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*
Malaria Program, Naval Medical Research Institute, Bethesda, MD 20889;
Section of Retroviral Immunology, Division of Viral Products, Center for Biologics Research and Evaluation, Food and Drug Administration, Bethesda, MD 20892;
University of Maryland, Baltimore, MD 21201; and
§
Vical, Inc., San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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responses of CD8+ T cells to the PyCSP 280–288 amino acid
epitope increased but CTL activity did not change. The most dramatic
changes after adding GM-CSF plasmid were increases in Ag-specific IL-2
production and CD4+ T cell proliferation. We hypothesize
that GM-CSF may act on dendritic cells to enhance presentation of the
PyCSP Ag, with enhanced IL-2 production and CD4+ T cell
activation driving the increases in Abs and CD8+ T cell
function. Recombinant GM-CSF is already used in humans for medical
purposes, and GM-CSF protein or plasmids may be useful as enhancers of
DNA vaccines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(8), and nitric oxide
(11, 12, 13). Several Py proteins have been identified as targets of these
immune responses, among them is Py circumsporozoite protein (PyCSP)
(14). This protein has well-characterized epitopes for Abs (15, 16),
CD4+ (4), and CD8+ (14) T cells, making it a
useful tool for monitoring a range of immune responses. In addition,
one DNA plasmid vaccine encoding the PyCSP fused to 82 amino acids (aa)
of IL-2 protected 54% of BALB/c mice against sporozoite
infection (17). Granulocyte-macrophage CSF (GM-CSF) is a glycoprotein of 127 aa in humans that was first described as a growth factor for stem cells of the granulocyte and macrophage lineages (18). Subsequently, GM-CSF has been found to have effects on many cell types, both bone marrow derived and somatic. In particular, GM-CSF enhances the maturation of dendritic cell precursors (19). GM-CSF recombinant protein has been used as a vaccine adjuvant with hepatitis B vaccine in humans, where it led to Ab production after a single immunization (20). GM-CSF has also been been studied as an adjuvant of DNA vaccines in rodent models (21, 22, 23, 24, 25). However, in none of the published studies was in vivo protection measured as well as the range of Ab and T cell effects of GM-CSF. We now report that mixing GM-CSF plasmid with the PyCSP plasmid vaccine enhances protection against malaria and increases B cell, CD8+ T cell, and especially CD4+ T cell responses to PyCSP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PyCSP encoding plasmid used in these studies, PyCSP1012, was created by PCR amplification of the DNA sequence encoding the PyCSP from the plasmid nkCMVintPyCSP.1 (17) and ligation into the plasmid VR1012 (26). Transfection of UM449 human melanoma cells with plasmid VR2507 led to expression of PyCSP, as assessed by Western immunoblotting analysis using an anti-PyCSP mAb NYS1 (27) (data not shown). The plasmid encoding murine GM-CSF was produced in the VR1019 plasmid, which is a version of the VR1012 plasmid with the addition of a leader element from rat preproinsulin II (28). The plasmid encoding mouse GM-CSF mutated at amino acids 15 (H to A) and 21 (E to A) was produced by cloning cDNA provided by Dr. M. Prystowsky (29, 30) into the VR1019 plasmid. Plasmids for immunization were purified by double cesium banding and diluted in normal saline. Endotoxin levels were less than 0.6 EU/mg.
In vitro expression
UM449 cells were provided by Dr. Peter Hobart (Vical, Inc., San Diego, CA). Transient transfections with plasmid constructs for Western blot analysis were performed as described previously (31). Ab and positive control protein for GM-CSF studies were polyclonal rabbit anti-mouse GM-CSF Ab and recombinant mouse GM-CSF produced in yeast, both from (Genzyme, Cambridge, MA). We used the M-NFS-60 cell line as a assay of bioactive murine GM-CSF (32), a gift of Dr. Drew Pardoll (Johns Hopkins University, Baltimore MD).
Cell lines
P815, EL4, and A20 cell lines were purchased from the American Type Culture Collection (Manassas VA).
Animals
BALB/cByJ, B10.D2, and B10.Q female mice were purchased from the The Jackson Laboratories (Bar Harbor, ME). Mice received their first immunization at 4 to 6 wk of age.
Immunization procedure
Mice were immunized two times at 6-wk intervals. A total volume of 50 µl was injected into the tibialis anterior muscle of each leg. Plasmids were mixed and injected in the same syringe. Unless otherwise mentioned, immunizations were with 50 µg of PyCSP1012 plasmid and 50 µg of GM-CSF plasmid at each site.
Sporozoite infection
Three weeks after the second DNA immunization, mice were challenged by i.v. injection with 100 Py sporozoites. To document malaria infection, Giemsa-stained blood films were examined on days 7, 11, and 14 after challenge.
Recombinant protein and synthetic peptides
All peptides were kindly provided by Dr. G. Corradin (University of Lausanne, Epalinges, Switzerland). Peptides corresponding to PyCSP 57–70 aa (KIYNRNIVNRLLGD), 58–67 aa(IYNRNVRL), 280–295 aa (SYVPSAEQILEFVKQI), and 280–288 aa (SYVPSAEQI) were used for in vitro T cell studies (4, 14). For capture Ags in ELISAs of serum Abs, we used the PyCSP repeat peptide (QGPGAP)x4 (27) and the PyCS.1 recombinant protein comprising PyCSP 64–321 aa fused to 81 aa of the nonstructural protein of influenza A (15).
In vitro T cell responses
Spleen were harvested 3 wk after the second DNA immunization unless otherwise stated. Depletion of CD4+ and CD8+ cells was accomplished by negative selection using Ab coated magnetic beads from Dynal (Lake Success, NY) or Miltenyi Biotec (Auburn CA), using the manufacturer’s methods.
Lymphocyte proliferation studies
Spleen cells were cultured in 96-well flat-bottom plates at 2.5 x 105 cells per well at 37°C and 5% CO2. Medium was DMEM supplemented with 10% FCS, 5000 units penicillin/streptomycin, and L-glutamine. After 5 days of culture, 1 µCi of tritiated thymidine in 10 µl volume was added to each well, and on day 6 cultures were harvested and radioactivity incorporated into DNA was measured by scintillation counting.
Enzyme-linked immunospot (ELISPOT)
ELISPOT assays for production of cytokines by T cells were
performed by using three different methods. 1) Ag-specific IFN-
secreting cells were detected by culturing spleen cells overnight with
P815 cells pulsed with PyCSP peptide (aa 280–288) as previously
published (33). 2) Lymph node or spleen cells were taken directly from
mice and tested for spontaneous IFN- and IL-4 production as
previously published (34, 35, 36). 3) Spleen cells were cultured with
soluble PyCSP peptide (aa 280–295 or aa 57–70) for 6 h, and
cells were assayed by ELISPOT for production of IL-2, IL-4, IL-6,
IL-10, IL-12, IFN-, and GM-CSF in a modification of a previously
published assay (34) Briefly, serial 5-fold dilutions of a single cell
suspension starting from 1 x 106 cells/well were
incubated with or without 3 µM of synthetic PyCSP peptide or Con A
for 6 h at 37°C, 5% CO2. Culture was in 96-well
plates coated with anti-cytokine Abs. The wells were overlaid with
0.05 ml of 1 µg/ml biotinylated anti-cytokine Ab for 2 h,
then developed by treated with avidin conjugated alkaline phosphatase
and 5-bromo-4-chloro-3-indolylphosphate/nitroblue
tetrazolium to reveal spots corresponding to the position of cytokine
secreting cells.
ELISA
ELISAs for detection of Abs to PyCSP repeat peptide (QGPGAP)x4
(27) and the PyCS.1 recombinant protein (15) were as previously
described. ELISAs for detection of IL-4 and IFN- after in vitro
restimulation were as previously described (37). Briefly, in a 96-well
flat-bottom plate, 1.5 million spleen cells per well were cultured for
3 days with synthetic PyCSP peptides (aa 280–295 or aa 57–70) at a
concentration of 3 µM. Con A or medium were used in control wells.
After 72 h incubation at 37°C, 5% CO2, supernatants
were collected and diluted 1:5 with a solution of 1% BSA in PBS. These
supernatants were then used in ELISA assays for IFN- and IL-4. In
some experiments, spleen cells were depleted of CD8+ or
CD4+ cells before culture.
CTL assays
CTL assays using the PyCSP 280–288 aa Kd-restricted epitope were performed as previously described (14). Briefly, 5 million spleen cells were cultured at 37°C and 5% CO2 in wells of a 24 well plate in 2 ml of medium containing DMEM supplemented with 10% FCS, 5000 units penicillin/streptomycin, and L-glutamine. PyCSP 280–295 aa peptide was added to a 2 µM concentration. Forty-eight hours later, 20 units per ml of recombinant human IL-2 (Cetus, San Francisco, CA) was added, and cultures continued for an additional 5 days, when they were used as effector cells. Target cells were P815, A20, or EL4 cells cultured overnight with Cr-51 and 1 µM PyCSP 280–288 aa peptide. CTL assays using the PyCSP 57–70 and 58–67 aa epitopes were run by using these peptides in the same protocol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were immunized with the PyCSP1012 plasmid
vaccine alone or mixed with GM-CSF plasmid. Mice were challenged with a
large inoculum of 100 Py sporozoites, as in our experience 1 to 10
sporoites is enough to infect 50% of normal mice. Animals were
followed through day 14 for the development of parasitemia. As is usual
with Py, infected mice developed parasitemias of 20% whereas protected
mice had no detectable parasites. Although the fraction of mice
protected from sporozoite challenge varied between experiments
(probably due to differences in parasite viability/virulence), the
inclusion of GM-CSF with the vaccine invariably improved protection,
nearly doubling efficacy (Table I
). When
the fraction of protected mice in each experimental group was summed
over the six experiments, there was a statistically significant
enhancement of protection when GM-CSF and PyCSP1020 were
mixed together (p < 0.0001 
2,
Yates corrected). The addition of plasmid encoding mutated GM-CSF did
not increase protection compared with PyCSP1020 plus
control (p = 0.55, Fisher’s exact test). The
GM-CSF-encoding plasmid alone did not protect.
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A), yet only the native form
of GM-CSF showed activity in the bioassay for GM-CSF (Fig. 1B). When coinjected with the PyCSP1012 plasmid,
the plasmid encoding the mutated form of GM-CSF did not increase
protective immunity in vivo (Table I
). From these experiments we
conclude that increased protection is not due to inherent stimulatory
properties of the injected DNA itself, but depends on the encoded
GM-CSF protein.
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A, as little as 3 µg of
plasmid enhanced protection.
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B).
B10.Q mice are known to be poor responders to the PyCSP DNA vaccine
(11), although they are protected by Py sporozoite immunization and
make Abs against the parasite (42). B10.Q mice were not protected by
the combination of PyCSP1012 plus GM-CSF plasmids (Fig. 2B), nor did they make Abs to PyCSP (data not shown). In vitro studies of the effects of GM-CSF plasmid
Because GM-CSF plasmid enhanced protection, we began a series of in vitro studies to determine which aspects of the immune response were influenced by adding this plasmid. Using previously defined epitopes on the PyCSP, we looked at CD8+ and CD4+ T cell responses, cytokine production, and Ab levels.
Ag-specific CD8+ T cell responses were measured by
two methods: IFN- ELISPOT and chromium release assay. In the IFN-
assay, spleen cells from mice immunized with or without GM-CSF plasmid
were cultured in vitro overnight with syngeneic P815 cells pulsed with
the PyCSP 280–288 aa peptide which binds to MHC class I
Kd. Mice immunized with both GM-CSF plus PyCSP
plasmids had 3- to 4-fold more cells secreting IFN- than did mice
immunized with the PyCSP1012 plasmid alone or mixed with
control plasmid p = 0.039 (Fig. 3A). When spleen cells were
depleted before culture using magnetic beads coated with anti-CD4
or anti-CD8 Abs, IFN- production was entirely associated with
the CD8+ cell population (data not shown). In all cases,
cells cultured with P815 cells that had been pulsed with an irrelevant
(control) peptide were not stimulated to secrete IFN- (data not
shown).
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B). Lytic activity was almost
entirely due to CD8+ T cells, as determined by magnetic
bead depletion of effector cell cultures. Immunization with GM-CSF
plasmid did not enhance the amount of specific lysis measured. Although
Figure 3B shows only data for effector:target ratio =
80:1, specific lysis at 40:1 and 20:1 also unchanged (data not shown).
T cell proliferation to the PyCSP 57–70 aa peptide was greatly
increased by the inclusion of GM-CSF plasmid (Fig. 4). This proliferation was dependent on
CD4+ T cells, as magnetic bead depletion of
CD8+ cells at the start of culture had no effect on
thymidine uptake, while depletion of CD4+ cells or
Th1+ cells eliminated all thymidine uptake (data not
shown). There was no detectable thymidine uptake in cultures with the
PyCSP 280–295 aa peptide (data not shown).
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, we measured cytokine secretion from unfractionated spleen
cells after 6-h in vitro culture with PyCSP 57–70 aa or PyCSP 280–295
aa peptides in solution (Fig. 5). No
Ag-specific responses were seen for IL-6, IL-12, or GM-CSF (data not
shown). Small increases in Ag-specific IL-4 were detected only in mice
immunized with PyCSP1012 plus GM-CSF plasmids, and only
with the PyCSP 57–70 aa peptide. Immunization with
PyCSP1012 plasmid either alone or with GM-CSF led to IL-10
production which was decreased by the restimulation with either
peptide. A 2-fold increase in numbers of cells secreting Ag-specific
IFN- to either peptide was detected in mice immunized with the
addition of GM-CSF plasmid. Adding GM-CSF plasmid caused a large
increase in the numbers of IL-2 secreting cells present after
restimulation with either peptide.
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after peptide
stimulation, the ELISPOT experiments were repeated after magnetic bead
depletion of cell subsets (data not shown). Bead depletion of
CD8+ cells completely eliminated IL-2 and IFN- producing
cells after PyCSP 280–295 peptide restimulation, but only reduced by
half the numbers of IL-2 and IFN- producers from PyCSP 57–70
stimulated cultures This is consistent with the hypothesis that
280–295 is primarily an epitope recognized by class I-restricted
CD8+ T cells, whereas 57–70 contains both class
I4 and class II-restricted
epitopes for CD8+ and CD4+ cells, respectively.
This interpretation was confirmed by IFN- ELISA assay. Spleen cells
from mice immunized with PyCSP1012 plasmid alone or with
GM-CSF plasmid were depleted of CD4+ or CD8+
cells, and cultured with peptide PyCSP 280–295 aa or PyCSP 57–70 aa
at 3 µM final concentration for 72 h. Culture supernatants were
assayed for IFN- by ELISA. In accordance with the ELISPOT results,
coimmunization with GM-CSF plasmid increased Ag-specific IFN-
production after stimulation with either peptide (data not shown).
Depletion of CD8+ cells but not CD4+ cells
eliminated IFN- production with PyCSP 280–295 aa peptide. Depletion
of either CD4+ or CD8+ cells eliminated only
half of the IFN- produced after stimulation with PyCSP 57–70 aa
peptide.
To examine the kinetics of T cell activity after immunization, the
number of spleen cells spontaneously secreting IL-4 or IFN- were
monitored by ELISPOT assay (Fig. 6). Mice
immunized with the mixture of PyCSP1012 and GM-CSF plasmids
had larger numbers of IFN- secreting cells that remained elevated
longer after the second immunization. As we previously described (26),
IL-4 producing cells were present after the first immunization and then
declined in all groups (data not shown).
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).
Administration of PyCSP1012 plasmid alone induced only a
modest increase in IgG Abs 3 wk after primary immunization and an
anamnestic response 2 wk postboost. By comparison, coadministration of
the GM-CSF plasmid significantly increased total IgG, IgG1, and IgG2a
anti-CS.1 serum titers. IgG2b was also increased but IgG3 and IgM
were not (data not shown). A similar increase in PyCSP-specific Ab
titer in mice coimmunized with PyCSP1012 plasmid plus
GM-CSF plasmid was seen using the (QGPGAP)x4 repeat peptide in the
ELISA assay (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Coadministering plasmid encoding murine GM-CSF with
PyCSP1012 plasmid reproducibly enhanced protection in
BALB/c mice. Initial screening experiments mixing PyCSP1012
plasmid with other plasmids encoding murine IL-2, IL-4, IFN-, IL-12,
IL-15, B7.1, or B7.2 showed no large or consistent increases in the
number of mice protected (W. R. Weiss, unpublished observations).
We therefore focused our work on GM-CSF plasmid.
Our initial questions regarded the mechanism by which the addition of GM-CSF plasmid enhanced protection. One possibility was that immunostimulatory DNA sequences in the GM-CSF plasmid were increasing responses to the PyCSP1012 plasmid. In this case, enhancing effects would not depend on the production of bioactive GM-CSF in vivo, but only on the primary structure of the DNA. To test this hypothesis, we constructed a plasmid encoding an altered GM-CSF, identical to the first plasmid except for changes at two amino acids required for binding to the GM-CSF receptor. The mutant GM-CSF plasmid produced a protein product in in vitro transfections; however, mixing the mutant GM-CSF plasmid with PyCSP1012 plasmid gave no increased protection against malaria infection. We believe that these data largely eliminate the possibility that direct stimulatory effects of the plasmid DNA are responsible for enhanced protection. We infer that it is the production of bioactive GM-CSF protein after injection which is critical for the enhancing effect.
In vivo production of GM-CSF might increase protection of a PyCSP1012 plasmid vaccine by either of two mechanisms: GM-CSF could be killing the parasite by its direct action, or it could be increasing the immune response to PyCSP1012. To measure the direct effect of GM-CSF on the parasite, we injected mice with GM-CSF plasmid alone and found that it gave no protection against a challenge with 100 Py sporozoites. Although this is not definitive evidence, it suggests that GM-CSF does not directly kill the malaria parasite at the levels produced by plasmid injection. We are currently studying the effects of recombinant mouse GM-CSF on protection against malaria, but our hypothesis now is that GM-CSF has its most important effect as a modulator of the immune response to PyCSP1012 plasmid.
Immunization with PyCSP encoding plasmid alone induces protection
dependent on CD8+ effector T cells (17). We have not yet
defined the immune responses that are responsible for protection after
immunization with PyCSP1012 plus GM-CSF. However, in vitro
studies provide insights into the possible mechanisms of increased
protection. Inclusion of GM-CSF plasmid stimulated a rapid increase in
the number of spleen cells capable of secreting Ag-specific IL-2 and
IFN-. IL-4 production was increased early and then declined, whereas
IL-6, IL-10, IL-12, and GM-CSF production were not altered. Despite the
enhancement of Th1-type cytokines, serum Abs of IgG1, IgG2a, and IgG2b
isotypes were equally increased, indicating that the effect of GM-CSF
on Ab production is complex and cannot be explained by the Th1 vs Th2
dichotomy. CD4+ T cell proliferation and cytokine responses
to the PyCSP 57–70 aa epitope became prominent, increasing 10- to
100-fold. Finally, the addition of GM-CSF more than tripled the
frequency of CD8+ T cells responding to the PyCSP 280–288
aa epitope as measured by IFN- or IL-2 ELISPOT. However, CTL
activity of CD8+ T cells to the same 280–288 aa epitope
was not changed by the inclusion of GM-CSF plasmid. If this is not a
technical artifact due to the different sensitivities of the two
assays, it may indicate that there are two distinct populations of
Ag-specific CD8+ T cells, and that enhanced protection is
associated with expansion of the subpopulation making cytokines- but
without lytic activity. This is consistent with data showing that in
many models of pre-erythrocytic malaria immunity, protection can be
eliminated by neutralizing CD8+ T cells (8, 9), IFN-
(8), or nitric oxide (11, 12, 13).
Kinetic studies of cytokine production in spleen showed that GM-CSF
both accelerates and increases the magnitude of immune responses.
Coimmunization stimulated a more rapid rise in the number of cells
secreting IFN– in vivo, and a higher and prolonged response to in
vitro restimulation with a PyCSP peptide. Similarly, Ab levels to PyCSP
appeared earlier and at higher titers in animals receiving GM-CSF
plasmid.
There have been several recent reports involving the immune-enhancing properties of GM-CSF plasmid. Xiang and Ertl (21) coinjected the GM-CSF plasmid with a plasmid encoding a rabies protein and found enhanced Ab production and increased protection in mice. That report did not examine CTL or Th responses. Iwasaki et al. (23) injected GM-CSF plasmid plus a plasmid encoding influenza NP and found that CTL responses were enhanced but did not report on Ab or Th responses. Kim et al. (22) mixed GM-CSF plasmid with plasmids encoding proteins from HIV-1 and found increased Ab production and T cell proliferation, but no increase in CTL. Geissler et al. (24) administered hepatitis C core protein with GM-CSF encoding plasmid and found an increase in Abs, a small increase in T cell proliferation, but no change in cytokine secretion or CTL activity. Okada et al. (25) used GM-CSF plasmid along with plasmid encoding HIV env protein given intranasally in liposomes and found enhancement of both Ab and CTL activity. Thus, the literature on GM-CSF plasmid immunization consistently describes increases in Abs and T cell proliferation, whereas the enhancement of other T cell responses has been inconsistent.
GM-CSF can act on many cell types (reviewed in 19 . Macrophages, dendritic cells, Langerhans cells, eosinophils, granulocytes, megakaryocytes, fibroblasts, and red blood cell precursors among others can respond to GM-CSF. We hypothesize that GM-CSF is activating dendritic cells, leading to the enhanced presentation of PyCSP epitopes. It is known that bone marrow-derived cells (such as the dendritic cell) are required for presentation of DNA plasmid Ags after i.m. injection (43, 44, 45). We suggest that GM-CSF-activated dendritic cells are better able to present Ag to naive T cells. We are particularly impressed by the enhanced CD4+ T cell responses we have measured. It is possible that better CD4+ T cell induction would lead to increased B cell and CD8+ T cell responses against some epitopes. However, we do not understand why some CD8+ T cell responses are boosted and some are not. Neither do we understand how MHC controls GM-CSF responsiveness, as seen in our experiments with B10.Q(H-2q) mice. We are currently investigating these aspects of GM-CSF enhancement.
We believe that a plasmid encoding human GM-CSF may be clinically useful in enhancing DNA vaccines in humans. Recombinant human GM-CSF is used in patients to stimulate cell growth from bone marrow cells in several clinical settings (46), and its potential toxicities are well defined (20, 46, 47, 48). Human GM-CSF has already been tested as an adjuvant in the immunotherapy of human cancer (49) and to enhance Ab responses to recombinant hepatitis B vaccine (20). We have seen no evidence of ill effects in mice given GM-CSF plasmids. If our plasmid dose/response studies in mice are a guide, it should be possible to avoid systemic toxicity by using a very small amount of human GM-CSF plasmid to achieve adjuvant effects. If safety concerns related to injecting a human cDNA can be addressed, we are optimistic that DNA vaccines in humans can be enhanced by the addition of either plasmids encoding GM-CSF or recombinant GM-CSF cytokine.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Walter R. Weiss, Naval Medical Research Institute, 12300 Washington Avenue, Rockville, MD 20852.
3 Abbreviations used in this paper: Py, Plasmodium yoelii; PyCSP, Py circumsporozoite protein; aa, amino acid(s); GM-CSF, granulocyte-macrophage CSF; ELISPOT, enzyme-linked immunospot.
4 E. D. Franke, A. Sette, J. Sacci, Jr., S. Southwood, G. Corradin, and S. L. Hoffman. A subdominant CD8+ CTL epitope from the Plasmodium yoelii circumsporozoite protein induces CTL activity and partial protection against sporozoite challenge. Submitted for publication.
Received for publication January 27, 1998. Accepted for publication April 23, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent elimination of infected hepatocytes. J. Immunol. 157:4061.[Abstract]
-Interferon, CD8+ T cells, and antibodies required for immunity to malaria sporozoites. Nature 330:664.[Medline]
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. Proc. Natl. Acad. Sci. USA 93:2879.This article has been cited by other articles:
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 K. Teramoto, K. Kontani, Y. Ozaki, S. Sawai, N. Tezuka, T. Nagata, S. Fujino, Y. Itoh, O. Taguchi, Y. Koide, et al. Deoxyribonucleic Acid (DNA) Encoding a Pan-Major Histocompatibility Complex Class II Peptide Analogue Augmented Antigen-specific Cellular Immunity and Suppressive Effects on Tumor Growth Elicited by DNA Vaccine Immunotherapy Cancer Res., November 15, 2003; 63(22): 7920 - 7925. [Abstract] [Full Text] [PDF]
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A. B. Ismael, D. Sekkai, C. Collin, D. Bout, and M.-N. Mevelec The MIC3 Gene of Toxoplasma gondii Is a Novel Potent Vaccine Candidate against Toxoplasmosis Infect. Immun., November 1, 2003; 71(11): 6222 - 6228. [Abstract] [Full Text] [PDF]
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M. Sedegah, M. Belmonte, J. E. Epstein, C.-A. Siegrist, W. R. Weiss, T. R. Jones, M. Lu, D. J. Carucci, and S. L. Hoffman Successful Induction of CD8 T Cell-Dependent Protection Against Malaria by Sequential Immunization with DNA and Recombinant Poxvirus of Neonatal Mice Born to Immune Mothers J. Immunol., September 15, 2003; 171(6): 3148 - 3153. [Abstract] [Full Text] [PDF]
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D. H. Barouch, P. F. McKay, S. M. Sumida, S. Santra, S. S. Jackson, D. A. Gorgone, M. A. Lifton, B. K. Chakrabarti, L. Xu, G. J. Nabel, et al. Plasmid Chemokines and Colony-Stimulating Factors Enhance the Immunogenicity of DNA Priming-Viral Vector Boosting Human Immunodeficiency Virus Type 1 Vaccines J. Virol., August 15, 2003; 77(16): 8729 - 8735. [Abstract] [Full Text] [PDF]
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A. A. Siddiqui, T. Phillips, H. Charest, R. B. Podesta, M. L. Quinlin, J. R. Pinkston, J. D. Lloyd, M. Paz, R. M. Villalovos, and J. Pompa Induction of Protective Immunity against Schistosoma mansoni via DNA Priming and Boosting with the Large Subunit of Calpain (Sm-p80): Adjuvant Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-4 Infect. Immun., July 1, 2003; 71(7): 3844 - 3851. [Abstract] [Full Text] [PDF]
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W. Mwangi, W. C. Brown, H. A. Lewin, C. J. Howard, J. C. Hope, T. V. Baszler, P. Caplazi, J. Abbott, and G. H. Palmer DNA-Encoded Fetal Liver Tyrosine Kinase 3 Ligand and Granulocyte Macrophage-Colony-Stimulating Factor Increase Dendritic Cell Recruitment to the Inoculation Site and Enhance Antigen-Specific CD4+ T Cell Responses Induced by DNA Vaccination of Outbred Animals J. Immunol., October 1, 2002; 169(7): 3837 - 3846. [Abstract] [Full Text] [PDF]
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W. O. Rogers, W. R. Weiss, A. Kumar, J. C. Aguiar, J. A. Tine, R. Gwadz, J. G. Harre, K. Gowda, D. Rathore, S. Kumar, et al. Protection of Rhesus Macaques against Lethal Plasmodium knowlesi Malaria by a Heterologous DNA Priming and Poxvirus Boosting Immunization Regimen Infect. Immun., August 1, 2002; 70(8): 4329 - 4335. [Abstract] [Full Text] [PDF]
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M. Sedegah, G. T. Brice, W. O. Rogers, D. L. Doolan, Y. Charoenvit, T. R. Jones, V. F. Majam, A. Belmonte, M. Lu, M. Belmonte, et al. Persistence of Protective Immunity to Malaria Induced by DNA Priming and Poxvirus Boosting: Characterization of Effector and Memory CD8+-T-Cell Populations Infect. Immun., July 1, 2002; 70(7): 3493 - 3499. [Abstract] [Full Text] [PDF]
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D. H. Barouch, S. Santra, K. Tenner-Racz, P. Racz, M. J. Kuroda, J. E. Schmitz, S. S. Jackson, M. A. Lifton, D. C. Freed, H. C. Perry, et al. Potent CD4+ T Cell Responses Elicited by a Bicistronic HIV-1 DNA Vaccine Expressing gp120 and GM-CSF J. Immunol., January 15, 2002; 168(2): 562 - 568. [Abstract] [Full Text] [PDF]
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W. O. Rogers, J. K. Baird, A. Kumar, J. A. Tine, W. Weiss, J. C. Aguiar, K. Gowda, R. Gwadz, S. Kumar, M. Gold, et al. Multistage Multiantigen Heterologous Prime Boost Vaccine for Plasmodium knowlesi Malaria Provides Partial Protection in Rhesus Macaques Infect. Immun., September 1, 2001; 69(9): 5565 - 5572. [Abstract] [Full Text] [PDF]
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O. Bruna-Romero and A. Rodriguez Dendritic Cells Can Initiate Protective Immune Responses against Malaria Infect. Immun., August 1, 2001; 69(8): 5173 - 5176. [Abstract] [Full Text] [PDF]
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 J. D. Ahlers, I. M. Belyakov, S. Matsui, and J. A. Berzofsky Mechanisms of cytokine synergy essential for vaccine protection against viral challenge Int. Immunol., July 1, 2001; 13(7): 897 - 908. [Abstract] [Full Text] [PDF]
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G. I. Sanchez, M. Sedegah, W. O. Rogers, T. R. Jones, J. Sacci, A. Witney, D. J. Carucci, N. Kumar, and S. L. Hoffman Immunogenicity and Protective Efficacy of a Plasmodium yoelii Hsp60 DNA Vaccine in BALB/c Mice Infect. Immun., June 1, 2001; 69(6): 3897 - 3905. [Abstract] [Full Text] [PDF]
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M. Biondo, Z. Nasa, A. Marshall, B. Hock Toh, and F. Alderuccio Local Transgenic Expression of Granulocyte Macrophage-Colony Stimulating Factor Initiates Autoimmunity J. Immunol., February 1, 2001; 166(3): 2090 - 2099. [Abstract] [Full Text] [PDF]
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J. Riopel, M. Tam, K. Mohan, M. W. Marino, and M. M. Stevenson Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice Have Impaired Resistance to Blood-Stage Malaria Infect. Immun., January 1, 2001; 69(1): 129 - 136. [Abstract] [Full Text] [PDF]
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E. Kass, D. L. Panicali, G. Mazzara, J. Schlom, and J. W. Greiner Granulocyte/Macrophage-Colony Stimulating Factor Produced by Recombinant Avian Poxviruses Enriches the Regional Lymph Nodes with Antigen-presenting Cells and Acts as an Immunoadjuvant Cancer Res., January 1, 2001; 61(1): 206 - 214. [Abstract] [Full Text]
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G. Wunderlich, I. C. Moura, and H. A. del Portillo Genetic Immunization of BALB/c mice with a Plasmid Bearing the Gene Coding for a Hybrid Merozoite Surface Protein 1-Hepatitis B Virus Surface Protein Fusion Protects Mice against Lethal Plasmodium chabaudi chabaudi PC1 Infection Infect. Immun., October 1, 2000; 68(10): 5839 - 5845. [Abstract] [Full Text] [PDF]
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R. Weiss, W. W. Leitner, S. Scheiblhofer, D. Chen, A. Bernhaupt, S. Mostbock, J. Thalhamer, and J. A. Lyon Genetic Vaccination against Malaria Infection by Intradermal and Epidermal Injections of a Plasmid Containing the Gene Encoding the Plasmodium berghei Circumsporozoite Protein Infect. Immun., October 1, 2000; 68(10): 5914 - 5919. [Abstract] [Full Text] [PDF]
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D. Haddad, J. Ramprakash, M. Sedegah, Y. Charoenvit, R. Baumgartner, S. Kumar, S. L. Hoffman, and W. R. Weiss Plasmid Vaccine Expressing Granulocyte-Macrophage Colony-Stimulating Factor Attracts Infiltrates Including Immature Dendritic Cells into Injected Muscles J. Immunol., October 1, 2000; 165(7): 3772 - 3781. [Abstract] [Full Text] [PDF]
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G. Delogu, A. Howard, F. M. Collins, and S. L. Morris DNA Vaccination against Tuberculosis: Expression of a Ubiquitin-Conjugated Tuberculosis Protein Enhances Antimycobacterial Immunity Infect. Immun., June 1, 2000; 68(6): 3097 - 3102. [Abstract] [Full Text] [PDF]
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M. Sedegah, W. Weiss, J. B. Sacci Jr., Y. Charoenvit, R. Hedstrom, K. Gowda, V. F. Majam, J. Tine, S. Kumar, P. Hobart, et al. Improving Protective Immunity Induced by DNA-Based Immunization: Priming with Antigen and GM-CSF-Encoding Plasmid DNA and Boosting with Antigen-Expressing Recombinant Poxvirus J. Immunol., June 1, 2000; 164(11): 5905 - 5912. [Abstract] [Full Text] [PDF]
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C.-H. Chen, T.-L. Wang, C.-F. Hung, Y. Yang, R. A. Young, D. M. Pardoll, and T-C. Wu Enhancement of DNA Vaccine Potency by Linkage of Antigen Gene to an HSP70 Gene Cancer Res., February 1, 2000; 60(4): 1035 - 1042. [Abstract] [Full Text]
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A. Lobell, R. Weissert, S. Eltayeb, C. Svanholm, T. Olsson, and H. Wigzell Presence of CpG DNA and the Local Cytokine Milieu Determine the Efficacy of Suppressive DNA Vaccination in Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 1999; 163(9): 4754 - 4762. [Abstract] [Full Text] [PDF]
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, PyCSP plasmid with medium; 
,
PyCSP plus GM-CSF plasmids with medium; 
, PyCSP plasmid with peptide
stimulation; 
, PyCSP plus GM-CSF plasmids with peptide
stimulation.