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Dose Dependence of CTL Precursor
Frequency Induced by a DNA Vaccine and
Correlation with Protective Immunity Against
Influenza Virus Challenge
Tong-Ming Fu, Liming Guan, Arthur Friedman, Timothy L.
Schofield, Jeffrey B. Ulmer, Margaret A. Liu and John J.
Donnelly
J Immunol 1999; 162:4163-4170; ;
http://www.jimmunol.org/content/162/7/4163
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Copyright © 1999 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Dose Dependence of CTL Precursor Frequency Induced by a
DNA Vaccine and Correlation with Protective Immunity
Against Influenza Virus Challenge
Tong-Ming Fu,1* Liming Guan,* Arthur Friedman,* Timothy L. Schofield,† Jeffrey B. Ulmer,2*
Margaret A. Liu,2* and John J. Donnelly2*
he ability of CD81 CTL to recognize viral Ags in the
form of 8- to 11-amino acid peptides presented by MHC
class I molecules on the cell surface (1–3) has suggested
that they play a role in the clearance of intracellular virus during a
viral infection (4 – 6). The role of CD81 T cell responses in immunity against influenza virus in mice was established by early
studies of adoptive lymphocyte transfer (7–10). It also has been
demonstrated that CD41 T cell responses can be induced against
influenza virus infection, and in many cases these CD41 T lymphocytes are protective when adoptively transferred into naive
mice (11, 12). In some instances, CD41 MHC class II-restricted
CTL have been observed (6, 12). Therefore, although the specific
in vivo effector functions of CD81 or CD41 T cells are still topics
for debate, cell-mediated immunity, especially the cytotoxic T cell
response, apparently plays a role in the clearance of influenza virus
infection in mice (6, 13).
DNA vaccination represents a novel method for induction of
both humoral and cellular immune responses (14). Intramuscular
injection of naked plasmid DNA results in efficient expression of
Ag in muscle cells (15). The initiation of immune responses, especially CTL responses, requires the participation of bone marrowderived professional APCs (16 –18) and may include an Ag transfer process from muscle cells to professional APCs, similar to
T
Departments of *Virus and Cell Biology and †Biometrics, Merck Research Laboratories, West Point, PA 19486
Received for publication July 30, 1998. Accepted for publication January 4, 1998.
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
Address correspondence and reprint requests to Dr. Tong-Ming Fu, Merck Research
Laboratory, WP26-246, West Point, PA 19486. E-mail address: tong-ming_fu@
merck.com
2
Current address: Chiron Corp., 4560 Horton St., Emeryville, CA 94608.
Copyright © 1999 by The American Association of Immunologists
cross-priming (17). Other studies from our laboratory demonstrated that i.m. injection of a plasmid DNA encoding influenza
virus nucleoprotein (NP),3 derived from A/PR/8/34 (H1N1), can
induce a robust CTL response specific for an H-2Kd-restricted
NP147–155 epitope in BALB/c mice (19). In these studies immunization with NP DNA conferred protection against a lethal dose
challenge with the influenza virus A/HK/68 (H3N2), a virus strain
that conserves the sequence encoding NP including the H-2Kdrestricted CTL epitope 147–155, but contains significant changes
in the viral surface glycoproteins (hemagglutinin (HA) and neuraminidase) compared with A/PR/8/34 (H1N1).
Although DNA immunization is being explored as a vaccine
strategy in both preclinical animal studies and clinical trials, the
immunogenicity of DNA vaccines for the induction of CTL responses has not been directly compared with natural viral infection. Moreover, different aspects of immune responses, especially
CTL precursor (CTLp) frequency, as a function of the dose of
DNA vaccine administered have not been fully characterized. Furthermore, the relationship between the frequency of DNA vaccineinduced Ag-specific CTLp and protective efficacy has not been
addressed in the influenza virus challenge model. The primary objectives of this study are 1) to compare the immunogenicity of the
NP DNA vaccine with that of influenza virus infection and to
document the kinetics of NP147–155-specific CTLp frequency
changes following DNA immunization vs influenza virus infection; 2) to characterize the dose response of the NP DNA vaccine
in inducing CTLp, T cell proliferation, and Ab responses in mice;
and 3) to determine whether a relationship exists between the
NP147–155-specific CTLp frequency and the NP DNA-induced protective immunity observed in our cross-strain virus challenge
3
Abbreviations used in this paper: NP, nucleoprotein; CTLp, cytotoxic T lymphocyte
precursor; HA, hemagglutinin; LDA, limiting dilution analysis.
0022-1767/99/$02.00
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Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1)
provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kdrestricted epitope NP147–155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness
of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the
frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus
and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with
influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were
injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not
proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of
NP147–155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP
DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated
lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain
influenza virus challenge. The Journal of Immunology, 1999, 162: 4163– 4170.
4164
model. In this paper we directly compare the immunogenicity of
NP DNA vaccine with that of influenza virus infection in inducing
NP147–155-specific CTLp in BALB/c mice, demonstrate a dosedependent relationship between the amount of NP DNA administered and the frequency of NP147–155-specific CTLp elicited in
mice, and present a correlation between the CTLp frequency and
the protective efficacy in mice against cross-strain influenza virus
challenge. This correlation provides a minimal estimate of the
NP147–155-specific reciprocal CTLp frequency of 100,000. namely,
one CTLp in every 100,000 spleen cells, that is associated with
protection from death or significant morbidity after cross-strain
influenza challenge in the presence of an intact immune system
that also is capable of CD41 T cell responses to NP.
Materials and Methods
DNA constructs
Animals
Four- to eight-week-old female BALB/c mice were purchased from
Charles River Laboratories (Raleigh, NC). The animals were maintained in
the animal facility of Merck Research Laboratories (West Point, PA). All
experiments were performed in accordance with the published procedures
of the institutional animal care and use committee.
ELISA for measuring anti-NP Ab
Maxisorb plates (Nunc, Naperville, IL) were coated with 100 ml/well overnight at 4°C with the purified recombinant NP protein at 7.5 mg/ml (total
protein) in PBS. To reduce background, plates were blocked with PBS
containing Tween-20 (0.05%, v/v) and BSA (1%, w/v) for 2 h at room
temperature. Serum samples and peroxidase-conjugated rabbit anti-mouse
IgG (Zymed, San Francisco, CA) were diluted in blocking buffer (1/100 to
1/1,000,000) and incubated sequentially on the plates for 2 h at room temperature, with extensive washing between each incubation. For color development, 0.1 M citrate buffer (pH 4.5) containing hydrogen peroxide
(0.012%, v/v) and O-phenylenediamine (1 mg/ml) was added for 10 –30
min, at which point the reaction was stopped with 1 M sulfuric acid. Plates
were read spectrophotometrically at 490 nm. For end-point titer determinations, a positive was scored as any well with an absorbance greater than
3 SD above background (calculated using .20 wells containing no primary
Ab).
Immunization, challenge, and adoptive transfer protocol
For immunization, mice under anesthesia induced by the administration of
ketamine-xylazine were injected with plasmid DNA dissolved in 100 ml of
sterile saline three times at 3-wk intervals. Fifty microliters of DNA was
injected bilaterally into the quadriceps muscles each time. To prime mice
with live influenza virus, mice were infected intranasally with 20 ml of
A/HK/68 (H3N2) or A/PR/8/34 (H1N1) at 102.5 of the 50% tissue culture
infective dose while awake. These doses administered to fully alert mice
will not result in death (22). For challenge studies mice were fully anesthetized and intranasally inoculated with 20 ml of A/HK/68 or A/PR/8/34
at doses of 102.5 and 103 of the 50% tissue culture infective dose, respectively. This generally results in over 70% death in unimmunized mice (19,
20, 22, 23). In some instances environmental factors, particularly ambient
temperature, may increase the survival rates of nonimmunized mice. Variations in the age of the mice at the time of challenge also can affect the
outcome of the challenge, with older mice generally being more resistant
to death from influenza infection while experiencing comparable loss of
body mass as younger mice. For this reason both survival and body mass
of the challenged mice were monitored for up to 4 wk. In studies in which
significant numbers of deaths were not observed, comparison of body mass
provided an alternative estimate of protective efficacy (22).
The adoptive transfer protocol has been described previously (23). The
spleen cells harvested from the donor mice were cultured in vitro and
restimulated with irradiated syngeneic cells pulsed with NP147–155 peptide
or infected with A/PR/8/34 for 7 days, and 10 U/ml of IL-2 was added on
the second day of culture. Recombinant human IL-2 was purchased from
Cellular Products (Buffalo, NY). Lymphocytes were washed with PBS
three times and resuspended at desired concentration in PBS. The recipient
mice, challenged with influenza virus A/HK/68 4 h previously, were adoptively transferred a 0.2-ml volume of lymphocytes through the tail vein,
which had been dilated by immersion in warm water (;50°C). Survival
and weight loss were monitored for up to 4 wk.
Lymphocyte proliferation
Spleen cells were plated in round-bottom 96-well plates at 1 3 105 cells/
well in 200 ml of complete RPMI 1640 medium in quadruplicate. Each well
also contained purified recombinant NP at 10 mg/ml. The cultures were
incubated for 3 days at 37°C in the presence of 5% CO2 and then pulsed
with 1 mCi/well [methyl-3H]thymidine overnight. The cultures were harvested onto glass-fiber filter mats using a Tomtec Cell Harvester (Orange,
CT), and radioactivity was measured in a liquid scintillation counter (Betaplate, Wallac, Finland).
Target cells and CTL cultures
P815 (H-2d) cells, routinely used as target cells for the cytotoxicity assay,
were maintained in DMEM supplemented with 10% heat-inactivated FBS,
2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 5 mM
HEPES, and 0.075% sodium bicarbonate. All cell culture reagents were
purchased from Life Technologies (Grand Island, NY).
Spleen cells were cultured to generate effector CTL as previously described (19, 23). Spleen cell cultures were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 5 3 1025 M 2-ME,
25 mg/ml pyruvic acid, 100 U/ml penicillin, 100 mg/ml streptomycin, 5
mM HEPES, and 0.0225% sodium bicarbonate. Irradiated syngeneic cells,
pulsed with the NP147–155 peptide (10 mM), were used as stimulator cells.
Cytotoxicity assay
Cytotoxicity assays were performed as described previously (19, 23).
Briefly, target cells labeled with Na51CrO4 (Amersham, Arlington Heights,
IL) were pulsed with synthetic peptide NP147–155 at a concentration of 10
mM for 1–2 h at 37°C. The target cells were then washed thoroughly and
mixed with CTL at designated E:T ratios in 96-well plates and incubated
at 37°C for 4 h in the presence of 5% CO2. A 20-ml sample of supernatant
from each cell mixture was counted to determine the amount of 51Cr released from target cells. The percentage of specific lysis was calculated
using the formula: percent specific lysis 5 (E 2 S)/(M 2 S), where E
represents the average counts per minute released from target cells in the
presence of effector cells, S is the spontaneous counts per minute released
in the presence of medium only, and M is the maximum counts per minute
released in the presence of 2% Triton X-100.
Limiting dilution analysis (LDA) for CTL
The LDA protocol was modified from a previously published method (24).
Briefly, splenocytes were cultured in twofold serial dilutions
(1,000 –128,000 cells/well) in 24 replicates in U-bottom 96-well plates in
a total of 200 ml of complete RPMI 1640 medium supplemented with 10%
Rat T-stim conditioned media (Collaborative Biomedical, Bedford, MA),
0.05 M methyl-a-D-mannopyranoside, and 5 U/ml IL-2 (Cellular Products,
Buffalo, NY). Each well also contained 1 3 105 gamma-irradiated (2000
rad) syngeneic spleen cells as filler cells and 1 3 104 gamma-irradiated
syngeneic cells pulsed with NP147–155 synthetic peptide as restimulator
cells. The cultures were incubated at 37°C in the presence of 5% CO2 for
7 days. One hundred microliters of cultured cells from each well were then
transferred to 96-well plates and incubated with 1 3 104 51Cr-labeled P815
cells that had been pulsed with NP147–155 peptide in a standard 4-h cytotoxicity assay (see above). A well was considered positive (presumably
containing at least one CTLp) if the specific lysis was .20%. The CTL
precursor frequency was estimated by the minimal x2 method (25) using an
application program provided by R. H. Bonneau, Pennsylvania State University (Hershey, PA).
Statistical methods
Analysis of covariance (26) was utilized on data from experiments showing
the relationship of reciprocal CTLp frequency with dosage on injected NP
DNA to establish the similarity of the dose-response relationship among
experiments and to estimate the overall effect. A Spearman-Karber correlation coefficient (27) was calculated to represent the association between
CTLp frequency and survival rate in BALB/c mice. A repeated measures
analysis of variance (28) was performed on percent of initial weight in
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The expression vector used in the study, V1J, has been described previously (20). Briefly, it contains the human CMV immediate early gene enhancer and promoter, the intron A sequence, multiple restriction sites for
cloning the gene of interest, and the bovine growth hormone polyadenylation sequence. The NP gene from influenza virus A/PR/8/34 (H1N1) was
cloned into the BglII and SalI sites. The plasmid expressing the HA of
A/PR/8/34 has also been described previously (20). The plasmid DNA used
for immunization was purified from Escherichia coli by a modified alkaline
lysis procedure, and DNA was banded twice in CsCl2 gradients (21).
CORRELATION OF CTLp WITH ANTIVIRAL IMMUNITY
The Journal of Immunology
BALB/c mice to determine a difference in this measurement among groups
of animals injected with HA DNA, Vector DNA, and NP DNA.
Results
Comparison of CTLp frequency after influenza virus infection
and NP DNA immunization
Previous studies from our laboratory demonstrated that i.m. injection of plasmid DNA encoding NP derived from influenza virus
A/PR/8/34 in BALB/c mice can induce a vigorous CTL response
against an H-2Kd-restricted epitope, NP147–155, as measured by the
percentage of specific lysis by the bulk CTL culture in a cytotoxicity assay (19, 23). However, we found that 51Cr release assays
with bulk cultured splenocytes from NP DNA-immunized mice
can rarely be used to distinguish the efficacy or document the dose
relationships of DNA vaccines. Immunization of mice with 1 mg of
NP DNA can easily yield a splenocyte CTL culture rendering similar percentages of specific lysis of target cells as those for mice
receiving a 100-mg NP DNA inoculation or mice recovered from
influenza virus infection (results not shown). Furthermore, a recent
paper by Rodriguez et al. (29) has suggested that CTLp frequency,
rather than the presence of lytic activity in bulk CTL culture upon
restimulation, is a better correlation for protective immunity. Thus,
we decided to quantitatively measure the functionality of CTL in
immunized mice by performing LDA of CTLp frequency.
The efficacy as well as the kinetics of the CTL response following i.m. injection of NP DNA are shown longitudinally in Fig. 1
for BALB/c mice that had been either intranasally infected with
influenza virus A/HK/68 or injected i.m. with NP DNA. Spleen
cells obtained from the mice at different time points after virus
infection or NP DNA injection were restimulated in limiting dilution cultures with syngeneic spleen cells pulsed with NP147–155
epitope peptide for 7 days and then tested against P815 cells pulsed
with NP147–155 peptide in the standard cytotoxicity assay. The cultures from individual wells were considered positive if the specific
lysis of the target cells was .20% (see Materials and Methods for
details). As shown in Fig. 1, mice infected with influenza virus
rapidly mounted a CTL response against the NP147–155 epitope,
marked as a decline in the reciprocal CTLp frequency, i.e., a rise
in total numbers of CTLp specific for NP147–155 peptide (Fig. 1,
upper panel). By 2 wk postinfection, the reciprocal CTLp frequency had reached 40,000 –50,000, namely, one CTLp in every
40,000 –50,000 spleen cells, compared with fewer than one CTLp
in 200,000 spleen cells before infection in these mice. The reciprocal CTLp frequency in these infected mice remained at the lower
levels until 10 wk postinfection, with the lowest reciprocal frequency being close to 20,000 (one CTLp in every 20,000 splenocytes). A reciprocal CTLp frequency of 20,000 – 60,000 can be
detected in these mice even months after infection (results not
shown). The NP DNA-immunized mice, on the other hand, displayed a slow rise in the number of CTLp, reflected in a gradual
decline in the reciprocal CTLp frequency after injection (Fig. 1,
lower panel). Boosting injections at 3 and 6 wk improved the CTL
immune response, and the reciprocal CTLp frequency reached levels similar to those induced by viral infection by 8 –10 wk. These
results indicated that three i.m. injections of 30 mg of NP DNA can
induce comparable numbers of CTLp against the NP147–155
epitope as those induced by the viral infection in BALB/c mice,
but after multiple exposures and with slower kinetics.
Dose dependence of the immune responses induced by NP DNA
To further assess the immunogenicity of the NP DNA vaccine and
define the dose dependence of the immune response to DNA vaccines, we titrated the injection dose of NP DNA in mice and measured different parameters of NP-specific immune responses. First,
we tested the NP147–155-specific CTLp frequency in mice immunized three times with different doses of NP DNA. Repeated injections were conducted to minimize any injection variation that
might occur. The reciprocal CTLp frequency in correlation with
the dose of injected NP DNA is depicted in Fig. 2A. The reciprocal
frequency of the CTLp against the NP147–155 peptide was inversely
correlated with the dose of NP DNA injected, with the higher
injection dose of NP DNA corresponding to the lower reciprocal
frequency of the NP147–155-specific CTLp in mice, i.e., injecting a
higher dosage of NP DNA elicited more CTLp in mice. Injection
of 1 mg or more of NP DNA consistently yielded a reciprocal
CTLp frequency ,100,000 (i.e., more than one CTLp in every
100,000 splenocytes), whereas injection of 0.03 mg yielded a reciprocal CTLp frequency close to that detected in naive mice (generally with a reciprocal CTLp frequency .200,000, namely, less
than one CTLp in every 200,000 splenocytes; see Fig. 1). The
results from three experiments demonstrated very similar inverse
relationships. An analysis of variance was performed on the log of
the reciprocal CTLp frequency vs the log of the dose of injected
NP DNA. There is no evidence of a difference in this relationship
among the replicate experiments ( p 5 0.33); therefore, the data
were pooled to determine the rate of decrease in CTL with dose.
The slope of the relationship is 20.27 (95% confidence interval,
20.19 to 20.35), which corresponds to a 32% decrease in reciprocal CTLp frequency per 10-fold increase in dose (95% confidence interval, 21– 43%). This represents a statistically significant
dose-response relationship due to the fact that the confidence interval on the rate of decrease excludes zero ( p , 0.05).
Second, we measured the anti-NP Ab responses in an end-point
titration ELISA (Fig. 2B). The immunogenicity of NP DNA for
humoral responses also was found to be generally correlated with
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FIGURE 1. Kinetics of the CTLp frequency change after viral infection
(upper panel) or NP DNA injections (lower panel). Spleen cells from mice
infected with influenza virus A/HK/68 (marked by the arrow) or injected
with NP DNA (30 mg/mouse) one, two, or three times (marked by arrows)
were cultured in limiting dilution cultures in 24 replicates with syngeneic
spleen cells pulsed with NP147–155 peptide for 7 days and tested against
P815 cells pulsed with NP147–155 peptide in a 4-h cytotoxicity assay. The
splenocytes used in the culture were pooled from three or four mice in the
same dose or immunization group. A well in the LDA culture is scored
positive if its counts per minute value is greater than that corresponding to
20% specific lysis (see Materials and Methods for details).
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CORRELATION OF CTLp WITH ANTIVIRAL IMMUNITY
Last, we measured the T cell proliferative responses in each
dose group. The T cell proliferative responses were measured as
[3H]thymidine incorporation by spleen cells in culture upon stimulation with recombinant NP protein. The proliferative responses
of T cells from these mice, however, were found not to exhibit the
linear dose dependence seen with Ab and CTLp responses (Fig.
2C). Rather, a threshold effect was observed, in which only the
group injected with a 30-mg dose showed a significant level of
proliferation in response to NP protein stimulation in culture.
Much weaker positive responses were seen at immunization doses
from 0.1 to 10 mg. Spleen cells from all groups demonstrated significant proliferative responses when stimulated with Con A (results not shown). These results are consistent with our previous
observations that proliferative responses and cytokine secretion by
CD41 lymphocyte cultures were most vigorous when a large
quantity of NP DNA (50 –200 mg/i.m. injection) was inoculated (30).
Restimulated NP DNA-primed lymphocytes can confer
protection in naive mice
FIGURE 2. Correlation of NP-specific immune responses with injection
doses of NP DNA. Groups of mice injected with different doses of NP
DNA were 1) sacrificed, and the spleen cells were restimulated with syngeneic spleen cells pulsed with NP147–155 peptide in limiting dilution cultures in 24 replicates and tested against P815 pulsed with NP147–155 peptide
for assessment of the CTLp frequency (A); 2) were bled for sera and tested
for anti-NP Ab responses in an end-point ELISA (B); or 3) were sacrificed
and the spleen cells were incubated in quadruplicate with recombinant NP
protein (10 mg/ml) for 4 days for detecting [3H]thymidine incorporation
(C). The splenocytes used in the experiments shown in A and C were
pooled from three or four mice 3 wk after the last immunization. The sera
assayed in the experiment shown in B were collected from the mice (groups
of 10) before they were challenged with live influenza virus (results shown
in Fig. 5). The error bars represent the SEMs.
the injection dose. Increasing the injection dose of NP DNA resulted in higher ELISA end-point titers in mice, and an injection
dose of 0.03 mg yielded a titer very close to that detected in naive
mice (,100).
We previously showed that NP DNA-primed spleen cells, upon in
vitro restimulation with influenza virus A/PR/8/34, can confer protection against lethal cross-strain challenge with influenza virus
A/HK/68 when adoptively transferred into naive mice (22). To
more precisely assess the role of NP147–155 peptide-specific lymphocytes in the protection, we restimulated spleen cells from mice
immunized with NP DNA or infected with influenza virus with
syngeneic spleen cells pulsed with NP147–155 synthetic peptide for
7 days. This condition hypothetically would only expand the
NP147–155 peptide-specific CTL in culture, since the peptide consists of amino acids of a minimum H-2Kd-restricted CTL epitope
(31, 32), and the recognition of this epitope by NP-specific CTL
requires the presentation by H-2Kd class I molecules (33). It is also
possible that restimulation of NP147–155-specific CD81 T cells
may result in the production of cytokines that may, in turn, activate
other antiviral effector cells during the in vitro restimulation period. As shown in Fig. 3, naive mice that received these NP147–155
peptide-restimulated splenocytes from the NP DNA-immunized or
A/PR/8/34 virus-primed mice were fully protected, whereas the
naive mice that received no cells succumbed to the challenge.
These results indicated the ability of the NP147–155-specific T cells
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FIGURE 3. NP147–155-restimulated splenocytes are protective against
lethal influenza virus challenge. Spleen cells from groups of 20 mice
primed with influenza virus A/PR/8/34 infection or NP DNA injection (3 3
100 mg/mouse) were in vitro restimulated with syngeneic spleen cells
pulsed with NP147–155 peptide for 7 days and adoptively transferred into
groups of 10 naive mice at 5 3 107 cells/mouse. The recipient mice and
naive control mice were infected with a lethal dose of A/HK/68 before the
transfer of splenocytes. The percentage of survival and weight change (not
shown) were monitored up to 4 wk.
The Journal of Immunology
induced by NP DNA immunization to contribute to protection
from cross-strain influenza challenge.
To estimate the number of NP DNA-primed and in vitro fully
activated lymphocytes needed for complete protection in naive
mice, we titrated the cultured spleen cells in an adoptive transfer
experiment. These cells had been activated by restimulation with
influenza virus in vitro and were cytolytic against NP147–155 peptide-pulsed target cells at the time of the transfer (results not
shown). Three groups of 10 mice that had been challenged with
influenza virus A/HK/68 received 5 3 107, 1.7 3 107, or 5 3 106
cells/mouse, respectively. As shown in Fig. 4A, only the group that
received 5 3 107 cells was completely protected (100% survival),
whereas the group that received 1.7 3 107 showed an 80% survival
rate, and the group that received 5 3 106 showed almost no protection. The control group of naive mice was not protected (this
challenge was performed at the same time as that shown in Fig. 3;
refer to Fig. 3 for the survival curve of the control group). The
change in the body mass of these mice also reflected significant
morbidity (Fig. 4B). Even though all the mice in the group that
received 5 3 107 cells survived challenge, they suffered, on the
average, a loss of .10% of body mass on day 7 postinfection
before recovering from the infection. These findings indicated that
survival of the mice may depend on the number of NP DNAprimed lymphocytes present at the time of infection. The results
also showed that not even the fully activated lymphocytes adoptively transferred into naive mice shortly after the infection could
FIGURE 5. Correlation of CTLp frequency with protective immunity
induced by NP DNA injection. Groups of 10 mice that were injected with
different doses of NP DNA were challenged with a lethal dose of influenza
virus A/HK/68 and monitored for weight loss and survival up to 4 wk (A).
The survival rates from these experiments were plotted against their corresponding CTLp frequency, shown in Fig. 2A (B). The correlation coefficient (r) was determined for all the correlates in three experiments.
prevent the establishment of viral infection or eliminate the virus
from the host before the onset of morbidity. This experiment, however, was not designed to offer an accurate estimate of how many
NP147–155-specific CTLp are needed for optimal protection, since
all the NP-specific lymphocytes transferred would have been fully
activated by in vitro restimulation with infectious virus.
NP147–155-specific CTLp frequency as a correlate of protection
To study the association of NP147–155 CTLp frequency and crossstrain protection by NP DNA immunization, we challenged the
groups of mice that had been injected with different doses of NP
DNA (immunized in parallel with the groups assayed in the experiments shown in Fig. 2) with a lethal dose of influenza virus
A/HK/68. The mortality and morbidity of these mice were recorded as survival rate and group weight loss up to 4 wk postinfection (Fig. 5A). The mice that received 1 mg or more of NP DNA
largely survived the challenge (.80% survival), whereas other
groups generally succumbed to influenza virus challenge. Nonetheless, all the surviving mice suffered similar levels of morbidity,
as indicated by an average 10 –20% weight loss by day 7 postinfection (Fig. 5A). A repeat of the challenge experiment yielded
similar observations (results not shown), and the survival rates of
the groups are incorporated in Fig. 5B (see below).
After plotting the group survival rate against the corresponding
reciprocal CTLp frequency determined in the three experiments
shown in Fig. 2A, the survival rate was found to inversely correlate
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FIGURE 4. Number of NP DNA-induced fully activated CTL needed to
achieve optimal protection in naive mice. Spleen cells from groups of 20
mice injected with NP DNA (3 3 100 mg/mouse) were in vitro restimulated with syngeneic spleen cells infected with A/PR/8/34 for 7 days and
adoptively transferred into groups of 10 naive mice at the indicated cell
number per mouse. The recipient mice were infected with a lethal dose of
A/HK/68 before cell transfer, and survival (A) and mass loss (B) were
monitored for up to 4 wk. This challenge was performed at the same time
as the challenge shown in Fig. 3; therefore, the results for control group of
naive mice in this experiment can be found in Fig. 3.
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CORRELATION OF CTLp WITH ANTIVIRAL IMMUNITY
with the reciprocal frequency of the NP147–155-specific CTLp (Fig.
5B). The Spearman correlation coefficient between CTLp frequency and survival rate was 20.81 ( p , 0.05). Thus, there is
evidence of a significant association between CTLp frequency and
survival rate. The group of mice with a reciprocal CTLp frequency
,100,000, i.e., having more than one CTLp in every 100,000
splenocytes, generally survived the lethal cross-strain challenge
with influenza virus A/HK/68 (.80% survival), whereas the group
with a higher reciprocal CTLp frequency (.1/120,000, namely,
having less than one CTLp in every 120,000 splenocytes) exhibited suboptimal or no protection in the challenge experiments.
These results indicated that the minimal reciprocal CTLp frequency associated with optimal cross-strain protection is 100,000,
namely, one NP147–155 epitope-specific CTLp in every 100,000
splenocytes. This does not mean that the function of CTLp is directly correlated with the protection we documented in the challenge experiment, but only indicates that the NP147–155-specific
CTLp frequency in NP DNA-immunized mice can serve as a surrogate marker for an individual’s chance to survive the lethal challenge.
To illustrate the difference between protective immunities directed
against the variable surface protein HA and those against the conserved internal protein NP, we challenged two groups of mice that
had been injected three times with 100 mg of A/PR/8/34 HA DNA
or 100 mg of blank vector DNA, respectively, with a lethal dose of
influenza virus A/PR/8/34 (H1N1). The group that received A/PR/
8/34 HA DNA achieved a 100% survival rate against the homologous strain virus challenge, while none of the mice in the group
that received blank vector DNA survived (Fig. 6A). Noticeably, the
immunized group suffered no significant weight loss at any time
point monitored. However, the mice immunized with A/PR/8/34
HA DNA were not protected when challenged with a heterologous
strain of influenza virus of a different subtype, A/HK/68 (H3N2).
As shown in Fig. 6B, the group that received the A/PR/8/34 HA
DNA showed similar levels of weight loss as the group that received blank vector DNA, whereas the group immunized with NP
DNA clearly demonstrated improved recovery from weight loss
resulting from influenza virus infection. The group weight change
between the groups illustrated the morbidity suffered by the mice,
although in this particular experiment the majority of the mice
survived the challenge. A repeated measures analysis of variance
was performed on percentage of initial weight. Missing values for
the animals that had died were imputed from the information retained from the surviving animals. There is a statistically significant difference among treatment groups in the average percentage
of initial weight through 20 days ( p , 0.001), where this outcome
is the result of the difference between groups receiving HA DNA
and vector DNA relative to the group receiving NP DNA. A graph
of the average percentage of initial weight for the three groups is
presented in Fig. 6A. Error bars represent pairwise comparisons of
the groups at each time point. A significant difference between
groups at a time point is signified in error bars that do not overlap
( p , 0.05). Significant differences in the percentage of initial
weight were observed between groups receiving HA DNA or vector DNA relative to that in the group receiving NP DNA from 9
days postinfection on. These data indicated that the predominantly
strain-specific Ab-based protective immunity can prevent both
mortality and morbidity inflicted by influenza virus infection with
the homologous strain virus (A/PR/8/34), but offers no protection
against infection with heterologous strain virus A/HK/68 (H3N2),
whereas cellular immunity induced by NP DNA immunization can
provide cross-strain protection against mortality and morbidity
caused by influenza virus infection.
FIGURE 6. Protection of mice immunized with HA DNA against lethal
challenge with homologous and heterologous strains of influenza virus.
Two groups of 10 mice immunized with a DNA vaccine expressing A/PR/
8/34 HA or a blank vector DNA were challenged with a lethal dose of
influenza virus A/PR/8/34 (H1N1; A), or three groups of 10 mice injected
with DNA vaccines expressing A/PR/8/34 HA, A/PR/8/34 NP, or blank
vector DNA were challenged with a lethal dose of influenza virus A/HK/68
(H3N2; B) and monitored for mass loss for up to 3 wk. Significant differences in the percentage of initial weight were observed between groups
receiving HA DNA or vector DNA relative to the group receiving NP DNA
from 9 days postinfection and thereafter (p , 0.001).
Discussion
The present study was designed to use influenza virus NP as a
model Ag to quantitatively assess the immunogenicity of DNA
vaccines. The i.m. injection of a plasmid DNA encoding NP from
influenza virus A/PR/8/34 (H1N1) can induce a robust CTL response against the H-2Kd-restricted epitope 147–155, and the immune response induced by NP DNA immunization can provide
protection in mice against a cross-strain challenge with influenza
virus A/HK/68 (H3N2) (19, 23). Here we initially recorded the
kinetics of the CTLp response following i.m. injection of NP DNA
compared with those of natural influenza virus infection. Second,
we documented the dose dependency of NP147–155-specific CTLp
frequency following i.m. injection of titrated doses of NP DNA in
mice. Third, we demonstrated an association between NP147–155specific CTLp frequency and survival rate in mice and assessed the
minimal CTLp frequency associated with protective immunity
against cross-strain influenza virus challenge. Last, we illustrated
the difference in cross-strain immunities between immunization
with NP DNA and A/PR/8/34 HA DNA. Although HA DNA provided sterilizing immunity in mice against challenge with the homologous (H1N1) strain of influenza virus, it offered no protection
against the challenge with a heterologous (H3N2) strain of influenza virus even when the challenge was capable of causing only
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Protection mediated by immune responses against HA
The Journal of Immunology
other methods, such as the enzyme-linked immunospot assay and
the tetramer-staining assay (40 – 44); and 3) the effector cells estimated in this study were splenic CTL precursors induced by
DNA immunization and thus were probably predominantly or exclusively memory T cells and were different from the lymphocytes
detected in mice recovering from an acute viral infection or suffering a persistent viral infection. They were also different from the
lymphocytes used in the adoptive transfer experiments, which
were fully activated by in vitro restimulation and were highly cytolytic in vitro (i.e., functional effector cells). Although the conclusions from adoptive transfer experiments are generally definitive about the roles of CD41 or CD81 T cells in protective
immunity (7, 8, 12), the infusion of large quantities of active cytolytic cells in recipients may not necessarily be reflective of the
natural protective responses induced by immunization. Therefore,
the approach taken in the present study not only provides a quantitative estimate of host-generated cytotoxic T cell immune responses and protective efficacy by NP DNA immunization, but
also offers a physiological assessment of the levels of CTLp associated with cross-strain protection when other immune factors
associated with host anti-viral immunity are present.
In vivo depletion studies have shown that CD41 T cells contribute to cross-strain protection in mice immunized with NP
DNA, and T cell subset adoptive transfer experiments have shown
that activated CD41 T cells can provide cross-strain protective
immunity in mice (30). The roles of CD41 T cells in antiviral
immunity are complex and may include production of antiviral
cytokines, lysis of virus-infected host cells, amplification of CD81
T cell responses, and helper function for Ab production (6, 13).
Our results, showing little if any correlation of spleen cell proliferative responses with injection doses of NP DNA (Fig. 2C),
should not be considered evidence against the role of CD41 T cells
in the protective immunity induced by NP DNA immunization.
Rather, our results indicate that bulk spleen cell proliferative responses are probably not an appropriate surrogate marker to assess
the antiviral immunity in this system. T cell proliferative responses
in bulk cultures, like the percentage of specific lysis recorded with
bulk CTL culture, may serve primarily as a semiquantitative measure for CD41 lymphocyte responses. A more quantitative
method, such as the enzyme-linked immunospot assay, should be
considered in the future to assess CD41 T cell responses.
NP DNA-induced cellular immunity can accelerate the viral
clearance and improve the host’s chance for survival. The virus
titers recovered from the lung were much lower in NP DNA-immunized mice than in the mice injected with blank vector DNA
(19). However, NP DNA-induced cellular immunity is by no
means a sterilizing immunity, and the mice generally suffered
about 10 –20% weight loss before recovering from viral infection
(Figs. 4B and 5A). In contrast, the mice injected with A/PR/8/34
HA DNA, in which the HA type-specific Ab responses were elicited (results not shown), were completely protected against lethal
challenge with the homologous strain of influenza virus in terms of
both mortality and morbidity, but were provided no protection
against even a relatively mild cross-strain challenge with the heterosubtypic influenza virus A/HK/68 (Fig. 6). Thus, an optimal
prophylactic vaccine against influenza virus should include means
to induce both Ab responses for type-specific protection and cytotoxic T cell responses for broad cross-strain protection (13).
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CORRELATION OF CTLp WITH ANTIVIRAL IMMUNITY