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The Coooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, Brisbane, Queensland;
John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory;
Centre for Immunology and Cancer Research, University of Queensland Department of Medicine, Princess Alexandra Hospital, Brisbane; and
§
Department of Microbiology and Immunology, University of Melbourne, Victoria, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß CTL epitope-based vaccines requires an
effective strategy capable of co-delivering large numbers of CTL
epitopes. Here we describe a DNA plasmid encoding a polyepitope or
"polytope" protein, which contained multiple contiguous minimal
murine CTL epitopes. Mice vaccinated with this plasmid made
MHC-restricted CTL responses to each of the epitopes, and protective
CTL were demonstrated in recombinant vaccinia virus, influenza virus,
and tumor challenge models. CTL responses generated by polytope DNA
plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering
a gene for granulocyte-macrophage CSF, and appeared to be induced in
the absence of CD4 T cell-mediated help. The ability to deliver large
numbers of CTL epitopes using relatively small polytope constructs and
DNA vaccination technology should find application in the design of
human epitope-based CTL vaccines, in particular in vaccines against
EBV, HIV, and certain cancers. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
CTL are known as important mediators of protective immunity against
many viruses (1, 2), tumors (3), intracellular bacteria (4), and
parasites (5). Vaccination modalities capable of safely and effectively
inducing CTL in humans have not proven easy to develop (6). The advent
of DNA-based vaccination strategies not only offers a relatively safe
modality capable of inducing both CTL and Ab, but also allows the
engineering of artificial immunogens and coexpression of
immunomodulatory proteins (7). A conventional DNA vaccination plasmid
might code for a single Ag; however, this may not offer the ideal
strategy for CTL vaccination, as single Ags often do not contain
sufficient numbers of CTL epitopes to span the HLA diversity of a
target population (1). Many pathogens also have multiple strain
variants with altered CTL epitopes (5). In addition, a vaccine that can
induce broad CTL responses against multiple antigenic targets is likely
to be of benefit in diseases such as melanoma, in which individual
target Ags may be down-regulated (3, 8), and HIV, in which individual
epitopes may mutate (9). Such considerations would require a vaccine to
deliver multiple recombinant Ags. Unfortunately, the construction of
single DNA plasmids expressing large numbers of Ags is complicated by
construction and transcriptional interference problems. An alternative
to using large mixtures of individual plasmids is to use an
epitope-based approach whereby minigenes, which only code for an
epitope sequence, are expressed in vaccination plasmids (10, 11).
However, to offer a clear advantage, the minigene strategy needs to be
capable of co-delivering large numbers of epitopes encoded by a single
plasmid. Here, we show that vaccination with a DNA plasmid expressing an artificial polyepitope or "polytope" protein comprising a series of contiguous minimal CTL epitopes was capable of inducing multiple independent MHC-restricted CTL responses. The CTL responses induced by polytope DNA vaccination were shown to be functional in challenge systems, were maintained for 1 yr in vivo, could be enhanced by covaccination with granulocyte-macrophage CSF (GM-CSF),4 and were generated in the absence of an obvious source of CD4 T cell help. A polytope DNA vaccination approach, perhaps combined with an immunomodulatory cytokine, thus offers an ideal strategy for the design of epitope-based CTL vaccines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three plasmids were constructed for DNA vaccination experiments
(Fig. 1
A). The control plasmid, pDNAVacc, was
constructed by removing the mammalian expression cassette from
pCIS.CXXNH (12) using EcoRI and cloning it into
EcoRI-cut pUC8 (13). The expression cassette in pDNA Vacc
contains the CMV promoter, a synthetic intron, a multiple cloning site
(MCS), and the SV40 poly(A) signal sequence. The polytope plasmid,
pSTMPDV, encoded an artificial murine polytope protein (Fig. 1B) comprising 10 contiguous CD8 CTL epitopes (Table I). This plasmid was constructed by
removing the murine polytope gene from pBSMP (14) using SalI
and cloning it into XhoI-cut pDNAVacc. The construction of
the murine polytope gene and its cloning into pBluescript to generate
the plasmid pBSMP has been described previously (14). The GM-CSF
plasmid, pRSVMGMCSF, was constructed by removing the murine GM-CSF cDNA
from the plasmid pZenGMCSF (15) with XhoI and cloning it
into SalI-cut pRSVHygro (16).
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Mice
Specific pathogen-free 6- to 8-wk-old female BALB/c (H-2d) and C57BL/6 (H-2b) mice were obtained from the Animal Resource Centre, Perth, Western Australia.
DNA vaccination of mice
Plasmid DNA for DNA vaccination was prepared by double CsCl gradient purification. Two methods were used to vaccinate mice with DNA, either i.m. injection or particle bombardment, using the Acell II particle bombardment device (gene gun) (Agracetus, Middleton, WI) (18). Mice vaccinated by i.m. injection were first anesthetized and then given a single injection of 50 µg of plasmid DNA (in 50 µl PBS) in the quadriceps muscles, followed 2 to 3 wk later by an identical booster injection. Mice were vaccinated by particle bombardment as described previously (18). Briefly, plasmid DNA was precipitated onto gold beads, then coated onto mylar sheets. The gold particles were then delivered into the abdominal skin of anesthetized mice. In experiments in which two plasmids were co-delivered, the plasmids were mixed at the indicated ratios before precipitation onto gold beads. Mice were vaccinated with 4 µg of plasmid DNA followed by a booster vaccination 2 wk later with 1 µg of plasmid DNA.
Chromium release assays
Three weeks after the booster vaccination (unless stated otherwise), splenocytes from each vaccinated mouse were restimulated for 6 days in vitro by the addition of 1 µg/ml of the indicated synthetic peptide (Chiron Mimotopes, Melbourne, Australia) for BALB/c effectors or, for C57BL/6 mice, by the addition of irradiated (8000 rad), peptide-sensitized (10 µg/ml, 1 h, 37°C), washed EL4 cells (effector:stimulator ratio, 20:1). These effectors were used in standard (6-h) 51Cr release assays against peptide-sensitized target cells. Target cells for effectors from BALB/c (H-2d) and C57BL/6 (H-2b) mice were P815 cells and the EL4 murine thymoma cell line, respectively. Target cells were sensitized for 1 h with 10 µg/ml peptide at 37°C (performed at the same time as chromium labeling) followed by two washes.
Protection assays
Tumor challenge model. Two groups (n = 10) of 6-wk-old female C57BL/6 mice were vaccinated by i.m. injection as described above with either the control or polytope plasmids. Six weeks later, vaccinated mice (n = 5) were injected s.c. in the nuchal region with 1 x 107 cells of either the EL4 murine thymoma line or the E.G7 line (EL4 transfected with OVA) (19) and observed for 10 days. At day 10, the challenged mice were killed and their tumors dissected and weighed.
Vaccinia virus challenge. Groups (n = 5) of 6-wk-old female BALB/c mice were vaccinated with either the control plasmid (i.m. injection), polytope plasmid (gene gun), or the polytope plasmid plus the influenza HA-expressing plasmid (gene gun, ratio 1:1). Three weeks after the booster vaccination, mice were challenged with a recombinant vaccinia virus (1 x 107 pfu in 100 µl PBS i.p.) that expressed the last five CTL epitopes in the murine polytope protein. This vaccinia was constructed as described previously (14) by removing the sequence coding for the first five epitopes by restriction enzymes from the plasmid pSTMOUSEPOLY using XbaI/SpeI and re-ligating before cloning into the vaccinia shuttle vector (14). The relevant epitopes presented by this recombinant vaccinia in this BALB/c challenge system were thus SYIPSAEKI (H-2Kd) and RPQASGVYM (H-2Ld). At day 4 postinfection, both ovaries were removed and homogenized in 1 ml of PBS using an electric grinder. The ovary vaccinia virus titers were then determined by plaque assay on confluent CV1 cells.
Influenza virus challenge. Groups of 6-wk-old BALB/c mice were vaccinated by i.m. injection with either control plasmid (n = 10) or polytope plasmid (n = 10). Three weeks after the booster vaccination, the mice were anesthetized (ketamine:xylazine:water, 1:1:12 i.p.) and infected intranasally with a sublethal dose of the reassortant influenza virus, Mem71 (4.5 x 105 pfu in 50 µl PBS), which has the HA of A/Memphis/1/71 (H3) and the neuraminidase of A/Bellamy/42 (N1)(CSL Ltd., Melbourne, Australia). At day 5, lungs and spleens were removed, and viral titers in each lung were determined in duplicate on the Madin-Darby canine kidney cell line using a standard influenza plaque assay (20).
HA titer determination
Sera of mice vaccinated with the plasmid expressing A/PR/8/34 influenza HA plus the polytope plasmid and mice vaccinated with the polytope plasmid alone were tested for reactivity against A/Puerto Rico/8/34 using an IgG-specific ELISA, described previously (21).
Depletion of CD4 T cells with mAb
BALB/c mice (6–8 wk old) were treated with rat anti-murine CD4 mAb GK1.5 mAb (purified by ammonium sulfate precipitation from ascites fluid) as described previously (22). Control IgG was prepared from rat serum using the same protocol. Purified Ab (1 mg in 2 ml of PBS) was injected i.p. 5 days before i.m. polytope plasmid immunization with 100 µg of DNA. After a further 18 days, the mice were again treated with the mAb, and 5 days later the animals were given a booster dose of 100 µg of polytope plasmid. Three weeks later, the splenocytes were removed and restimulated in vitro for CTL assays as described above. FACS analysis was performed on parallel animals 5 days after Ab treatment.
Parallel mice were immunized twice s.c. in the base of the tail with a recombinant protein in the presence (n = 4) and absence (n = 4) of CD4 depletion, as described above, to monitor the effect of CD4 depletion on CD4-dependent IgG Ab production. The Ag used was the recombinant E7-GST fusion protein (50 µg/mouse) from papilloma virus formulated with Quil A (Spikoside; Iscotec, Uppsala, Sweden) (10 µg/mouse) in 100 µl of saline (23). Serum IgG at 3 wk postboosting were measured by standard ELISA using E7-GST-coated plates, with serum from parallel animals immunized with OVA (Sigma) (n = 4) used as a negative control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasmids that were constructed and used for DNA vaccination
are described in Figure 1
A.
The polytope plasmid encoded the polytope protein (Fig. 1B). The polytope protein comprised 10 contiguous
minimal CTL epitopes, which were derived from five viruses, a parasite,
and a tumor model (Table I). The GM-CSF plasmid encoded murine
GM-CSF.
Induction of CTL by the polytope plasmid
To determine whether vaccination with the polytope plasmid could
induce CTL responses in vivo to the individual epitopes contained
within the polytope protein, groups of mice were vaccinated by i.m.
injection with either the control plasmid or the polytope plasmid. The
splenocytes from individual mice were restimulated with the indicated
peptide and used in chromium release assays against targets sensitized
with the same peptide (Fig. 2, closed
squares). Polytope plasmid-vaccinated BALB/c and C57BL/6 mice generated
effectors specific for each of the four epitopes presented in these two
mouse strains. Restimulated splenocytes from mice vaccinated with the
control plasmid gave no significant specific target cell lysis (Fig. 2, open triangles). These results demonstrated that vaccination with
plasmid DNA coding for a polytope protein, comprising only contiguous
CTL epitopes, could induce independent MHC-restricted CTL responses to
the multiple individual epitopes.
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Ab responses against the linear B cell epitope included at the end
of the murine polytope protein (Fig. 1B) could not be
detected in BALB/c mice vaccinated by i.m. injection with the polytope
plasmid. Western blot assays with sera from these mice did not detect a
GST fusion protein, described previously (24), which contained this B
cell epitope linked to a different (EBV) polytope protein. A mAb
specific for this epitope, 8E7/55 (25), did recognize the fusion
protein in these assays (data not shown). This epitope has previously
been shown to be highly immunogenic as a peptide coupled to diphtheria
toxin (26).
Challenge of polytope plasmid vaccinated mice
Three separate challenge models were used to demonstrate that CTL generated by DNA vaccination with the polytope plasmid were functional in vivo: a tumor, a recombinant vaccinia virus, and a Mem71 influenza virus challenge.
Tumor challenge.
C57BL/6 mice vaccinated with the polytope plasmid by i.m. injection and
given E.G7 cells failed to develop visible E.G7 tumors (Fig. 3A). Mice vaccinated
with the control plasmid developed 0.28 (SD ± 0.19)-gram E.G7
tumors (unpaired Student t test between two groups,
p = 0.014, n = 5). In contrast, mice
challenged with EL4 cells developed significant tumors irrespective of
the plasmid used for vaccination.
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B). Polytope-vaccinated mice had significantly
reduced ovary virus titers compared with mice vaccinated with the
control plasmid (Student t test, p = 0.025,
n = 5).
Influenza challenge.
Groups of BALB/c mice previously vaccinated i.m. with control plasmid
(n = 10) or polytope plasmid
(n = 10) were challenged intranasally with
influenza virus (Mem71, 4.5 x 105 pfu). Lung
influenza virus titers were assayed in duplicate on day 5 after
challenge. Polytope plasmid vaccination resulted in a moderate
(
30%) but significant (Student t test, p
= 0.02, n = 20) reduction in lung influenza virus
titers. This assay is distinct from the lethal dose challenge system
using the A/Puerto Rico/8/34 strain, in which BALB/c mice vaccinated
with the TYQRTRALV epitope failed to show significant protection from
influenza challenge (27, 28). The reassortant Mem 71 virus used in this
study is nonlethal and grows to high titers in mouse lungs before being
cleared by a mechanism that involves CTL (29).
These results demonstrated that SIINFEKL (H-2Kb) (tumor
challenge, Fig. 3A)-, SYIPSAEKI (H-2Kd)
and/or RPQASGVYM (H-2Ld) (vaccinia challenge, Fig. 3B)-, and TYQRTRALV (H-2Kd) (influenza
challenge)-specific CTL induced by DNA vaccination with the polytope
plasmid were active in vivo.
CTL memory in polytope DNA plasmid-vaccinated mice
To determine the longevity of CTL memory following vaccination
with murine polytope, C57BL/6 mice (n = 3) were
vaccinated with the polytope DNA plasmid by particle bombardment (gene
gun). After 12 mo, splenocytes were harvested and CTL assays performed
as shown in Figure 2. CTL responses to all four epitopes restricted by
H-2b were clearly present after this extended period (Fig. 4).
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The polytope plasmid was able to induce CTL responses despite the
absence of an obvious source of CD4 T cell-mediated help. To determine
whether addition of T cell helper epitopes would improve CTL responses,
BALB/c mice (n = 3) were vaccinated with either
the polytope plasmid or the polytope plasmid plus a plasmid coding for
influenza HA (ratio 1:1) by particle bombardment (gene gun). Mice were
vaccinated twice with a 2-wk interval, and splenocytes and sera were
taken 4 wk after the booter vaccination. The splenocytes from
individual mice were restimulated with the four
H-2d-restricted peptides and used in chromium release
assays against targets sensitized with matching peptides (as shown in
Fig. 2). Covaccination of BALB/c mice with the influenza HA plasmid
showed no improvement in the CTL responses over those seen following
vaccination with the murine polytope alone (data not shown).
Sera from HA plasmid-vaccinated animals developed end point IgG titers of 1/9600 ± 5386 (SE), whereas animals that received polytope plasmid alone developed end point titers of 1/40 ± 40 (SE). HA plasmid vaccination had therefore induced HA-specific IgG responses, and therefore presumably also CD4 T cell responses, in these experiments.
To determine whether the CTL generated following covaccination with
influenza HA were more effective in vivo, BALB/c mice
(n = 5) were vaccinated with the polytope
plasmid plus an influenza HA plasmid (ratio 1:1) or the polytope
plasmid alone. These mice were then challenged with recombinant
vaccinia virus. No significant improvement in the reduction of vaccinia
virus titers was observed following covaccination with HA (Fig. 3B).
Thus, addition of HA as a source of CD4 T cell helper epitopes did not significantly improve CTL responses or CTL-mediated protection, indicating that T cell help was not limiting for the generation of CTL responses with the polytope plasmid.
CD4 T cell help: depletion of CD4 T cells with mAb
To determine whether CD4 T cells were important for CTL induction
by the polytope plasmid mice, BALB/c mice (n =
3) were depleted of CD4 T cells twice, using a rat anti-CD4 mAb
(GK1.5), before two DNA vaccinations. FACS analysis showed that: 1)
<3% of CD4 cells remained in the spleens of parallel mice 5 days
after treatment with GK1.5; and 2) at the time of sacrifice, the CD4
numbers had recovered to 15% of normal (data not shown). No
significant overall effect on CTL lysis levels from polytope
DNA-vaccinated mice could be demonstrated following CD4 T cell
depletion (Fig. 5, A and
B). Control animals were treated with rat IgG (Fig. 5C).
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Co-delivery of GM-CSF improved CTL responses
Co-delivery of a plasmid coding for GM-CSF has recently been shown
to enhance CTL responses (30). To examine whether GM-CSF could enhance
CTL responses to multiple epitopes in the murine polytope DNA vaccine,
groups of BALB/c mice (n = 3) were vaccinated
with either the polytope plasmid or the polytope plasmid plus the
GM-CSF plasmid (ratio 4:1) by particle bombardment (gene gun).
Splenocytes from individual mice were restimulated and used as
effectors in CTL assays as described for Figure 2. The lysis values
obtained for SYIPSAEKI-specific effectors were: 11.2% ± 10.5 (SD) (at
an E:T ratio of 50:1), 3.1 ± 3.1 (10:1), and 1.1 ± 0.5
(2:1) for polytope plasmid-vaccinated animals; and 30.7% ± 7.8
(50:1), 10.1 ± 5.5 (10:1), and 3.0 ± 1.2 (2:1) for polytope
plus GM-CSF plasmid-vaccinated animals. For YPHFMPTNL effectors, these
values were 33.7% ± 21.4, 12.3 ± 11.7, 4.6 ± 4.8, and
48.3% ± 10.7, 18.3 ± 6.7, 7.1 ± 2.2 (for GM-CSF
co-delivery); and for RPQASGVYM effectors, 28.5% ± 20.8, 9.2 ±
7.6, 2 ± 1.4 and 36.7% ± 7.4, 10.2 ± 4.5, and 2.7 ±
0.1 (for GM-CSF), respectively. Thus, a moderate but significant
(p = 0.015 using a two-way analysis of variance
of all of the above data) increase (averaging 50%) in CTL responses
could be demonstrated in mice covaccinated with the GM-CSF plasmid.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
Mice vaccinated with the polytope plasmid also showed in vivo activity
in three challenge systems (Fig. 3) and retained multiple
polytope-induced CTL responses for 1 yr (Fig. 4). The ability of vaccination with the polytope DNA plasmid to induce multiple memory CTL responses lasting for 1 yr illustrated 1) that DNA vaccination can provide an excellent modality for inducing long term CTL memory and 2) that independent responses to multiple epitopes can be maintained for an extended period following polytope DNA vaccination. The longevity of these CTL responses may be due to the persistence of DNA vaccine in vivo; however, compelling evidence now indicates that CTL memory does not require such persisting Ag or CD4 T cell help (31, 32). The stability of the individual CTL responses also illustrated that immunodominance effects have not acted significantly over the 1-yr period to limit the responses to a reduced number of epitopes.
CTL responses were induced by the polytope DNA vaccine in the absence
of an obvious source of CD4 T cell help. Covaccination with a plasmid
coding for influenza virus HA as a source of CD4 T helper epitopes did
not improve the CTL responses to the polytope vaccine or the capacity
of vaccinated mice to resist challenge (Fig. 3B). The
possible source of help in the polytope DNA vaccine thus remains
unclear. De novo CD4 T cell epitopes may be generated by the joining of
two CD8 CTL epitopes; however, we have been unable to detect
presentation of CD4 T cell epitopes by this construct (data not shown)
or similar constructs containing known CD4 T cell
epitopes.5 Ags delivered into
cytoplasmic compartments tend not to induce class II-restricted
responses efficiently unless they are directed into endosomal
compartments by specific signal sequences (33). Conceivably, help may
be supplied by bacterial contaminants in the plasmid DNA. However, the
inability to significantly depress polytope CTL responses by depletion
of CD4 cells (Fig. 5) when Ab responses were depressed by 85% argues
that the induction of CTL by polytope DNA vaccination was not strongly
dependent on CD4 T cells. Complete elimination of all CD4 cells cannot
be achieved by this protocol (22), and thus absolute independence
cannot be claimed. The differences that were seen in Figure 5, B and C, in the behavior of RPQASGVYM- and
SYIDSAEKI-specific CTL may reflect a greater CD4 dependence for these
epitopes compared with TYQRTRALV and YPHFMPTNL. However, these
differences might reflect differences in the avidity of CTL for
individual epitopes and the related requirement for CD4 T cell activity
during in vitro restimulation (34). Although CD4 T cells may not always
be required for primary induction of CTL (11), they are required for
maintenance (35) and/or function of active CTL (36).
Primary CTL responses can be readily induced in CD4-deficient mice by virus infection, illustrating that help for CTL induction can be derived from sources other than CD4 T cells (36). The recently described activity of immunostimulatory sequences within plasmid DNA vaccines may be sufficient to generate the help required for CTL induction. These immunostimulatory sequences appeared necessary for successful DNA vaccination and may supply help by inducing IL-12 expression (37). Ligation of CD40 on dendritic cells (DC) by Th cells and the induction of autocrine IL-12 have been implicated as critical events in the activation of DC stimulatory capacity by Th cells (38). Interestingly, IL-12 could substitute for CD4 T cell help in experiments that used peptide-loaded DC to induce CD8 T cell responses (39).
Polytope DNA-induced CTL responses were moderately (averaging 50%)
enhanced by covaccination with a plasmid coding for the GM-CSF gene, in
agreement with recent reports (40), which also showed that co-delivery
of IL-2 could enhance CTL responses. More substantial increases in CTL
have recently been reported using co-delivered IL-12 expression
vectors (41).
Polytope proteins have been reported to be extremely unstable and may be rapidly degraded within the cytoplasm as a result of their limited secondary and tertiary structure (24). The current polytope protein could not, for instance, be detected using a mAb specific for the Ab epitope when the polytope protein was expressed by recombinant vaccinia (14). This instability may explain the inability to induce Ab responses to the Ab epitope included in polytope construct, since intact Ag or epitope may need to be transferred or released from the transfected muscle cells and be taken up by B cells (42). However, An and Whitton (43) have recently shown induction of Ab, CTL, and Th responses using a multivalent minigene recombinant vaccinia construct, suggesting that the ability to induce Ab responses may depend on the Ab epitope and/or the stability of an individual construct.
The ability to induce long term protective CTL responses against a large number of CTL epitopes using a relatively small construct, possibly in conjunction with an immunomodulatory cytokine, is likely to find application in the design of human epitope-based CTL vaccines. This DNA polytope approach may be particularly suitable for vaccines against 1) melanoma, in which multicomponent peptide epitope vaccines have already shown some success (8); 2) EBV-induced infectious mononucleosis and/or post-transplant lymphoproliferative disease, in which whole Ag vaccines would be potentially oncogenic (44); and/or 3) HIV, in which induction of CTL responses against multiple epitopes might foreshadow immune escape by the virus (45, 46).
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Acknowledgments |
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Footnotes |
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2 S.A.T. and M.A.S. contributed equally to this work and should be considered joint first authors.
3 Address correspondence and reprint requests to A. Suhrbier, The Cooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Qld. 4029, Australia. E-mail address:
4 Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; MCS, multiple cloning site; HA, hemagglutinin; pfu, plaque-forming unit; GST, glutathione S-transferase; DC, dendritic cells; RSV, Rous sarcoma virus.
5 S. A. Thomson, S. R. Burrows, B. E. Coupar, and R. Khanna. Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4 cytotoxic T cells: implications for antiviral vaccine design.
Received for publication March 17, 1997. Accepted for publication October 29, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 D. P. A. J. Fonseca, B. Benaissa-Trouw, M. van Engelen, C. A. Kraaijeveld, H. Snippe, and A. F. M. Verheul Induction of Cell-Mediated Immunity against Mycobacterium tuberculosis Using DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein Infect. Immun., August 1, 2001; 69(8): 4839 - 4845. [Abstract] [Full Text] [PDF]
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D. Loirat, F. A. Lemonnier, and M.-L. Michel Multiepitopic HLA-A*0201-Restricted Immune Response Against Hepatitis B Surface Antigen After DNA-Based Immunization J. Immunol., October 15, 2000; 165(8): 4748 - 4755. [Abstract] [Full Text] [PDF]
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D. E. Hassett, M. K. Slifka, J. Zhang, and J. L. Whitton Direct Ex Vivo Kinetic and Phenotypic Analyses of CD8+ T-Cell Responses Induced by DNA Immunization J. Virol., September 15, 2000; 74(18): 8286 - 8291. [Abstract] [Full Text]
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G. T. Belz, P. G. Stevenson, and P. C. Doherty Contemporary Analysis of MHC-Related Immunodominance Hierarchies in the CD8+ T Cell Response to Influenza A Viruses J. Immunol., September 1, 2000; 165(5): 2404 - 2409. [Abstract] [Full Text] [PDF]
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P. J. R. Goulder, C. Brander, K. Annamalai, N. Mngqundaniso, U. Govender, Y. Tang, S. He, K. E. Hartman, C. A. O'Callaghan, G. S. Ogg, et al. Differential Narrow Focusing of Immunodominant Human Immunodeficiency Virus Gag-Specific Cytotoxic T-Lymphocyte Responses in Infected African and Caucasoid Adults and Children J. Virol., June 15, 2000; 74(12): 5679 - 5690. [Abstract] [Full Text]
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J. Fensterle, L. Grode, J. Hess, and S. H. E. Kaufmann Effective DNA Vaccination Against Listeriosis by Prime/Boost Inoculation with the Gene Gun J. Immunol., October 15, 1999; 163(8): 4510 - 4518. [Abstract] [Full Text] [PDF]
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L. Mateo, J. Gardner, Q. Chen, C. Schmidt, M. Down, S. L. Elliott, S. J. Pye, H. Firat, F. A. Lemonnier, J. Cebon, et al. An HLA-A2 Polyepitope Vaccine for Melanoma Immunotherapy J. Immunol., October 1, 1999; 163(7): 4058 - 4063. [Abstract] [Full Text] [PDF]
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 E. Desmézières, Y. Jacob, M.-F. Saron, F. Delpeyroux, N. Tordo, and P. Perrin Lyssavirus glycoproteins expressing immunologically potent foreign B cell and cytotoxic T lymphocyte epitopes as prototypes for multivalent vaccines J. Gen. Virol., September 1, 1999; 80(9): 2343 - 2351. [Abstract] [Full Text]
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T. Woodberry, J. Gardner, L. Mateo, D. Eisen, J. Medveczky, I. A. Ramshaw, S. A. Thomson, R. A. Ffrench, S. L. Elliott, H. Firat, et al. Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+ Cytotoxic T-Cell Epitopes J. Virol., July 1, 1999; 73(7): 5320 - 5325. [Abstract] [Full Text]
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G. Y. Ishioka, J. Fikes, G. Hermanson, B. Livingston, C. Crimi, M. Qin, M. France del Guercio, C. Oseroff, C. Dahlberg, J. Alexander, et al. Utilization of MHC Class I Transgenic Mice for Development of Minigene DNA Vaccines Encoding Multiple HLA-Restricted CTL Epitopes J. Immunol., April 1, 1999; 162(7): 3915 - 3925. [Abstract] [Full Text] [PDF]
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C. Castelli, P. Tarsini, A. Mazzocchi, F. Rini, L. Rivoltini, F. Ravagnani, F. Gallino, F. Belli, and G. Parmiani Novel HLA-Cw8-Restricted T Cell Epitopes Derived from Tyrosinase-Related Protein-2 and gp100 Melanoma Antigens J. Immunol., February 1, 1999; 162(3): 1739 - 1748. [Abstract] [Full Text] [PDF]
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H. T. Maecker, D. T. Umetsu, R. H. DeKruyff, and S. Levy Cytotoxic T Cell Responses to DNA Vaccination: Dependence on Antigen Presentation via Class II MHC J. Immunol., December 15, 1998; 161(12): 6532 - 6536. [Abstract] [Full Text] [PDF]
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B. Wizel, N. Garg, and R. L. Tarleton Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection Infect. Immun., November 1, 1998; 66(11): 5073 - 5081. [Abstract] [Full Text] [PDF]
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G. J. P. Fernando, T. J. Stewart, R. W. Tindle, and I. H. Frazer Th2-Type CD4+ Cells Neither Enhance nor Suppress Antitumor CTL Activity in a Mouse Tumor Model J. Immunol., September 1, 1998; 161(5): 2421 - 2427. [Abstract] [Full Text] [PDF]
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 D J Moss, A Suhrbier, and S L Elliott Candidate vaccines for Epstein-Barr virus BMJ, August 15, 1998; 317(7156): 423 - 424. [Full Text]
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 B. Minev, J. Hipp, H. Firat, J. D. Schmidt, P. Langlade-Demoyen, and M. Zanetti Cytotoxic T cell immunity against telomerase reverse transcriptase in humans PNAS, April 25, 2000; 97(9): 4796 - 4801. [Abstract] [Full Text] [PDF]
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