains a major public health problem in the world (20, 21, 22). It is estimated that there are 2 to 3 million deaths due to malaria and at least 300 million cases of clinical malaria each year. However, there are no effective malaria vaccines available. Since malarial parasites change both antigenically and biochemically during their life cycle, an effective malarial vaccine should ideally induce different types of immune responses against multiple targets from different stages in the parasite’s life cycle. DNA vaccines seem to be well suited for this purpose (23, 24, 25) because a DNA vaccine against malaria could be designed to express Ags from both preerythrocytic and erythrocytic stages and thereby elicit immune responses against multiple life cycle stages. Recently, there has been significant progress in DNA vaccine development against preerythrocytic stages of malaria (8, 23, 24, 25). DNA vaccines encoding preerythrocytic Ags from Plasmodium yoelii, including PyCSP, PyHEP17, and PySSP2, have been shown to induce protective CD8+ T cell responses against sporozoite challenge, but not blood-stage challenge, in a genetically restricted manner in rodent models (23, 24, 25). Moreover, combination of the individual DNA vaccines circumvented the genetic restriction of protection against P. yoelii sporozoite challenge (26). In addition, Ag-specific CTL and humoral responses have also been induced in nonhuman primates following immunization with plasmid DNA encoding four Plasmodium falciparum preerythrocytic stage Ags (24). However, DNA vaccination has not yet been successful in inducing humoral immune responses required for protective immunity against a blood-stage challenge with malaria parasites.
One of the most promising vaccine candidates against blood stages of malaria is the merozoite surface protein-1 (MSP-1). Immunization with intact, affinity-purified MSP-1 protein can induce protective responses against challenge in nonhuman primate and rodent models of malarial infection (27, 28, 29, 30), and these responses are primarily directed to the C-terminal region of MSP-1 (31). The C-terminal 19-kDa fragment contains a series of cysteine residues that are conserved among different species of plasmodia infecting humans, primates, and rodents (32), as well as between different isolates of P. falciparum (33, 34, 35). It has been suggested that these cysteine residues are arranged as two putative epidermal growth factor-like domains (36). We have previously shown that, when expressed as a fusion protein with glutathione S-transferase (GST) of Schistosoma japonicum, the C-terminal region of the P. yoelii MSP-1 (designated PyC2) can be recognized by a protective mAb (mAb302) (37) and can induce protective immune responses against an otherwise lethal blood-stage challenge infection in several adjuvant systems (38, 39). These results were confirmed by others (40, 41) and have been extended to the homologous region of P. falciparum in Aotus monkeys (42, 43). In rodents, we have shown that these protective effector functions are predominantly mediated by Abs because the passive transfer of immunized sera or purified Ig can protect naive mice against lethal blood-stage challenge with P. yoelii (39). In addition, no effector role for CD8+ T cells could be identified after challenge infection in immune mice selectively depleted of these cells (39).
In the present study, we have characterized the malaria-specific humoral immune responses induced by a DNA vaccine that contains the DNA sequence encoding GST-PyC2 and compared them to the corresponding protein vaccine, GST-PyC2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-wk-old, male BALB/c ByJ mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and maintained in our American Association for the Accreditation of Laboratory Animal Care-approved facilities. The lethal P. y. yoelii 17XL strain was originally obtained from Dr. John Finerty (National Institutes of Health, Bethesda, MD) and maintained as cloned cryopreserved stabilates. Blood-stage infections were initiated by i.v. injection of 1 x 104 P. yoelii 17XL parasitized erythrocytes (E). Resulting parasitemias were monitored by enumerating parasitized RBC in thin tail-blood smears stained with Leukostat (Fisher Diagnostic, Pittsburgh, PA).
Fusion protein construct and protein isolation
The fusion protein GST-PyC2 was described previously (39). Briefly, the C-terminal region of the P. yoelii 17XL MSP-1 gene was PCR amplified and joined in frame to the 3' end of the S. japonicum GST gene within the pGEX/2T vector. The resultant fusion protein, designated GST-PyC2, was expressed in recombinant Escherichia coli and isolated by affinity chromatography.
Construction of PyMSP-1 based DNA vaccine
The DNA fragment encoding GST-PyC2 was PCR amplified from the pGEX/2T construct, and BamHI sites were added to each end of the PCR product. This PCR product was cloned into a BamHI site in the expression vector VR1020 (Vical), which contains a tissue-type plasminogen activator (TPA) leader sequence, a CMV promoter, a bovine growth hormone poly(A) terminus, and a kanamycin resistance marker. The sequences of the junction region and the insert DNA encoding GST-PyC2 were confirmed by DNA sequencing. This DNA vaccine was designated as V3 and used to immunize mice. The V3 plasmid was prepared in large scale with the Endo-Mega kit (Qiagen, Hilden, Germany). The endotoxin level in the plasmid DNA was <3 EU/ml as measured by the E-TOXATE (Limulus amebocyte lysate) kit (Sigma, St. Louis, MO). Plasmid DNA was then analyzed by agarose gel electrophoresis and quantified by spectrophotometry (OD260/OD280 ratio > 1.8). The isolated DNA was diluted in endotoxin-free PBS and was then ready for immunization.
Transient transfection of Cos 7 cells in vitro
Protein expression from the V3 plasmid was evaluated in vitro by transiently transfecting Cos 7 cells (American Type Culture Collection (ATCC), Manassas, VA) using the LIPOFECTIN Reagent (Life Technologies, Grand Island, NY) following the instructions from the manufacturer. Briefly, Cos 7 cells were grown in DMEM supplemented with 10% FBS overnight. In a 6-well (35-mm) tissue culture plate, 1 x 105 Cos 7 cells in 2 ml complete DMEM were seeded and incubated in a 10% CO2 incubator at 37°C overnight until the cells were 40 to 60% confluent. For each transfection, 4 µg of V3 plasmid DNA was diluted in 100 µl of serum-free medium (OPTI-MEM I reduced serum medium (OPTI-MEM); Life Technologies), and 10 µl of LIPOFECTIN reagent was diluted in OPTI-MEM. The diluted DNA and diluted LIPOFECTIN reagent were combined, mixed gently, and incubated at room temperature for 15 min. The mixture was then added to 0.8 ml OPTI-MEM, mixed gently, and overlaid onto the attached Cos 7 cells. After the cells were incubated in a 10% CO2 incubator for 24 h, 1 ml of complete DMEM containing 20% FBS was added to each well, and the cells were incubated for an additional 36 h. The cells were then washed once with methionine-free DMEM medium (Life Technologies) and incubated in 1 ml of methionine-free DMEM containing 500 uCi [35S] Protein Labeling Mix (NEN Life Science Products, Boston, MA) for another 8 h. Following the labeling, the medium from each of the wells was collected for immunoprecipitation experiments, and the cell pellet was resuspended in solubilization buffer containing 20 mM Tris, 50 mM NaCl, 5 mM EDTA, 1% Brij58, 0.2 mM tosylphenylchloromethylketone, 0.2 mM 1,10 phenanthroline, 2 mM PMSF, 2 mM tosyllysylchloromethylketone, and 1 mM iodoacetamide. The suspension was incubated on ice for 30 min with gentle mixing. Following incubation, the mixture was transferred to a polyallomer centrifuge tube (Beckman Instruments, Palo Alto, CA) and centrifuged at 85,000 x g for 1 h. The supernatant fluids were collected and used as soluble Ags from the pellet of transfected cells. TCA-precipitable proteins (1 x 105 cpm) from the supernatant fluids or cell pellets from transfected cells were used for each reaction in the immunoprecipitation experiments.
Treatment of transfected COS 7 cells with tunicamycin
Eight hours after transfection of COS 7 cells with V3 plasmid as above, 1 ml of complete DMEM supplemented with 20% FBS was added to each well, and the cells were incubated for 12 h. Tunicamycin was added to each well to a final concentration 1 µg/ml, and the cells were incubated for another 24 h (44). Following tunicamycin treatment, the cells were radiolabeled, and supernatants and the cell pellets were collected and treated as described above. Both supernatants and cell extracts were then used as Ags in immunoprecipitation experiments with antiserum.
Immunoprecipitation assay
Mice were infected with P. yoelii 17XL, and blood was collected when parasitemia reached approximately 30%. Plasmodial proteins were then metabolically labeled with [35S] Protein Labeling Mix as described (37). Immunoprecipitation assays were performed as described previously (45). Briefly, 2 µl of sera from pooled GST-PyC2 protein-immunized mice, V3 DNA-immunized mice, or 25 µl of mAb302 hybridoma tissue culture fluid were mixed with approximately 1 x 105 cpm of TCA-precipitable soluble parasite proteins or soluble Ags generated from transfected cell cultures as described above. The mixtures were then incubated for 1 h on ice. One microliter of rabbit anti-mouse IgG, IgM, IgA secondary Abs (Zymed Immunochemicals, San Francisco, CA) was added to the tube, mixed, and incubated for 30 min on ice. Twenty microliters of a 10% suspension of heat-killed Staphylococcus aureus Protein A (Boehringer Mannheim, Indianapolis, IN) were added to each tube, mixed thoroughly, and incubated on ice for 30 min. The mixture was then underlaid with 200 µl of 1 M sucrose in precipitation buffer and centrifuged at 12,000 rpm for 3 min. The supernatant was carefully removed and discarded, and the pellet was washed three times with precipitation buffer. Finally the pellet was resuspended in 20 µl of 2x SDS-PAGE sample buffer. Samples were boiled and subjected to analysis by SDS-PAGE.
Immunization, sera collection, and challenge infection
Groups of four 8- to 10-wk-old BALB/c mice were immunized with V3 plasmid DNA five times at 3-wk intervals either i.m. in the anterior tibialis muscles (two sites) or intradermally (i.d.) on the back skin of mice (three sites). The dosage for each immunization was 200 µg for the first and 100 µg for all subsequent doses in a volume of 100 µl. The negative control groups were immunized with the parental plasmid (VR1020) without insert (designated as W) either i.m. or i.d. Another group of 4 BALB/c mice was immunized with GST-PyC2 recombinant fusion protein in Ribi adjuvant system (RAS; Ribi Immunization Research, Hamilton, MT) as described previously (46). Briefly, 8- to 10-week-old BALB/c mice were immunized with 60 µg of GST-PyC2 (providing 20 µg of PyC2). Fusion protein was first administered s.c. in 200 µl (two sites, 100 µl each site) of RAS adjuvant, suspended in 20 mM NaH2PO4/Na2HPO4-15 mM NaCl, pH 7.4 (PBS). This dose was repeated s.c. after 3 wk and again at 6 wk after the initial injection, but administered i.p. Mice were bled through the tail vein one day before each immunization to obtain sera for analysis. Two weeks after the final immunization, each group was challenged by i.v. injection of 104 E infected with the lethal variant P. yoelii 17XL as described (39). The course of infection was monitored by microscopic examination of stained blood films.
ELISA for measurement of Ab titers and isotypes
ELISAs were performed as described (38). Briefly, GST-PyC2 fusion protein was isolated by affinity chromatography, cleaved with thrombin, and purified such that free GST and uncleaved fusion protein were removed from the preparations. The C-terminal MSP-1 portion of the fusion protein (PyC2) was used to coat the wells of Maxi-sorb immunoplates (Nunc, Naperville, IL) at 0.5 µg/ml in carbonate buffer, pH 9.6. Wells were then blocked with 0.2% Tween 20 (Sigma) in 25 mM Tris-HCl (pH 8.0)-150 mM NaCl (Tris-buffered saline, TBS). Antisera collected from individual mice from each group were serially diluted in 0.2% Tween 20 in TBS. Ab dilutions were added to the appropriate wells in duplicate and followed by biotinylated rabbit anti-mouse Abs, including anti-IgG, IgM, IgA, Heavy and Light chain (Zymed), streptavidin-alkaline phosphatase, and p-nitrophenyl phosphate substrate (Sigma 104). The reaction was terminated by adding 50 µl of 5 N NaOH, and OD was measured at 405 nm using a Flow Titertek Multiscan Plus plate reader. For isotype analysis, the bound Abs were detected with affinity-purified, biotinylated, rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, or IgM Abs (Zymed), avidin-alkaline phosphatase (Zymed), and p-nitrophenyl phosphate (Sigma 104), and the OD value was measured at 405 nm as described above.
mAb302 epitope competition assay
This assay was utilized to assess the ability of immune sera to compete with protective mAb302 for binding to MSP-1 (P. A. Calvo and C. A. Long, unpublished observations). Briefly, sera were serially diluted in 0.2% Tween 20 in TBS and incubated in individual wells of Maxi-immunoplates coated with PyC2 Ag. The Ab dilutions were incubated for 45 min at room temperature. Following incubation, approximately 1 x 105 TCA-precipitable counts of metabolically labeled mAb302 tissue culture fluid was added to each well and allowed to incubate for another 45 min. Following the second incubation, the supernatants were discarded, and wells were washed 3 times with 0.2% Tween in TBS and two times with TBS alone. Subsequently, 100 µl of 1% SDS was added to individual wells and incubated for 30 min. After incubation, the SDS solution in each well was carefully removed from the individual wells, transferred to scintillation vials containing 3 ml of Scintiverse II (Fisher Scientific, Fair Lawn, NJ), and radioactivity was measured using a Beckman LS7000 Scintillation Counter.
Ab avidity analysis
An ELISA-based Ab avidity assay was performed as described previously (47). PyC2 was used to coat the wells of Maxi-sorb immunoplates, and the tested sera were diluted in 0.2% Tween 20 in TBS and added to each well as described for ELISA. Following incubation at room temperature for 1 h, the individual wells were washed 3 times with 0.2% Tween 20 in TBS. The Ag-Ab complexes bound to the plate were then subjected to incubation with either 0.2% Tween 20 in TBS or increasing concentrations of ammonium thiocyanate for 30 min at room temperature. The concentrations of NH4SCN used in this experiment were 0 M, 1 M, 2 M, 3 M, 4 M, and 6 M. Following extensive washes, the wells were incubated with biotinylated rabbit anti-mouse IgA, IgG, and IgM, streptavidin-alkaline phosphatase, and p-nitrophenyl phosphate substrate, and OD value was determined at 405 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DNA fragment encoding GST-PyC2 was cloned into a
BamHI site downstream from the TPA leader sequence in the
VR1020 vector, which also contains a CMV promoter, a bovine growth
hormone poly(A) terminus, and a kanamycin resistance marker. The
sequences of the junction region and the insert were confirmed by DNA
sequencing. This DNA construct encoding GST-PyC2 and its parental
plasmid VR1020 without insert were designated as V3 and W,
respectively. To investigate expression in mammalian cells, Cos 7 cells
were transiently transfected with either V3 or W plasmids in vitro and
labeled metabolically with [35S]methionine. An amount
equal to 1 x 105 cpm of TCA-precipitable soluble Ags
from both supernatants and cell pellets was immunoprecipitated with
mAb302, which recognizes a conformational epitope in the PyC2 region,
polyclonal mouse anti-GST-PyC2, or control sera. The precipitates
were analyzed by SDS-PAGE. While normal mouse sera (NMS) did not
recognize any proteins, both anti-GST-PyC2 and mAb302 recognized
two polypeptides from both supernatants (Fig. 1
) and pellets (data not shown) of the
transfected Cos 7 cells. The bands from the pellets precipitated by
mAb302 were much weaker than those precipitated by anti-GST-PyC2
sera (data not shown), suggesting that not all of the intracellular
GST-PyC2 expressed the epitope recognized by mAb302. However, the two
polypeptides in the supernatant fluids were equally recognized by the
two Abs, indicating that they carried the conformational mAb302
epitope. No proteins were recognized by either anti-GST-PyC2 or
mAb302 from W-transfected cell cultures (Fig. 1), establishing that the
precipitated polypeptides in V3-transfected cell cultures were products
expressed from the insert in V3, not from the vector itself.
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These results suggest that a proportion (about half) of the protein
expressed in V3-transfected Cos 7 cells undergoes
N-glycosylation. However, both the glycosylated and the
nonglycosylated polypeptides expressed in V3-transfected Cos 7 cells
maintained the conformational epitope recognized by mAb302. Immunization of mice with V3 vaccine elicits specific Abs against PyC2
Two groups of 8- to 10-wk-old BALB/c mice were immunized either i.m. or i.d. with the V3 plasmid DNA in PBS. The W plasmid was used as a negative DNA control. Five immunizations were given to each group of mice at 3-wk intervals. In addition, another group of BALB/c mice was immunized with the corresponding recombinant protein GST-PyC2 in RAS three times at 3-wk intervals as described previously (46). Blood samples were collected 1 day before the next immunization or parasite challenge, and anti-PyC2 Abs were examined by ELISA using PyC2 cleaved from GST-PyC2 as the target Ag.
As shown in Figure 2A, while
there were no PyC2-specific Abs induced by the W DNA control, both i.m.
and i.d. V3-immunized mice produced specific Abs against PyC2. The
titers of anti-PyC2 Abs from the DNA i.m. group were similar to
those of the DNA i.d. group after the fifth immunization
(p > 0.05), but they were significantly lower
than those of protein-immunized mice after the third inoculation
(p < 0.05) (Fig. 2A). In addition,
the relative concentrations of anti-PyC2 Abs after each
immunization from both DNA- and protein-immunized mice were measured at
a dilution of 1:400. As shown in Figure 2B, the
anti-PyC2 Abs in both DNA groups became detectable after the third
immunization, then increased after the fourth inoculation. There was no
further increase in relative concentration of anti-PyC2 Abs after
the fourth immunization in both DNA groups (p
> 0.05). This indicates that the DNA vaccination takes longer to
generate detectable specific Abs in mice than protein vaccination, in
which the anti-PyC2 Abs became detectable after the first
immunization (Fig. 2B).
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The isotype distribution of anti-PyC2 Abs in prechallenge sera
from DNA- and protein-immunized mice was determined by ELISA. As shown
in Figure 3, GST-PyC2 immune sera (P-3)
contained predominantly IgG1, IgG2a, and IgG2b, and lower levels of
IgG3 and IgM Abs. However, sera from DNA-immunized mice showed a
narrower distribution of isotypes, predominantly IgG1 and IgG2a for V3
i.m. group and almost exclusively IgG1 for the V3 i.d. group. In
addition, the ratio of IgG1 to IgG2a for V3 i.d. group (5.58) was
significantly higher than that of the V3 i.m. group (1.20)
(p < 0.01). This suggests that V3 i.d. and V3
i.m. immunizations may elicit different Th-type immune responses, with
V3 i.d. predominantly of the Th2 type while V3 i.m. may elicit both
Th1- and Th2-type immune responses.
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The specificity of the anti-PyC2 Abs induced by DNA
immunization was assessed by immunoprecipitation of metabolically
labeled total parasite proteins with sera from immunized mice taken
before challenge infection. As shown in Figure 4, the pooled sera from i.d. V3- and i.m.
V3-immunized mice, as well as the protein-immunized sera,
immunoprecipitated radiolabeled native PyMSP-1 (apparent molecular mass
220 kDa), indicating that the Abs induced by V3 plasmid immunization
recognize native parasite proteins. However, the sera from V3-immunized
mice precipitated much less native PyMSP-1 than those from
protein-immunized mice at the same dilution (1:50) (Fig. 4). To
investigate whether this differential ability of Abs to
immunoprecipitate native protein was due to the different levels of
anti-PyC2 Abs in sera, pooled sera from protein-immunized mice were
further diluted to 1:1400, at which they showed the same OD value with
a 1:50 dilution of sera from either i.m. V3 DNA- or i.d. V3
DNA-immunized mice. When the final dilution of 1:1400 protein-immunized
sera was used, the bands immunoprecipitated by pooled protein-immunized
sera (lane 4, Figure 4) showed similar intensity to
those obtained with 1:50 diluted, pooled DNA-immunized sera
(lane 2 and lane 3, Figure 4). These
results suggest that anti-PyC2 Abs from both DNA- and
protein-immunized mice have similar ability to react with native
parasite proteins after normalizing titers against PyC2.
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Since both the GST-PyC2 fusion protein and the polypeptides
expressed in V3-transfected Cos 7 cells contain the conformational
mAb302 epitope, the fine specificity of the elicited Abs was measured
using an mAb302 competition ELISA assay. The diluted antisera were
incubated in immunoplates coated with PyC2 for 45 min, and radiolabeled
mAb302 was subsequently added to each well and incubated for another 45
min. After extensive washing, bound mAb302 was dissolved from each
well, and the radioactivity was measured. NMS and unlabeled ascites
fluid from mice carrying mAb302 hybridoma cells were used as negative
and positive controls, respectively. As illustrated in Figure 5, while NMS did not compete with
[35S]mAb302, mAb302 did compete completely with
[35S]mAb302. This assay was used to assess the relative
level of the subpopulation of anti-PyC2 Abs in immune sera, which
can compete with [35S]mAb302 for binding its epitope.
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After normalizing the level of anti-PyC2 Abs, the different
dilutions of individual sera from each group were assayed for their
ability to compete with [35S]mAb302 for its epitope. As
shown in Table I, there were no significant differences in bound
[35S]mAb302 for sera from i.m. DNA-, i.d. DNA-, and
protein-immunized mice (p > 0.05). These data
indicate that anti-PyC2 Abs induced by DNA (both i.m. and i.d.) and
protein immunization have the same fine specificity for the mAb302
epitope in the range of concentrations tested.
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The avidity of anti-PyC2 Abs from both DNA- and protein-immunized mice was measured by an ELISA-based assay using thiocyanate elution (47). This method was based on the susceptibility of Ag-Ab complexes to dissociation by the chaotropic thiocyanate ion. Sera that had been diluted 1:400 were added to immunoplates coated with PyC2, and then increasing concentrations of NH4SCN were added to each well to disrupt the bound immune complexes. After extensive washing, the specific Abs bound to PyC2 were measured by ELISA. The absorbance readings in the absence of thiocyanate were assumed to represent effective total binding of specific Abs, and subsequent absorbance readings in the presence of increasing concentrations of thiocyanate were converted to the appropriate percentage of the total bound Abs. The avidity index represents the molar concentration of thiocyanate required to reduce the initial OD value by 50% (47). The avidity index has been reported to be independent of the level of specific Abs in sera (47), and we also determined that the avidity index changed minimally from 2.6 to 2.1 when a tertiary serum from protein-immunized mice was diluted from 1:250 to 1:10,000 (data not shown). These data suggest that the level of anti-PyC2 Abs has little effect on the avidity index over a relatively wide range.
We then compared the avidity indices of anti-PyC2 Abs from DNA- and
protein-immunized mice. As shown in Figure 6A, the avidity index of
anti-PyC2 tertiary sera from protein-immunized mice was
significantly greater than that of anti-PyC2 sera after the fifth
immunization with V3 plasmid (both i.m. and i.d.). There was no
significant difference in avidity indices of anti-PyC2 Abs between
V3 i.m. and V3 i.d. groups. The same results were obtained even after
normalizing levels of anti-PyC2 Abs from different groups (Table I). As shown in Table I, the avidity indices of anti-PyC2 Abs from
protein-immunized mice were still significantly higher than those from
both DNA-immunized groups (p = 0.016 for i.m.
group and p = 0.001 for i.d. group), and there was no
difference in avidity indices of anti-PyC2 Abs between DNA i.m. and
DNA i.d. groups (p > 0.05, Table I). These
results indicate that the avidity of anti-PyC2 Abs from
protein-immunized mice is significantly higher than that from
DNA-immunized mice (both groups).
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B). These data suggest
that there may be little or no affinitymaturation of
anti-PyC2 in DNA-immunized mice. To expand this observation to
avidity of Abs with the other specificities, the avidity index of
anti-GST Abs in the same sera from the V3 i.m. group and protein
group was also measured. As shown in Figure 6C, in contrast
to protein immunization, there was no significant increase in the
avidity indices of pooled anti-GST Abs during the course of DNA
immunization, either. Thus there may be little or no affinity
maturation in both anti-PyC2 and anti-GST Ab populations in
DNA-immunized mice. To our knowledge, this is the first demonstration
of the lack of avidity maturation of Abs during DNA immunization. DNA and protein immunization elicit Abs with different protective capability
Two weeks after the fifth immunization with V3 in PBS and after
the third immunization with GST-PyC2 in RAS, mice were challenged with
104 P. yoelii 17XL parasitized E. As shown in
Figure 7, W plasmid-immunized mice,
unimmunized infection controls, and all the V3-immunized mice died by
day 8. However, the GST-PyC2 protein-immunized mice had a peak
parasitemia at day 14 and cleared the i.v. infections by day 23. This
result indicates that the humoral immune responses induced by the
protein but not by the DNA immunization protected mice against the
lethal blood-stage challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have shown that the configuration of the immunizing Ag is critical for the induction of protective humoral immune responses in this model (45, 48). Therefore, it was important to determine initially whether the DNA vaccine could direct synthesis of the encoded polypeptide correctly in mammalian cells. The conformation of the expressed polypeptides in a V3-transfected Cos 7 cell line was examined in immunoprecipitation experiments with mAb302 and anti-GST-PyC2. Our results demonstrated that, although approximately half of the protein secreted from V3-transfected Cos 7 cells had undergone N-glycosylation, the mAb302 epitope is still available on both N-glycosylated and nonglycosylated polypeptides expressed from transfected Cos 7 cells. This lends support to the native configuration of the C-terminal region of PyMSP-1. This observation was also important since there is evidence that plasmodia do not N-glycosylate their proteins (49) and that the GST-PyC2 produced by recombinant E. coli would also not be N-glycosylated.
Our results demonstrated that V3 DNA vaccination successfully induced production of anti-PyC2 Abs in BALB/c mice, while the negative W plasmid control elicited no specific Abs. However, the induction of anti-PyC2 Abs by DNA immunization took 6 wk longer, and the titer was considerably lower than that elicited by the protein immunization. This result is similar to preliminary data from Gardner et al., who reported lower anti-PyC2 Ab titers after immunization with a DNA vaccine encoding the C-terminus of PyMSP-1 (25). Moreover, we also established that switching to IgG isotypes does occur during the course of V3 DNA immunization.
Two approaches to examination of the specificity of these Abs were taken. First, pooled sera from DNA- (both i.d. and i.m.) and protein-immunized mice were shown to immunoprecipitate native parasite protein at similar levels after normalizing their titers to PyC2. Second, the fine specificity of anti-PyC2 Abs to mAb302 epitope was evaluated by the mAb302 competition assay. We found that both DNA- and protein-immunized sera partially prevented mAb302 from binding its epitope, but protein-immunized sera competed with mAb302 better than DNA-immunized sera. However, after adjustment for relative concentration, the anti-PyC2 Abs induced by DNA and protein immunizations have the same ability to compete with mAb302 for binding to its epitope. These data demonstrated that, although the titer of anti-PyC2 Abs induced by the DNA inoculation was lower, the anti-PyC2 Abs resulting from DNA immunization showed the same ability to recognize native parasite protein and the mAb302 epitope.
Another important parameter for measuring humoral immune responses is
Ab affinity. Because of the difficulties involved in measurement of
true Ab affinity, Ab avidity was determined using an ELISA-based
thiocyanate elution assay (47). The avidity index, representing the
molar concentration of thiocyanate required to reduce the initial OD by
50%, was quantified. Importantly, the avidity index obtained from this
assay was reported to be relatively independent of the level of
specific Abs (47). Our data also showed that the avidity index of
anti-PyC2 Abs in prechallenge serum from protein-immunized mice
varied by just 0.5 over a 40-fold concentration (data not shown). As
assessed by this assay, the avidity index of anti-PyC2 Abs from
DNA-immunized mice (both groups) is considerably lower than that from
protein-immunized mice. This is also true even after normalizing the
titers of anti-PyC2 Abs from DNA- and protein-immunized mice (Table I). Surprisingly, while a clear increase in the avidity index of
anti-PyC2 Abs was observed during the course of GST-PyC2 protein
immunization, there was no significant increase in the avidity indices
of either the anti-PyC2 population or the anti-GST Abs during
the course of DNA immunization. Since affinity of Abs is primarily
determined by their dissociation rates, this observation suggests that
there is little or no affinity maturation of Abs induced by DNA
immunization. To our knowledge, this is the first report determining
the avidities of Abs induced by DNA vaccines and demonstrating a lack
of maturation of avidity of Abs during DNA vaccinations in mice.
Avidity maturation of Abs elicited by immunization with haptens and protein Ags has been well documented in animal models. In addition, infection of humans with pathogens such as rubella (50), cytomegalovirus (51), and Toxoplasma gondii (52) also elicits Abs whose avidities increase with time. For example, the avidity of anti-Toxoplasma IgG is considered as a criterion in the first half of pregnancy to discriminate between primary infection acquired in early pregnancy (low avidity) and infection that occurred before pregnancy (high avidity) (52). However, a few exceptions have been noted. Neutralizing Abs against vesicular stomatitis virus (VSV) seem not to undergo affinity maturation during acute infection in mice (53). In addition, severe measles occurs in immunized and nonimmunized HIV-infected individuals, and the impaired immune responses in these patients seem to be associated with defective avidity maturation of anti-measles Abs (54).
Affinity maturation is the consequence of somatic hypermutation of the Ig genes followed by selection of B cells with high affinity surface Ig in germinal centers (GCs), and depends upon the interaction of activated B cells with follicular dendritic cells (55). We do not know whether the apparent lack of affinity maturation of specific Abs during DNA immunization is the result of deficiencies in somatic hypermutation of Ig genes or in the selection of higher affinity B cells in germinal centers. It is also unclear whether the lack of avididty maturation during DNA immunization occurs only with particular Ags or in certain strains of mice.
We have considered several possibilities that might explain our observations. First, the glycosylation of the Ag in vivo may alter its localization in germinal centers. Using the same expression vector, DNA-based immunizations for HA in an influenza model were much more effective than DNA immunizations for Env in a human immunodeficiency model (56). Further study showed that the Env protein had been more heavily glycosylated than the HA (56). In our case, about half of the secreted protein had been glycosylated in V3-transfected monkey cells. Although the N-glycosylation of the Ag did not change the mAb302 epitope, the difference in physical structure between glycosylated and nonglycosylated Ag expressed in vivo may result in differences in the efficiency with which the Ag-specific B cells enter into GCs for hypermutation and affinity maturation of specific Abs.
Localization of Ag within GCs not only is important for affinity maturation but also may affect specific B cell memory. The role of follicular dendritic cells in driving the maturation of the humoral immune responses depends chiefly on their ability to retain Ags on their surface for long periods of time. Only B cells with higher affinity surface Ig survive and differentiate into either plasma cells or memory B cells. Therefore, deficiency in affinity maturation not only results in generation of Abs with lower affinity but also may affect specific B cell memory.
Another possible explanation for the lack of avidity maturation is the schedule chosen for DNA vaccination. Injection with DNA vaccines leads to lower but longer in vivo expression of the encoded Ags, and, since DNA vaccination takes longer to induce Ab immune responses, it may take longer for avidity increases to become apparent. In support of this possibility, Lyon and his colleague recently reported that increasing the interval between DNA immunization of mice increased the levels of Abs produced (57). To address the issue of longer intervals between DNA injections, we have done an experiment with the V3 DNA vaccine, immunizing three times at weeks 0, 12, and 32. Despite this very extended schedule, the avidities of the Abs produced were similar to those reported here with more frequent immunization (data not shown).
Additionally, it is of interest that the production of these low avidity Abs after DNA vaccination is similar to the phenomenon observed in mice for a protein Ag injected without adjuvant (58). While the protein Ag administered in CFA induced specific Abs with higher affinity, the same Ag in PBS elicited lower affinity Abs (58). In our case, the V3 DNA vaccine was given in PBS, and thus it is possible that the absence of appropriate adjuvant may contribute to the lack of maturation of Abs during DNA immunization. We are therefore currently investigating the effect of adjuvants on DNA vaccination in this model.
Finally, when DNA- and protein-immunized mice were tested for protection against lethal challenge of P. yoelii infection, the results showed that all the protein-immunized mice resolved the parasitemias, whereas the DNA-immunized mice died of infection. The data from our laboratory as well as others indicates that isotype is not a critical parameter for protection in this P. yoelii model (46, 59). Thus, we believe that the significantly lower titer and lower avidity of anti-PyC2 Abs induced by DNA immunizations are responsible for the lack of protection against lethal challenge in DNA-immunized mice. We have also seen a similar correlation of Ab avidity and titer with protection in mice immunized with GST-PyC2 protein in CFA (P. A. Calvo and C. A. Long, unpublished observations). C57BL/10 mice produced high titer Abs with an avidity index comparable to the protein-immunized BALB/c mice reported here (2.35) and were protected. In contrast, B10.BR mice produced Abs of both lower titer and avidity (1.55) comparable to those reported here for DNA immunization and were not protected.
In summary, V3 DNA vaccine encoding GST-PyC2 successfully elicited anti-PyC2 Abs in BALB/c mice, which recognized native MSP-1 protein and had similar specificity for the epitope recognized by mAb302 as Abs from protein-immunized mice. However, such Abs had significantly lower titer and lower avidity, different isotype profiles, and different protective capacity against lethal challenge. More importantly, our results suggest that there may be little or no affinity maturation of specific Abs during the course of V3 DNA immunization. Further understanding of the process by which DNA vaccines elicit humoral immune responses in vivo may allow us to obtain protective immune responses through this strategy.
Note added in proof. Lack of avidity maturation of anti-PyC2 antibodies was also observed in other strains of mice, including C57BL/10 and Swiss Webster mice, which were immunized with V3 DNA vaccine.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
3 Address correspondence and reprint requests to Dr. Carole A. Long, Department of Microbiology and Immunology, Allegheny University of the Health Sciences, 2900 Queen Lane, Philadelphia, PA 19129.
4 Abbreviations used in this paper:HA, hemagglutinin; MSP, merozoite surface protein; GST, glutathione S-transferase; PyC2, C-terminal region of the P. yoelii MSP-1; OPTI-MEM, OPTI-MEM I Reduced Serum Medium; i.d., intradermally; RAS, Ribi adjuvant system; NMS, normal mouse sera; GC, germinal center; TPA, tissue-type plasminogen activator.
Received for publication March 30, 1998. Accepted for publication June 22, 1998.
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-, and nitric oxide-dependent immunity. J. Exp. Med. 183:1739.This article has been cited by other articles:
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J.-P. Sauzet, B.-L. Perlaza, K. Brahimi, P. Daubersies, and P. Druilhe DNA Immunization by Plasmodium falciparum Liver-Stage Antigen 3 Induces Protection against Plasmodium yoelii Sporozoite Challenge Infect. Immun., February 1, 2001; 69(2): 1202 - 1206. [Abstract] [Full Text] [PDF]
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N. Ahlborg, I. T. Ling, A. A. Holder, and E. M. Riley Linkage of Exogenous T-cell Epitopes to the 19-Kilodalton Region of Plasmodium yoelii Merozoite Surface Protein 1 (MSP119) Can Enhance Protective Immunity against Malaria and Modulate the Immunoglobulin Subclass Response to MSP119 Infect. Immun., April 1, 2000; 68(4): 2102 - 2109. [Abstract] [Full Text] [PDF]
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