Prime Boost Vaccination Strategies: CD8 T
Cell Numbers, Protection, and Th1 Bias
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J Immunol 2003; 170:2599-2604; ;
doi: 10.4049/jimmunol.170.5.2599
http://www.jimmunol.org/content/170/5/2599
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Copyright © 2003 by The American Association of
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References
Tonia Woodberry, Joy Gardner, Suzanne L. Elliott, Sonja
Leyrer, David M. Purdie, Paul Chaplin and Andreas Suhrbier
The Journal of Immunology
Prime Boost Vaccination Strategies: CD8 T Cell Numbers,
Protection, and Th1 Bias1
Tonia Woodberry,2* Joy Gardner,* Suzanne L. Elliott,* Sonja Leyrer,† David M. Purdie,*
Paul Chaplin,† and Andreas Suhrbier3*
V
accine strategies that involve primary vaccinations with
a DNA vaccine followed by boosting with a recombinant poxvirus vector encoding the same immunogen
have emerged as favored approaches for generating protective
CD8 T cell responses against a number of diseases including HIV
(1–3), malaria (4, 5), and cancer (6). The immunological mechanisms underpinning the seemingly unparalleled ability of DNA/
poxvirus vaccine combinations to induce enhanced levels of CD8
T cell-mediated protection have not been extensively explored.
Several explanations have been proposed, and they include the
induction by DNA/poxvirus combinations of immune responses
with enhanced IFN-␥ secretion phenotypes or Th1 bias (7). The
ability to avoid induction of antivector responses by priming with
a DNA vaccine has also been postulated to be important for an
effective outcome from this vaccination strategy (8). Recently, evidence has also been presented suggesting that DNA/poxvirus vaccinations are able to induce high avidity CD8 T cells (9).
To identify the factors underpinning the success of DNA/poxvirus vaccination strategies, we measured CD8 T cell numbers and
CD8 T cell-dependent protection induced by a series of prime
boost strategies using combinations of the following vaccine modalities: 1) a standard DNA plasmid vaccine (10), 2) a modified
vaccinia Ankara (MVA)4 replication deficient poxvirus vector, and
*Queensland Institute of Medical Research, Cooperative Research Center for Vaccine
Technology, Australian Center for International & Tropical Health & Nutrition, Brisbane, Australia; and †Bavarian Nordic Research Institute, Martinsried, Germany
Received for publication September 30, 2002. Accepted for publication December 26,
2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Dora Lush National Health and Medical Research
Council scholarship and the Queensland Cancer Fund, Australia.
2
Current address: Partners AIDS Research Center, Massachusetts General Hospital,
Harvard Medical School, Charlestown, MA 02129.
3
Address correspondence and reprint requests to Dr. Andreas Suhrbier, Queensland
Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland, 4029
Australia. E-mail address: [email protected]
4
Abbreviations used in this paper: MVA, modified vaccinia Ankara; sp, synthetic
promoter; TT, tetanus toxoid; TCID50, 50% tissue culture-infective dose; i.n., intranasal; RRV, Ross River Virus T48; CCID50, 50% cell culture-infective dose; TYQ,
Copyright © 2003 by The American Association of Immunologists, Inc.
3) synthetic peptide-based vaccines formulated with tetanus toxoid
(TT) in the water-in-oil adjuvant, Montanide ISA 720 (M720)
(11). The DNA and MVA vaccines both encode the murine polyepitope or polytope immunogen, which contains the following
CD8 T cell epitopes: TYQRTRALV (TYQ) from the influenza
nucleoprotein, RPQASGVYM (RPQ) from the lymphocytic choriomeningitis virus nucleoprotein, and SYIPSAEKI (SYI) from the
Plasmodium berghei circumsporozoite protein (10). Epitope-specific CD8 T cell numbers were enumerated using IFN-␥ ELISPOT
technology, and the size of the IFN-␥ spots produced by individual
T cells was taken as a measure of the amount of IFN-␥ secreted by
each T cell (12). An influenza protection model was used to investigate the effect of different vaccination strategies on the capacity of a given number of vaccine-induced CD8 T cells to mediate
protection. A crucial characteristic of this model is its relative insensitivity to IFN-␥ (13, 14). Because elevated CD8 T cell-derived
IFN-␥ secretion has been shown to improve CD8 T cell-mediated
protection in several systems (15, 16), this influenza challenge system was chosen to investigate whether DNA/MVA vaccination
generates CD8 T cells with enhanced IFN-␥-independent protective capabilities. Several reports have illustrated that enhanced
IFN-␥-independent protection can be mediated by CD8 T cells
with high avidity/affinity (17–19).
The ability to induce CD8 T cells with an enhanced IFN-␥ secretion phenotype was identified in these studies as the only advantage bestowed by the DNA/MVA vaccination strategy. The
influenza model failed to show that DNA/MVA vaccination induced CD8 T cells with improved IFN-␥-independent protection,
and other prime boost strategies were often able to generate higher
numbers of CD8 T cells than DNA/MVA vaccination. These experiments illustrate that alternative prime boost strategies could
rival DNA/poxvirus strategies for optimal induction of protective
CD8 T cells.
CD8 T cell epitope TYQRTRALV from the influenza nucleoprotein; RPQ, CD8 T
cell epitope RPQASGVYM from the lymphocytic choriomeningitis virus nucleoprotein; SYI, CD8 T cell epitope SYIPSAEKI from the Plasmodium berghei circumsporozoite protein; MVAsp, modified vaccinia Ankara synthetic promoter; CpG, cytidine-phosphate-guanosine; mpt, murine polytope; BN, Bavarian Nordic.
0022-1767/03/$02.00
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Vaccination strategies involving priming with DNA and boosting with a poxvirus vector have emerged as a preferred combination
for the induction of protective CD8 T cell immunity. Using IFN-␥ ELISPOT and a series of DNA plasmid, peptide, and modified
vaccinia Ankara (MVA) vaccine combinations, we demonstrate that the DNA/MVA combination was uniquely able to enhance
IFN-␥ secretion by Ag-specific CD8 T cells. However, CD8 T cell populations induced by DNA/MVA vaccination failed to show
an enhanced capability to mediate protection in an IFN-␥-independent influenza challenge model. The DNA/MVA vaccine strategy
was also not unique in its ability to induce high numbers of CD8 T cells, with optimal strategies simply requiring the use of vaccine
modalities that individually induce high numbers of CD8 T cells. These experiments argue that rivals to DNA/poxvirus vaccination
strategies for the induction of optimal protective CD8 T cell responses are likely to emerge. The Journal of Immunology, 2003,
170: 2599 –2604.
2600
PRIME BOOST VACCINATION STRATEGIES
Materials and Methods
MVA vaccines
The murine polytope (mpt) construct (20) was inserted into MVA-Bavarian
Nordic (BN) (patent WO 0242480) under the control of a natural p7.5
promoter (MVAp7.5(mpt)) (21) or a strong synthetic promoter (sp)
(MVAsp(mpt)) (22), which permits substantially enhanced Ag production
(23). MVA expressing human tyrosinase (a melanoma Ag) via the p7.5
promoter was used as a control MVA (MVA control) (24). MVA-BN titers
were determined using 50% tissue culture-infective dose (106 TCID50) as
described previously (24). In our hands TCID50 and PFU values were
broadly comparable for MVA-BN titers (data not shown). All MVA-BN
vaccines were constructed and supplied by Bavarian Nordic (Martinsried,
Germany).
Mice and vaccination
IFN-␥ ELISPOT
Three weeks after booster immunizations, mice were culled and splenocytes were analyzed directly ex vivo for epitope-specific CD8 T cell responses using an IFN-␥ ELISPOT assay as described previously (26). The
number and area of IFN-␥ spots were measured using the KS ELISPOT
reader (Zeiss, Halbermoos, Germany). In the analysis of spot areas, the
Student residuals statistical technique was used to identify outliers, with
each observation weighted by the inverse of the SE. A Bonferonii correction was applied to the p value to take account of the multiple tests (27).
Influenza challenge
Groups of mice were challenged by i.n. administration of influenza virus
Mem71, which contains the hemagglutinin of A/Memphis/1/71 (H3) and
the neuraminidase of A/Bellamy/42 (N1), as described previously (10).
Percent protection was calculated as the percentage reduction in lung influenza titers compared with control mice, which had been mock immunized with PBS. Using SAS for Windows (release 8.02), a Pearson’s correlation coefficient was calculated between mean values of the percentage
protection and the log of the spot number, weighting each mean by the
inverse of the variance of the measures.
Results
CD8 T cell responses from individual vaccine modalities
The vaccine modalities used in this study were initially tested at
different doses to determine the optimal doses and epitopes to be
used in future experiments. As observed previously with recombinant murine polytope vaccines (i.e., DNA and poxvirus), IFN-␥
ELISPOT analysis revealed a hierarchy of responses after vaccination that was not apparent after vaccination with peptide vaccines. Of the three epitopes, SYI emerged as the most immunodominant, followed by RPQ, with TYQ being relatively subdominant
(Ref. 26 and Fig. 1).
Murine vaccination experiments performed by others have used
doses of recombinant MVA of as much as 107–2 ⫻ 108 PFU (24,
28). At a single dose of 5 ⫻ 107 TCID50, MVA-BN encoding the
murine polytope under the synthetic promoter (MVAsp(mpt)) induced CD8 T cell responses of 250 –300 IFN-␥ spots per 106
splenocytes for the dominant epitopes (Fig. 1). These results are
FIGURE 1. Epitope-specific immune responses after single immunizations. BALB/c mice (n ⫽ 4 per group) were immunized with one of the
following: 1) (Peptide) a s.c. injection of 10 ug of each peptide, TYQ, SYI,
or RPQ formulated with Montanide ISA 720 (M720) and tetanus toxoid
(TT); or 2) (2⫻ DNA) two i.m. injections of 100 ug of DNA encoding the
mpt; or 3) (MVAsp) a single i.m. injection of 106 or 5 ⫻ 107 CCID50 of
MVAsp(mpt). After 3 wk, TYQ, SYI, and RPQ-specific CD8 T cell numbers were enumerated using ELISPOT.
broadly comparable with responses reported by others (4). A single
dose of 106 MVAsp(mpt) reliably induced TYQ responses of
10 –15 IFN-␥ spots per 106 splenocytes (Fig. 1). This relatively
subdominant TYQ epitope and this relatively low dose of MVA
were chosen for prime boost and protection studies so that enhancements in CD8 T cell activities might be readily observed.
The role of Th1 bias in prime boost immunization
DNA vaccines are reported to bias the immune response toward a
Th1 phenotype, with enhanced T cell-derived IFN-␥ production
(29). To investigate whether DNA prime and MVA boost immunizations induce Ag-specific CD8 T cells with enhanced IFN-␥
production, TYQ-specific CD8 T cells from the different prime
boost vaccination regimes were analyzed by IFN-␥ ELISPOT. To
avoid any potential interference from anti-MVA Abs, MVA
booster vaccinations were administered i.n. (30). The area of each
ELISPOT spot was measured and was taken to represent a measure
of the amount of IFN-␥ secreted by each TYQ-specific CD8 T cell
(12). Unlike conventional bulk IFN-␥ assays, which measure secretion from a population of cells, this ELISPOT-based method
allows IFN-␥ secretion profiles to be measured for individual Agspecific CD8 T cells.
Analysis of results from a series of prime boost combinations
illustrated a trend where the mean spot area increased with the
increasing number of Ag-specific CD8 T cells (Fig. 2). As the total
number of CD8 T cells increases, the stochastic probability of
having cells that secrete high amounts of IFN-␥ might be expected
to increase. Importantly, however, a clear exception to the trend
was observed for a DNA prime followed by a MVAsp(mpt) boost
immunization (Fig. 2, vaccination strategy 8), which showed a
significantly higher mean spot area compared with the other vaccination strategies ( p ⫽ 0.0055).
These results illustrate that a DNA prime and MVA boost immunization strategy, more than any other combination tested, increased the IFN-␥ secretion of individual Ag-specific CD8 T cells.
Analysis of CD8 T cell numbers and protection
DNA prime and poxvirus boost immunizations have been postulated to elicit high avidity/affinity CD8 T cells with enhanced protective capabilities (9). To investigate whether DNA/MVA vaccination strategies enhance the protective capacity of individual CD8
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Female BALB/c (H-2d) mice (6 – 8 wk) were purchased from Animal Resources (Perth, Australia). Mice were immunized with the following: 1)
recombinant MVA viruses at a dose of 106 TCID50 delivered by i.m. injection or intranasal (i.n.) delivery. For i.m. injections, mice were anesthetized with ketamine and xylazine (100 ␮l of ketamine 10 mg/ml: xylazine
2 mg/ml: water (1:1:12), per 10 g body weight), and 50 ␮l of MVA was
injected into each quadriceps muscle. For i.n. immunizations, mice were
anesthetized with methoxyflurane (Medical Developments, Victoria, Australia), and 25 ␮l of MVA was introduced into each nostril. 2) Peptide
vaccines were formulated with 10 ␮g of peptide (Mimotopes, Clayton,
Australia) and 2.5 ␮g/ml tetanus toxoid (CSL, Parkville, Australia) emulsified in Montanide ISA 720 (M720) (SEPPIC, Paris, France) and were
injected s.c. at the base of the tail as described (11). 3) A DNA vaccine
encoding the murine polytope was administered twice (100 ␮g i.m. 50 ␮l
into each quadriceps), 2 wk apart as described (20). Prime and boost immunizations were separated by a period of 3 wk (unless stated otherwise).
Ross River Virus T48 (RRV) (1000 50% cell culture-infective dose
(CCID50)) was delivered in a volume of 200 ␮l to mice by i.p. injection (25).
The Journal of Immunology
FIGURE 3. Correlation between protection against influenza Mem71
challenge and CD8 T cell numbers. Vaccine combinations and protocol
related to Fig. 2 plus (15) primary coimmunization with TYQ/M720/TT
and RRV with a MVAsp(mpt) boost, and (16) primary coimmunization
with TYQ/M720/TT and MVA(control) with a MVAsp(mpt) boost.
Splenocytes from immunized mice were analyzed by ELISPOT to enumerate the number of TYQ-specific CD8 T cells, and a parallel group of
immunized mice (n ⫽ 6 per group) were challenged with influenza
Mem71. The percentage protection refers to the reduction in lung influenza
titers compared with control mice. The correlation coefficient r ⫽ 0.85, p ⫽
0.005 was calculated with each mean weighted by the inverse of variance
of the measures.
CD8 T cells as assessed by IFN-␥ ELISPOT. In fact, priming with
peptide followed by MVA produced substantially higher numbers
of TYQ-specific CD8 T cells (Fig. 3, combinations 10 and 11),
perhaps largely because of the more efficient induction of TYQspecific CD8 T cells during priming (see Fig. 5).
The role of vector responses in prime and boost immunization
T cells, a series of vaccination regimes were compared for their
ability to induce CD8 T cells and to mediate protection against
influenza. The MEM71 i.n. influenza challenge system was chosen
because TYQ-specific CD8 T cell responses were previously
shown to be protective (31). Furthermore, IFN-␥ appears to have
little impact on the level of infection in this model (13, 14), thus
differences in IFN-␥ secretion by Ag-specific CD8 T cells (see Fig.
2) are unlikely to complicate this analysis significantly.
Sixteen different vaccination regimes were used to immunize
parallel groups of mice that were either: 1) challenged with
MEM71 influenza or 2) used to enumerate vaccine-induced TYQspecific CD8 T cell responses using IFN-␥ ELISPOT. A significant
correlation (r ⫽ 0.86, p ⫽ 0.005) emerged between the log of CD8
T cell numbers and vaccine-conferred protection against influenza
challenge (Fig. 3). Importantly, DNA prime and MVA boost immunizations did not appear to provide better protection for the
number of CD8 T cells than any other vaccination strategy (Fig. 3,
combinations 7 and 8). Thus, these data do not support the notion
that DNA prime and MVA boost immunizations induce CD8 T
cells with superior IFN-␥-independent protective capabilities. One
would expect vaccination strategies that produced CD8 T cells
with superior protective capabilities to plot significantly above the
correlation line, an outcome that might be expected if the immunization strategy induced CD8 T cells with high avidity/affinity.
It is also apparent from this analysis that DNA/MVA combinations do not have a monopoly on induction of high numbers of
The ability of DNA prime and MVA boost and peptide prime and
MVA boost to raise large numbers of CD8 T cells may be because
of the near exclusive induction during priming of responses specific for the immunogen rather than induction of antivector responses (32). The subsequent MVA boost might then preferentially expand the pre-existing immunogen-specific CD8 T cells
rather than expanding vector responses. To test whether avoidance
of vector responses is important for induction of large numbers of
CD8 T cells, a control MVA or an irrelevant virus, RRV, was
codelivered with a primary immunization of the peptide vaccine.
Neither codelivered MVA nor the contemporaneous virus infection significantly affected the efficiency of TYQ-specific CD8 T
cell induction by the peptide vaccine (Fig. 4, Prime). These findings argue that induction of anti-MVA vector responses do not
significantly compete with induction of immunogen-specific CD8
T cell responses during priming. Importantly, the addition of MVA
or RRV to the primary vaccination also did not significantly affect
the outcome after peptide prime and MVA boost (Fig. 4, Prime
boost). Thus, avoiding immune competition between vector and
immunogen during priming, by using modalities that induce few
antivector responses, does not appear to be a crucial component of
effective prime boost vaccination strategies. This experiment does
not address the separate and well established problem of antivector
Ab responses inhibiting subsequent immunizations with the same
vector. This issue was avoided in these experiments by i.n. delivery of MVA booster immunizations (30).
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FIGURE 2. Mean number of TYQ-specific IFN-␥ spots measured by
ELISPOT plotted against the mean spot area for BALB/c mice immunized
with different prime boost combinations. BALB/c mice (n ⫽ 4 per group)
were given primary immunizations with the following: 1) a single i.m.
injection of MVA (106 CCID50), 2) two i.m. injections of 100 ␮g of
DNA(mpt), 3) a single s.c. injection of TYQ/M720/TT, 4) CFA emulsified
with PBS but without peptide, and/or 5) PBS without peptide. Booster
immunizations were given after 3 wk, with MVA (106 CCID50) booster
immunizations given i.n. (106 CCID50). The vaccine combinations were as
follows: (0) 2⫻ DNA as prime and PBS boost (1) PBS prime and
MVAp7.5(mpt) boost, (2) PBS prime and MVAsp(mpt) boost, (3)
MVAsp(mpt) prime and MVAp7.5(mpt) boost, (4) MVAp7.5(mpt) prime
and MVAp7.5(mpt) boost, (5) MVAsp(mpt) prime and MVAsp(mpt)
boost, (6) MVAp7.5(mpt) prime and MVAsp(mpt) boost, (7) 2⫻
DNA(mpt) as prime and MVAp7.5(mpt) boost, (8) 2⫻ DNA as prime and
MVAsp(mpt) boost, (9) TYQ/M720/TT prime and PBS boost, (10) TYQ/
M720/TT prime and MVAp7.5(mpt) boost, (11) TYQ/M720/TT prime and
MVAsp(mpt) boost, (12) CFA prime and MVAp7.5(mpt) boost, (13) CFA
prime and MVAsp(mpt) boost, and (14) primary coimmunization with
TYQ/M720/TT and MVAp7.5(mpt) with a PBS boost. Three weeks after
the booster vaccination, TYQ-specific CD8 T cells in spleen cells were
enumerated using IFN-␥ ELISPOT. The area of 40 –50 spots was measured
for each immunization except for combination numbers 1 (n ⫽ 12), 2 (n ⫽
25) and 7 (n ⫽ 39). Combinations 0, 3, and 12 were excluded because they
produced too few spots and/or a high number of background spots.
2601
2602
PRIME BOOST VACCINATION STRATEGIES
Optimal induction of CD8 T cell numbers can be achieved using
different prime boost strategies
To determine whether the optimal prime boost strategy for induction of high numbers of CD8 T cells remains constant, irrespective
of the epitope being tested, mice were primed with the following:
1) a peptide vaccine containing a mixture of three peptides (TYQ,
SYI, and RPQ); or 2) DNA(mpt), which encodes these three
epitopes; or 3) MVAsp(mpt), which similarly encodes these
epitopes. All of the mice were then given a booster immunization
of MVAsp(mpt) delivered i.m. Surprisingly, the following occurred: 1) the TYQ-peptide prime and MVAsp(mpt) boost strategy
induced more TYQ-specific CD8 T cells than the other two prime
boost combinations (Fig. 5A, TYQ), 2) the 2⫻ DNA(mpt)/
MVAsp(mpt) induced more RPQ-specific CD8 T cells than the
other combinations (Fig. 5A, RPQ), and 3) MVAsp(mpt)/
MVAsp(mpt) induced the highest number of SYI-specific responses (Fig. 5A, SYI). Thus, the optimal prime boost strategy for
induction of high numbers of CD8 T cells appeared to depend on
the epitope being tested.
Can responses raised by single vaccine modalities predict the
outcome following prime boost?
Excluding CFA and coimmunization experiments, a total of 17
prime boost combinations have been tested (Figs. 2 and 5), and the
outcomes after immunization with each individual vaccine modality have also been measured (Fig. 1). A linear regression analysis
was undertaken to determine whether the outcome from prime
boost (P/B) immunizations might be predicted from knowledge of
the immune responses generated when the prime (P) and boost (B)
immunization modalities are given alone. Using the weighted least
squares method, the best equation to predict the outcome (P/B)
based on P and B emerged to be P/B ⫽ 1.34 ⫹ 1.48P ⫹ 0.99B,
with a resulting correlation coefficient between the observed and
predicted outcome of r ⫽ 0.87 ( p ⬍ 0.0001) (Fig. 5B). The 1.48
( p ⫽ 0.006) and 0.99 ( p ⫽ 0.012) coefficient values were not
significantly different from 1, with the 95% confidence limits being
0.51–2.46 and 0.26 –1.72, respectively. To test for a synergistic
effect between P and B, the linear regression was repeated with a
P ⫻ B term. The coefficient for the P ⫻ B term was ⫺0.00105, and
FIGURE 5. The immunodominance of the CD8 T cell epitope changes
the outcome following prime boost vaccination. A, Prime boost immunizations were delivered to BALB/c mice (n ⫽ 4 per group) using one of the
following: 1) s.c. injection with 10 ␮g of each peptide TYQ, SYI, and RPQ
in M720/TT, 2) two i.m. injections of 100 ␮g of DNA (2⫻ DNA), or 3) a
single i.m. injection of 106 CCID50 of MVAsp(mpt) (MVA). All prime
immunizations were boosted using an i.m. injection of 106 CCID50 of
MVAsp(mpt) (MVA). CD8 T cells specific for TYQ, SYI, and RPQ were
enumerated as above. B, Correlation between the outcome of prime boost
immunization and the mathematical sum of the responses observed when
prime and boost modality are used alone. The y-axis shows the observed
outcome (i.e., epitope-specific CD8 T cell responses enumerated using
IFN-␥ ELISPOT) of P/B immunizations shown in Figs. 2 and 5A. The
x-axis shows the mathematical sum of the observed IFN-␥ ELISPOT responses seen following use of the priming immunization alone (Fig. 1) plus
the observed responses seen following use of the boosting immunization
alone (Figs. 1 and 2). The prime boost combinations from Fig. 2 are numbered as before, and the prime boost combinations from Fig. 5 are labeled
(a) MVA/MVA (TYQ responses), (b) DNA/MVA (TYQ responses) (c)
peptide/MVA (TYQ responses), (d) MVA/MVA (RPQ responses), (e)
DNA/MVA (RPQ responses), (f) peptide/MVA (RPQ responses), (g)
MVA/MVA (SYI responses), (h) DNA/MVA (SYI responses) (i) peptide/
MVA (SYI responses). Linear regression analysis showed the best-fit equation to be P/B ⫽ 1.34 ⫹ 1.48P ⫹ 0.99B, with a correlation coefficient of
r ⫽ 0.87, p ⬍ 0.0001.
the associated p value was 0.8351. The data thus provided no evidence for the existence of a synergistic effect between P and B and
argue that CD8 T cell responses following prime boost may be
simply additive.
Discussion
This study indicates that the main advantage of the DNA prime
MVA boost vaccination strategy is the induction of CD8 T cells
with enhanced IFN-␥ secretion phenotypes. The DNA/MVA strategy did not have the monopoly on induction of high numbers of
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FIGURE 4. Coinduction of vector responses during priming does not
suppress the induction of immunogen-specific CD8 T cell response during
priming. Prime, BALB/c mice (n ⫽ 4 per group) were immunized using
one of the following: 1) a s.c. injection with 10 ␮g of TYQ/M720/TT
(TYQ), 2) a s.c. injection with 10 ␮g of TYQ/M720/TT and an i.m. injection of 106 CCID50 of MVA(control), or 3) a s.c. injection of 10 ␮g of
TYQ/M720/TT and an i.p. injection of 103 CCID50 of the alphavirus, RRV.
TYQ-specific CD8 T cell numbers were determined by ELISPOT 3 wk
later. Prime Boost, a parallel group of mice were immunized as described
under Prime and were given a single i.n. injection of 106 CCID50 of
MVAsp(mpt) as a booster immunization after 8 wk. TYQ-specific CD8 T
cells were enumerated as above.
The Journal of Immunology
The coinduction of antivector responses during priming did not
appear to significantly influence the generation of epitope-specific
CD8 T cell responses following priming or after prime boost immunizations (Fig. 5). These experiments do not support the notion
that for prime boost to be effective, the priming vaccine modality
must avoid induction of antivector responses. Recent use of recombinant adenovirus as an effective priming modality supports
this conclusion (41). The results shown in Fig. 5 do not address the
separate issue of whether antivector neutralizing Ab responses
generated by priming immunizations are able to inhibit effective
boosting by the same vector. This issue has been addressed by
others (42), and any such potential influences were avoided in the
current study by using i.n. MVA booster immunizations (30). In
addition, recent studies have shown that the 106 CCID50 MVA
dose used here fails to induce sufficient anti-MVA Ab responses to
interfere with subsequent MVA booster immunizations (P. Chaplin, unpublished observations).
The current study has demonstrated that there are other prime
boost strategies besides DNA/MVA that can induce high levels of
protective CD8 T cells. However, perhaps the most surprising outcome is the observation that the number of CD8 T cells induced
following prime boost immunizations approximates to the simple
addition of the responses seen when the prime and boost vaccination modalities were administered individually. This 1 ⫹ 1⬇2 concept contrasts with Ab responses following prime boost immunizations where substantial synergistic affects are obtained (7). The
1 ⫹ 1⬇2 notion is based on IFN-␥ ELISPOT data and might be
compromised because this assay most likely enumerates effector/
effector memory CD8 T cells rather than the central memory CD8
T cells, which are the cells believed to expand upon re-exposure to
Ag (43). However, the size of the effector/effector memory CD8 T
cell expansion often appears to be related to the number of central
memory CD8 T cells generated (44). Either way, the 1 ⫹ 1⬇2
concept might best be viewed as a broad approximation illustrating
the following: 1) prime boost strategies including DNA/MVA do
not produce exceptional CD8 T cell expansions, and 2) the best
method for induction of high numbers of CD8 T cells is to combine vaccine modalities that individually induce high levels of
CD8 T cells.
In the current study the priming and boosting vaccine modalities
did not share Ag-specific CD4 T cell epitopes. In contrast, prime
boost strategies using vaccines encoding whole Ags would also
amplify Ag-specific CD4 T cell responses, and such responses are
known to influence maintenance and recall of CD8 T cell responses and contribute to protection (45, 46). How Ag-specific
(and vaccine vector-specific) CD4 T cell responses influence the
outcome of different prime boost vaccination strategies remains to
be explored. In addition, recent simian-human immunodeficiency
virus studies have reported that protection after MVA only was
similar to protection seen after DNA/MVA vaccination, suggesting
that strategies that optimize postchallenge immune-cell trafficking
may be as valuable as those that induce high numbers of CD8 T
cells (47).
The increased regulatory and manufacturing complexities associated with the licensing and registering of a vaccine product comprising two different vaccination modalities may hinder the development of heterologous prime boost vaccine products. The current
study may facilitate the design of improved prime boost vaccine
strategies and aid in the development of effective vaccine products
that use only a single vaccination modality.
Acknowledgments
We thank T. T. T. Le (Queensland Institute of Medical Research), L. Mateo
(Bavarian Nordic), and Dr. G. Leggatt (Center for Immunology and Cancer
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CD8 T cells, with other vaccine strategies outperforming this combination (Figs. 2 and 3). An analysis of 16 different vaccine combinations also failed to provide any evidence for the induction by
DNA/MVA of CD8 T cells with improved IFN-␥-independent
protective capacities (Fig. 3), as might be expected if high avidity
T cells were induced by this strategy. Surprising was the observation that the best prediction for the number of CD8 T cells induced
by prime and boost immunizations was a simple sum of the responses induced when the prime was used alone plus the responses
induced when the boosting modality was used alone.
The ELISPOT assay was used to show that the IFN-␥ production per CD8 T cell was on average significantly higher after DNA/
MVA immunization than after other vaccination strategies. The
ability of DNA vaccines to induce CD8 T cells that produce more
IFN-␥ per cell can likely be attributed to the presence of cytidinephosphate-guanosine (CpG) motifs in the DNA plasmids (29). The
cytokine secretion phenotype of CD8 T cells may be determined
during priming, and importantly, the phenotype appears to be
largely retained upon expansion of the memory CD8 T cell pool
(33). Thus, DNA priming may establish a memory pool with enhanced Th1 bias, which is then effectively expanded by the poxvirus boost. Current evidence would indicate that use of immunostimulatory CpG oligonucleotides as adjuvants in primary
immunizations should impart the same Th1 enhancement as priming with DNA (34). However, it remains to be established whether
the Th1 bias imposed on CD8 T cells by DNA/MVA vaccination
or by CpG adjuvants is influenced by the following: 1) the immunodominance of the CD8⫹ epitope and/or 2) the nature of the CD4
T cell responses induced during vaccination. Although enhanced
IFN-␥ secretion appears to be of little protective value against
influenza (Fig. 2; confirming previous reports, Refs. 12 and 13),
there is little doubt that for other diseases including cancer, HIV,
and the malaria liver stage, enhanced CD8 T cell IFN-␥ secretion
is likely to mediate substantial improvements in protection
(35–37).
Several reports have shown that the avidity/affinity of CD8 T
cell populations can be manipulated in vitro (38, 17–19). By restimulating in vitro with low levels of peptide epitope, high avidity/affinity CD8 T cell populations can be expanded that show
enhanced IFN-␥-independent protective capabilities after adoptive
transfer into mice (17–19). Although we did not measure T cell
avidity/affinity directly, we showed that different vaccination strategies did not readily or significantly influence the IFN-␥-independent protective capabilities of CD8 T cell populations in vivo.
Epitope levels did not appear to influence the outcome, with CD8
T cell responses induced by DNA and MVAp7.5 vaccines (delivering low levels of Ag) or peptide and MVAsp vaccines (delivering relatively higher levels of Ag) failing to show significant differences in their protective capabilities. Assuming that high
avidity/affinity CD8 T cells should show enhanced protection in
the influenza challenge system, these experiments might argue that
different vaccine strategies do not readily influence the functional
avidity/affinity of CD8 T cell populations in vivo. Although this
view is not supported by conclusions from some studies (9, 39,
40), these latter investigations did not control for differences in T
cell numbers and/or IFN-␥ secretion and/or used readouts involving in vitro restimulation. Conceivably, the vaccine modalities
used in the current study already induce CD8 T cells with maximal
functional avidity/affinity. Thus, in the absence of any low avidity/
affinity CD8 T cell induction, we may have been unable to detect
differences in IFN-␥-independent protection. In either event, our
data does not support the view that DNA prime and MVA boost is
uniquely capable of inducing CD8 T cells with significantly improved IFN-␥-independent protective activities.
2603
2604
Research, Princess Alexandra Hospital, Brisbane, Australia) for their support and advice. Thanks also to Nirmala Pandeya for assistance with statistical analysis.
PRIME BOOST VACCINATION STRATEGIES
25.
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