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Malaria Program, Naval Medical Research Center, Silver Spring, MD 20910;
Department of Microbiology, University of Maryland School of Medicine, Baltimore, MD 21201;
Virogenetics Corp., Troy, NY 12180;
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Department of Molecular Microbiology and Immunology Johns Hopkins University, Baltimore, MD; and
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Vical Inc., San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting cells by enzyme-linked immunospot assay (1460 vs 280
IFN- spot-forming cells/106 cells). Priming with plasmid
vaccine plus pGMCSF and boosting with recombinant poxviruses strongly
improves the immunogenicity and protective efficacy of DNA vaccination
and allows for significant reduction of dose. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and nitric oxide and is
directed against the parasite-infected hepatocyte (2).
Protection ranges from 25 to 75% depending on the dosage regimen of
vaccine, infectivity, and dose of sporozoites used for challenge
(1, 2, 3). However, this level of protection is significantly
less than the >95% protection in BALB/c mice after immunization with
radiation-attenuated sporozoites (4, 5), and we have
continued to search for ways to improve the PyCSP plasmid
vaccine. We have recently reported that coadministration of
PyCSP DNA with a DNA plasmid that expresses murine GM-CSF
(pGMCSF) (3) improves vaccine efficacy. In a separate
paper, we described that boosting of pPyCSP DNA-primed
animals with recombinant poxvirus expressing the PyCSP
(rvPyCSP) (6) increases protective efficacy.
Both these strategies, boosting with recombinant poxvirus
(7, 8, 9, 10, 11, 12, 13) and the addition of pGMCSF (14, 15, 16, 17),
have been shown to enhance immunogenicity and protection of several
other DNA plasmid vaccines.
In an effort to further improve the protective efficacy of
pPyCSP DNA vaccine and to reduce the dose of DNA required
for immunization, we have combined these two approaches, first priming
with pPyCSP mixed with pGMCSF and then boosting with
rvPyCSP. The results demonstrate that this approach leads to
greater Ag-specific Ab, IFN-, and cytotoxic T cell activity and a
significant increase in protective efficacy. Furthermore, priming with
a low DNA dose of 1 µg pPyCSP and 1 µg pGMCSF gives
excellent protection, only slightly less than that achieved after
priming with high doses of 100 µg pPyCSP and 30 µg
pGMCSF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/cByJ (H-2d), A/J(H-2a), and C57BL/6 (H-2b)mice, 6–8 wk old, purchased from The Jackson Laboratory, Bar Harbor, ME, were used in all experiments.
Parasites
P. yoelii (17XNL) clone 1.1 parasites were used. Sporozoites for challenges were obtained from infected mosquito glands in M199 medium containing 5% normal mouse serum. All challenges were accomplished by injecting 50–100 sporozoites into the tail vein.
Plasmid immunization
The P. yoelii plasmid DNA construct, 1020PyCSP, designated here as pPyCSP and the murine GM-CSF plasmid, designated here as pGMCSF, used for all the immunizations have been described (3, 6). All DNA for injection was purified using cesium chloride-ethidium bromide density gradient centrifugation as previously described (6). Plasmids stored at -20°C were diluted in PBS (pH 7.2) to the appropriate concentration for injections. Priming with plasmids was done either with pPyCSP alone or mixed with pGMCSF. Plasmid DNA lacking the PyCSP gene was used as control plasmid and was added to other groups to equalize the total amount of injected plasmid. A 0.3-ml insulin syringe with a 29-gauge 0.5-inch-long needle was used for the i.m. injections in the tibialis anterior muscle of the two hind limbs.
Recombinant vaccinia virus immunization
The construction of P. yoelii recombinant vaccinia virus, designated (rvPyCSP) used for boosting has already been described (6). Mice were boosted i.p. with rvPyCSP at 3, 6, 9, or 12 wk after priming with DNA. The dose of virus was delivered in a total volume of 0.2 ml in PBS, pH 7.2.
Ab response
Indirect fluorescent Ab test (IFAT). The IFAT technique (1) was used to detect anti-P. yoelii sporozoite Abs in pooled sera from blood taken just before challenge.
ELISA. Serum Ab levels against recombinant protein or synthetic peptide were determined by standard ELISA protocols as previously described (18), using a full length recombinant PyCSP protein produced in yeast (rPyCSP) or linear synthetic peptide containing four copies of the major repeat of PyCSP protein, (QGPGAP)4, as solid phase Ags. Briefly, 50 µl of 0.1 µg/ml rPyCSP or 0.1 µg/ml (QGPGAP)4 in PBS were added into wells of Immunolon II ELISA plates (Dynatech Laboratory, Chantilly, VA) and incubated for 6 h at room temperature. The wells were washed three times with PBS containing 0.05% Tween 20 (washing buffer) and incubated overnight at 4°C with 100 µl 5% nonfat dry milk in PBS (blocking buffer). After three washes with washing buffer, the wells were incubated for 2 h with 50 µl of different dilutions of sera diluted in PBS containing 3% nonfat dry milk (diluting buffer). The wells were washed three times, incubated for 1 h with peroxidase-labeled goat anti-mouse IgG (Kirkegaard and Perry, Gaithersburg, MD) diluted 1:2000 in diluting buffer, then washed again three times. The wells were incubated for 20 min with 100 µl of a solution containing ABTS substrate (2,2'-azinodi(3-ethylbenzthiazoline sulfonate) (Kirkegaard and Perry)) and H2O2. Color reaction was measured in a microELISA automated reader (Dynatech, MR5000) at OD410 nm. All reaction steps except blocking were performed at room temperature. Means ± SD of the OD readings of quadruplicate assays were recorded. The results were reported as OD 0.5 units (the reciprocal of the serum dilution at which the mean OD reading was 0.5).
CTL assays
Restimulation CTL assay. 51Cr release assays using bulk cultures was performed as previously described (1). In this method, spleen cells (5 x 106) were stimulated in vitro for 7 days with a 16-amino acid peptide, PyCSP 280–295 (SYVPSAEQILEFVKQI), which contains the H-2Kd-restricted epitope, PyCSP 280–288 (SYVPSAEQI) in 24-well plates at peptide concentration of 2.5 µM. Culture medium used was RPMI supplemented with 10% heat-inactivated FCS, L-glutamine, 50 U/ml each of penicillin and streptomycin, and 2-ME at 5 x 10-5 M. P815 (H-2d) cells (American Type Culture Collection, Manassas, VA) labeled with 0.1 mCi 51Cr (DuPont-New England Nuclear, Boston, MA) and pulsed with the CTL peptide (PyCSP 280–288 (SYVPSAEQI)) at 0.025 µM were used as positive target cells. Control targets consisted of unpulsed 51Cr-labeled P815 cells. Varying numbers of effector cells were added to both positive and negative targets at 5000 target cells per well of 96-well U-bottom plates, and 5-h standard chromium release methods were followed. Percent lysis was calculated for both positive and negative targets by the formula: [(experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm)] x 100%. Spontaneous release was defined as background lysis of target cells in presence of medium alone (generally <10% of maximum lysis), and maximum lysis was defined as lysis in the presence of 5% Triton X-100. Net percent specific lysis was calculated as: % lysis of positive targets - % lysis of negative control targets.
Ex vivo CTL assay. Freshly isolated immune spleen cells were used without the 7-day in vitro restimulation period. In this method, varying numbers of freshly isolated spleen cells were added to 5000 peptide-pulsed, 51Cr-labeled P815 target cells with medium containing 2% rat T-cell stim (Collaborative Biomedical Products, Bedford, MA) in 96-well U-bottom plates. Both positive targets, 51Cr-labeled P815 cells pulsed with the CTL peptide (PyCSP 280–288 (SYVPSAEQI)) at 0.025 µM, and control targets, consisting of unpulsed 51Cr-labeled P815, cells were tested. A 12-h chromium release method was followed. Net percent specific lysis was calculated as: % lysis of positive target cells - % lysis of negative control target cells.
ELISPOT assay for detection of Ag-specific
IFN--secreting CD8+ T cells
The numbers of PyCTL epitope-specific
IFN--producing CD8+ T cells were determined
using freshly isolated spleen cells from mice that had received their
second immunization 14 days earlier. These measurements were made after
24–28 h incubation of spleen cells with irradiated P815 target cells
pulsed with a 1 µM concentration of the H-2d-restricted
PyCSP peptide SYVPSAEQI as previously described (6, 19). Nitrocellulose plates (96-well, Millipore, Bedford, MA)
were coated with 75 µl PBS containing 1 µg/ml purified rat
anti-mouse IFN- mAb (PharMingen, San Diego, CA). After overnight
incubation at room temperature, the wells were washed 6 times with
culture medium and incubated for 1 h with 100 µl culture medium
containing 10% FCS. Irradiated P815 target cells (100 µl of 2
x 105 cells/ml) were placed in the Ab-coated
wells. The starting concentration for the freshly isolated
unrestimulated effector cells was 1–4 x
106/ml, and 2-fold dilutions in duplicate were
assayed. One set of duplicate cells was cocultured with irradiated P815
cells, which had been incubated with 1 µM SYVPSAEQI. The other set of
cells was cocultured with irradiated P815 cells that had not been
incubated with peptide. After incubation at 37°C and 5%
CO2 for 24–28 h, the plates were washed six
times with PBS containing 0.05% Tween 20 (PBS/T). The wells were then
incubated with 100 µl of a solution of 1 µg/ml biotinylated
anti-mouse IFN--mAb (PharMingen) in PBS/T. After overnight
incubation at 4°C, wells were washed with PBS/T and 100 µl
peroxidase-labeled streptavidin (Kirkegaard and Perry) at a dilution of
1/1000 in PBS/T, was added to each well. After 1 h incubation at
room temperature, wells were washed twice with PBS/T and twice with
PBS. The spots were developed by following the instructions provided
with the DAB Reagent set (Kirkegaard and Perry Laboratories). After
10–15 min, the number of spots corresponding to IFN--producing
cells in wells containing the different spleen cell dilutions was
determined using a stereomicroscope. The results were expressed as the
number of IFN--secreting cells per 106 spleen
cells. Net spots per 106 spleen cells were
calculated as: number of spots with peptide pulsed targets -
number of spots with unpulsed targets.
Inhibition of liver stage development assay (ILSDA)
Cell culture. Mouse hepatocytes were obtained by in situ collagenase perfusion of mouse liver as previously described (20). Briefly, livers were perfused in situ sequentially with HBSS and a collagenase solution. The cell suspension generated was then centrifuged over a Percoll gradient to remove dead cells. The hepatocytes were then seeded onto 8-well Lab-Tek chamber slides in complete medium (EMEM with Earle’s balanced salts supplemented with 0.2% BSA (fraction 5), 10% FCS, 2% penicillin-streptomycin solution, insulin, 1% L-glutamine solution (100x), and 1% nonessential amino acids solution (100x)) at a concentration of 1 x 105 cells/well. The slides were incubated overnight at 37°C in a 5% CO2, 95% air environment. The medium was changed the following day and fresh media containing dexamethasone (7 x 10-4) was added to the cultures.
ILSDA: restimulated spleen cells. Hepatocyte cultures, which had been seeded onto 8-well chamber slides 24 h previously, were incubated with 7.5 x 104 P. yoelii sporozoites for 3 h. The cultures were then washed with medium and incubated for 24 h. Immune spleen cells, which had been restimulated with peptide PyCSP 280–295, were tested for their ability to inhibit the development of liver stage parasites. Spleen cells from naive mice were stimulated and tested in the same manner as the immunized mouse spleens and used as a control. Stimulated spleen cells (5 x 105) were added to the liver cultures and allowed to remain in culture with the infected hepatocytes for an additional 24 h. Assays were run in triplicate. The chamber slides were then fixed for 10 min in ice-cold absolute methanol and stained with NYLS3 mAb (21) and a FITC-labeled goat anti-mouse IgG. The stained parasites on the slides were then viewed and counted using an epifluorescence microscope. The slide reader was blinded and did not know which group received which vaccine combination. Percent inhibition was then calculated and expressed as a percentage according to the formula: % inhibition = [1 - (mean number of parasites in experimental wells/mean number of parasites in control wells)] x 100.
ILSDA: serum. The biological activity of the Abs produced by the various regimens was assessed in an ILSDA. Hepatocyte cultures, seeded onto 8-well chamber slides and allowed to attach overnight, were used. After 24 h of incubation at 37°C in an atmosphere of 5% CO2 in air, medium was removed, and 7.5 x 104 salivary gland dissected sporozoites suspended in 25 µl of medium, and 25 µl of varying dilutions of sera from immunized mice or medium were added. After 3 h incubation, the cultures were washed to remove sporozoites that did not invade hepatocytes, and fresh medium was added. Assays were run in triplicate. After a further 24 h, the medium was changed, and at 48 h the cultures were fixed and incubated with a mAb directed against liver stage parasites of P. yoelii (NYLS3) and a FITC-labeled goat anti-mouse IgG. The slide reader was blinded and did not know which group received which vaccine combination. The stained parasites on the slides were then viewed and counted as already described: % inhibition = [1 - (mean number of parasites in cultures with immune serum/mean number of parasites in cultures with medium control)] x 100.
Protection against challenge
Immunized mice were challenged i.v. with 50 to 100 infective P. yoelii sporozoites 2 wk after the second immunization, and protection was defined as absence of patent parasitemia in Giemsa-stained blood smears during the 14-day follow-up period.
Statistical analysis
Simple linear regression was performed using SPSS for Windows
version 8.0 (SPSS, Chicago, IL). Comparisons between groups were also
performed using the same SPSS for Windows version 8.0.
2 was used except when any cell contained
fewer than five observations; then Fisher’s exact test (two-tailed)
was substituted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous publication using DNA priming followed by
recombinant vaccinia boosting in the Py malaria model, we
used 1 x 107 rvPyCSP PFU as the
viral dose and were able to achieve 69% protection. We wished to know
whether increasing the viral dose would increase the immune response.
The results in Table I
show that larger
doses of virus provided better protection. Nine weeks after DNA
priming, boosting with 1 x 108 PFU
rvPyCSP resulted in a 100% protection, whereas a boost with
1 or 2 x 107 rvPyCSP PFU
resulted in 40 and 80% protection. As in our previous studies
(6), boosting with recombinant virus did not protect
unless there had been a previous immunization with
pPyCSP.
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Because pGMCSF can enhance DNA immune responses to
pPyCSP (3), we wished to know whether a mixture
of pPyCSP and pGMCSF would improve priming for
rvPyCSP. Our strategy was to use a suboptimal immunization
schedule giving lower levels of protection to easily detect immune
enhancement. We primed with a single dose of DNA followed 3 wk later by
a boost with 2 x 107 PFU of rvPyCSP, a less
than optimal amount of virus. Table II
shows the results of three separate protection experiments admixing
pGMCSF in the prime-boost regimen. In Experiment 1, using high
doses of plasmid (100 µg pPyCSP and 30 µg pGMCSF),
priming with admixtures of pGMCSF protected better than did priming
with pPyCSP alone. In Experiment 2, we compared smaller and
larger doses of DNA for priming. When a mix of the two plasmids was
used, protection was maintained although plasmid doses were reduced 30-
to 100-fold (1 µg pPyCSP plus 1 µg pGMCSF). In
Experiment 3, we systematically compared low and high plasmid doses and
extended the plasmid titration down to 0.001 µg of each DNA.
Experiment 3 confirmed the results of Experiments 1 and 2 and showed
that some protection was achieved with as little as 0.1 µg priming
doses of pPyCSP and pGMCSF. It thus appears that pGMCSF is a
powerful enhancer of pPyCSP at the priming step. Summing the
results of the three experiments, we found that priming with high dose
PyCSP plus GM-CSF DNA (DG-V) protected 23 of 28 mice (82%),
whereas priming with low dose PyCSP plus GM-CSF (dg-V)
protected 13 of 18 mice (72%) (comparison of DG-V vs dg-V
(p = 0.430, 2 test)).
This degree of protection with dg-V, 13 of 18 mice (72%) was higher
than that recorded for recombinant virus boosting with high dose
PyCSP plasmid priming D-V, 9 of 20 mice (45%)
(comparison of dg-V vs D-V, p = 0.089,
2 test). Furthermore, the degree of protection
by dg-V, 13 of 18 mice (72%), was significantly better than that
recorded for recombinant virus boosting with low dose PyCSP
plasmid priming alone, d-V, 0 of 10 mice (0%) (comparison of dg-V vs
d-V, p = 0.00023, Fisher’s exact test, two-tailed), or
two doses of high dose PyCSP plus GMCSF plasmid DG-DG, 3 of
20 mice (15%) (comparison of dg-V vs DG-DG, p =
0.0004, 2). This is an important improvement in
antimalarial immunity. Not only does the addition of pGMCSF to the
priming dose increase protection but it also allows a large decrease in
the amount of DNA required for immunization.
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Having determined that pGMCSF improved priming, we wished to know whether any in vitro measures of immunity correlated with increased protection. We measured T cell and Ab responses in mice immunized with or without pGMCSF at the priming dose.
T cell responses to the PyCSP 280–288 Kd-restricted epitope.
The results of an experiment comparing T cell responses to the
Kd-restricted epitope PyCSP 280–288
(SYVPSAEQI) among mice receiving the various immunizations are shown in
Fig. 1. Fig. 1A shows CTL
activity against targets cultured with peptide PyCSP
280–288 after 7 days of in vitro restimulation of splenocytes with
PyCSP 280–295 (SYVPSAEQILEFVKQI) peptide. Fig. 1B shows the number of cells producing IFN- in the same
spleens. In the ELISPOT assay, spleen cells were incubated for 24–28 h
with P815 cells pulsed with the PyCSP 280–288 peptide, and
IFN- spot-forming cells were counted. Spleens from two mice per
group were pooled for these assays. CTL activity and IFN--producing
cells was highest in the DG-V group with dg-V and D-V next highest in
that order.
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ILSDA is an in vitro assay system that quantifies the reduction of
malaria infected hepatocytes after the addition of cells or serum. In
mice from Experiment 2 in Table II
, splenocytes from two mice per group
were pooled and restimulated for 7 days with the PyCSP
280–295 peptide. These cultures were then divided; one-half were used
for a CTL assay, and one-half were used for ILSDA. Table III compares results for CTL, ILSDA, and
protection in these groups. All three measures of immunity showed a
consistent ranking of response with the highest levels of in vitro
activity seen in the mice primed with high doses of pPyCSP
plus pGMCSF plasmids.
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). Our experience with passive transfer
of protective mAbs (22) and immunization with synthetic
peptides (23) has indicated that only levels of
inhibition >90–95% have a high predictive value for protection.
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Table V is a summary of our analysis
of mice from Table II, Experiment 3. Abs to PyCSP were
measured three ways: by IFAT against P. yoelii sporozoites
(column 3), and by ELISA using as capture Ag either a full length
recombinant PyCSP protein produced in yeast (column 4), or a
synthetic 18-mer peptide representing three copies of the 6-aa
PyCSP repeat motif (column 5). T cell responses to the
PyCSP 280–288 epitope were measured using three methods:
ELISPOT measuring IFN- producing cells (column 6), and two CTL
assays measuring lysis from splenocytes, either directly from spleens
without restimulation (column 7), or after 7 days of culture with
PyCSP 288–295 peptide (column 8). High dose DNA priming
with pPyCSP + pGMCSF plasmids followed by boosting with
recombinant virus gave the highest protection, as well as the highest
levels of T cell and Ab responses by every measure. Other immunization
regimens, using less DNA with or without virus boosting, gave less
protection and lower Ab and T cell responses to the
Kd-restricted epitope, PyCSP 280–288
(SYVPSAEQI) in vitro. There is a strong association between protection,
and all measured immune responses as measured by simple linear
regression. From this analysis, we conclude that overall immunity is
increased by pPyCSP + pGMCSF DNA priming, but we cannot
determine which immune responses are responsible for protection.
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To understand the immune mechanisms responsible for protection in
the DNA prime-viral boost immunizations, we tried to abrogate
protection by injecting immunized mice with mAbs to IFN- and CD4 or
CD8. We first tested mice immunized without the addition of pGMCSF
(Table VI). Mice were primed with 100
µg pPyCSP and boosted with 2 x
107 PFU rvPyCSP 9 wk later.
Administration of an anti-CD8 mAb, which removed >99% of all
CD8+ T cells, eliminated protection
(p = 0.0001, Fisher’s exact test, two-tailed).
(Table VI, Experiment A). A single i.v. injection of 1 mg
anti-IFN- mAb at the time of challenge reduced protection from
80% to 30% (Table VI, Experiment B) (p =
0.069, Fisher’s exact test, two-tailed). These data indicated that the
mechanism of protection with this prime-boost regimen was similar to
that induced by pPyCSP immunization alone and probably
involved CD8+ T cells that recognize infected
hepatocytes and produced IFN-. Our previous data (2, 24, 25) indicate that the IFN- either induces the infected
hepatocyte to produce nitric oxide, which eliminates the infected
hepatocyte, or induces mononuclear cells to produce IL-12, which leads
to production of IFN- by other T cells or NK cells
(26).
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, Experiment C). Results revealed that after treatment with
the anti-CD4 mAb, CD4+ T cells were depleted
at least 98.3, 97.9, and 98.8% in the D-V, DG-V, and dg-V groups,
respectively. CD8+ T cells were depleted at least
99.3, 99.5, and 99.4% in the D-V, DG-V, and dg-V groups, respectively,
after treatment with the anti-CD8 mAb. In the low dose plasmid
group (dg-V), treatment with anti-CD8 mAb completely eliminated
protection (p = 0.0009, Fisher’s exact test,
two-tailed). However, in the high dose plasmid group (DG-V), this same
Ab reduced immunity, but 45% of the mice remained protected
(p = 0.414, Fisher’s exact test, two-tailed),
even though >99% of CD8+ T cells were removed
by the Ab treatment. Treatment with anti-IFN- mAb only reduced
protection to 83% in the DG-V group (p =
0.640, Fisher’s exact test, two-tailed), and to 58% in the dg-V group
(p = 0.155 Fisher’s exact test, two-tailed),
as compared with 30% in the previous D-V experiment. These data do not
allow an unequivocal assignment of immune effector function in
protection with the dg-V and DG-V prime-boost regimens. It appears that
immune effector function with low doses of pGMCSF DNA priming is
dependent on CD8+ T cells. However, with large
plasmid doses of pGMCSF DNA, the fact that we cannot completely
abrogate protection means either that the mAb treatment cannot
neutralize higher levels of CD8+ T cells and
IFN-, or that additional immune effector functions have come
into play. Genetic control of protection from prime/boost immunization
A/J and C57BL/6 mice were immunized with DNA i.m.,
boosted 12 wk later with DNA i.m. or recombinant vaccinia virus i.p.
Mice were bled for serum at week 14 and then challenged with P.
yoelii sporozoites (100 sporozoites for A/J mice and 50
sporozoites for C57BL/6 mice) to measure protection. Table VII shows protection data comparing the
prime-boost immunization to other regimens of immunization. In the
past, A/J mice have been difficult to protect with vaccination using
pPyCSP alone (2). It appears the DG-V regimen
is slightly more effective than other immunization regimens in A/J mice
(Table VII). Only the DG-V regimen was significantly better than its
control, CG-V (p = 0.0171, Fisher’s exact
test, two-tailed) The DG-V regimen elicited the highest levels of Abs
against sporozoites (Table VII), and sera from mice immunized with this
regimen had the highest inhibitory activity against sporozoite invasion
and development within hepatocytes in vitro (Table VIII). This supported the interpretation
that Abs might play a major role in the protection seen. However, in
efforts to determine a protective role for CD8+ T
cells in A/J mice, the nine mice that had been protected by the DG-V
regimen in Table VII were treated with a control Ab or an anti-CD8
mAb. In these mice, Ab injections for depletions were started at 68
days after boost with recombinant virus and these depleted mice were
rechallenged at 74 days after boost, and 57 days subsequent to first
challenge. Three of the five mice (60%) treated with the control Ab
were still protected, but none of the four mice treated with the
anti-CD8 mAb were protected. The numbers of animals are too few to
draw firm conclusions. However, the data support the interpretation
that CD8+ T cells were required for the
protection in the four previously protected A/J mice despite the high
levels of inhibitory Abs. C57BL/6 mice were not significantly protected
by any prime-boost regimen and may require immunization with additional
Ags to achieve T cell-mediated protection or further optimization of
the regimen.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induction of nitric oxide that apparently mediates killing
of developing liver stage parasites (2, 26). One method
for increasing the protection from a DNA vaccine is to include more
than one malarial Ag. With mixtures of plasmids, immune responses are
made independently to each plasmid Ag, and protection is apparently
additive (2). However, even with multiple Ags, we have not
consistently protected all animals and all mouse strains against
malaria. Therefore, a major goal has been to improve protection from
our P. yoelii DNA vaccines. Our second goal has been to increase the humoral response to DNA vaccines. Results of the first human trial of a DNA malaria vaccine showed no Ab responses in any volunteer despite the induction of CTL responses in the majority of vaccinees (27). In mice, Ab responses after i.m. plasmid immunization vary widely, from moderately strong to undetectable, depending on the encoded Ag (2). Abs to malaria Ags are an important source of protection, particularly against the blood stages of malaria, and we have tried to devise ways to boost the Ab response to DNA vaccines.
Our third goal has been to decrease the amount of plasmid needed for immunization. In our first clinical trial (27), volunteers received three doses of 20, 100, 500, or 2500 µg of Plasmodium falciparum CSP plasmid DNA. CTL assays were positive more often in the 2500- and 500-µg groups than in the 20- and 100-µg groups. Obviously, reducing the dose would reduce the cost of production of a vaccine that is intended in large part for population groups with few financial resources. Epidermal administration of DNA on gold beads by a gene gun has been shown to require less DNA per dose, often <4 µg (28) but requires a complex technology that may be impractical for a vaccine which will be used in the developing world. Furthermore, in a recent study of a simian HIV DNA vaccine, rhesus monkeys primed with DNA administered by the intradermal route and boosted with recombinant fowlpox were protected. However, monkeys primed with the same DNA delivered on gold particles by gene gun were not protected (29).
Recent publications from our laboratory have identified two methods for increasing both CD8 T+ cell responses and Abs to pPyCSP. When plasmid encoding murine GMCSF is mixed with PyCSP plasmid, protection is doubled and Abs to PyCSP increase 8-fold, and CD4+ and CD8+ T cell responses increase 4-fold (3). It appears that the GMCSF causes an influx of APC into the injected muscle, leading to stronger Ag-specific immune responses.5 The second novel approach we have described is combining plasmid vaccination as a first dose followed by a vaccination with a recombinant poxvirus expressing the same Ag as the second dose (6). This "DNA prime-viral boost" technique increases protective efficacy, Abs, and T cell responses. We do not understand why the combination of DNA followed by poxvirus is superior to two doses of DNA or two doses of virus, but recombinant poxviruses also boost immune responses primed by recombinant adenoviruses (30). Similar data with the DNA prime-viral boost have been generated by other laboratories studying malaria in mice (7, 8) and simian HIV in rhesus monkeys (29).
In this paper, we describe the combination of these two approaches: priming with pPyCSP + pGMCSF plasmids followed by boosting with recombinant poxvirus. The results are remarkable. Using this combined method, we have been able simultaneously to achieve all three of our goals for plasmid DNA vaccination: protective efficacy and T cell responses were increased; Abs were enhanced; and we were able to reduce the dose of DNA required for priming by 1–2 logs. If this same approach can be made to work in humans, it will be an important advance toward an effective malaria vaccine.
Despite these impressive results, there are important shortcomings of
the prime-boost vaccine, in terms of both our understanding and
application. As mentioned above, we are still unsure how GMCSF enhances
priming and why poxvirus boosting is so effective. Also, we have not
completely defined the immune effector mechanisms that are responsible
for immunity in the prime-boost model of PyCSP in BALB/c
mice. In all our previous experience, in vivo treatments of immunized
mice with mAbs to CD8+ T cells, or to IFN-
always abrogated immunity. This has allowed us to conclude
unambiguously that these two aspects of the immune response were most
significant in killing malaria parasites. However, these in vivo
depletions have not completely neutralized immunity in the prime-boost
experiments. This may indicate that additional arms of the protective
immune response such as specific Abs or CD4+ T
cells are playing an important role in protecting these mice.
Alternatively, our inability to abrogate the protective immune
responses with anti-CD8 and anti-IFN- mAbs may simply
reflect the very high levels of immune responses, which cannot be
completely neutralized by the in vivo Ab treatments we used.
Preliminary analysis of our data by multiple regression techniques also
seems to indicate that the high levels of specific Ab in our mice may
be contributing to the high levels of protection.
Although BALB/c mice have superior protection from the prime-boost PyCSP vaccine, protection in other strains of mice is much less complete. A/J mice have a modest level of protection (45%) from this same vaccine, whereas C57BL/6 mice show little if any protection. Although both the A/J and C57BL/6 of these strains show important increases in specific serum Abs from the prime-boost vaccine, the levels in the C57BL/6 mice are not as high. In the small number of animals tested, depletion of CD8+ T cells eliminated protection in the AJ mice previously protected, whereas 60% of mice receiving control Abs were still protected (>10 wk since the last immunization). Seeing protection in a previously unprotected strain (A/J) just by improving the immunization regimen suggests that further optimization of immunization regimens may increase this protection further. We are optimistic that the inclusion of several parasite Ags in the prime-boost format will allow an expanded vaccine to protect a wider range of mouse strains. An analogous multigene vaccine would hopefully be similarly effective in a wide range of human HLA types.
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Acknowledgments |
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Footnotes |
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2 The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy or the Naval service at large. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals (31).
3 Address correspondence and reprint requests to Dr. S. L. Hoffman, Malaria Program, Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500.
4 Abbreviations used in this paper: pPyCSP, circumsporozoite protein plasmid DNA; PyCSP(rvPyCSP), recombinant vaccinia virus expressing; pGMCSF, murine GM-CSF plasmid DNA; ELISPOT, enzyme-linked immunospot; IFAT, indirect fluorescent Ab test; ILSDA, inhibition of liver stage development assay.
5 D. Haddad. Granulocyte-macrophage colony-stimulating factor expressing plasmid vaccine elicits highly localized infiltrates of immature dendetric cells in injected muscles. Submitted for publication.
Received for publication December 3, 1999. Accepted for publication March 21, 2000.
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