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Departments of
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Virus and Cell Biology and
Biometrics, Merck Research Laboratories, West Point, PA 19486
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 marrow-derived professional APCs (16, 17, 18) and may include an Ag transfer process from muscle cells to professional APCs, similar to 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-2Kd-restricted 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 vaccine-induced 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 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 dose-dependent 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 CD4+ T cell responses to NP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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 µl/well overnight at 4°C with the purified recombinant NP protein at 7.5 µg/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 µl 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 µl 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 µl 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 x 105 cells/well in 200 µl of complete RPMI 1640 medium in quadruplicate. Each well also contained purified recombinant NP at 10 µg/ml. The cultures were incubated for 3 days at 37°C in the presence of 5% CO2 and then pulsed with 1 µCi/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 µg/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 x 10-5 M 2-ME, 25 µg/ml pyruvic acid, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mM HEPES, and 0.0225% sodium bicarbonate. Irradiated syngeneic cells, pulsed with the NP147–155 peptide (10 µM), 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 µM 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-µl 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 = (E - S)/(M - 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 µl of complete RPMI 1640 medium supplemented with
10% Rat T-stim conditioned media (Collaborative Biomedical, Bedford,
MA), 0.05 M methyl-
-D-mannopyranoside, and 5 U/ml IL-2
(Cellular Products, Buffalo, NY). Each well also contained 1 x
105 gamma-irradiated (2000 rad) syngeneic spleen cells as
filler cells and 1 x 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 x 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 
2 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 BALB/c mice to determine a difference in this measurement among groups of animals injected with HA DNA, Vector DNA, and NP DNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg 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-µg 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 µg
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.
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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 µg 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 µg 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 = 0.33); therefore, the data
were pooled to determine the rate of decrease in CTL with dose. The
slope of the relationship is -0.27 (95% confidence interval, -0.19
to -0.35), 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).
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B). The immunogenicity of NP DNA for
humoral responses also was found to be generally correlated with 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
µg yielded a titer very close to that detected in naive mice (<100).
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-µg 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 µg. 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 CD4+
lymphocyte cultures were most vigorous when a large quantity of
NP DNA (50–200 µg/i.m. injection) was inoculated (30).
Restimulated NP DNA-primed lymphocytes can confer protection in naive mice
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
CD8+ 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 induced by NP DNA
immunization to contribute to protection from cross-strain influenza
challenge.
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A, only the group that
received 5 x 107 cells was completely protected
(100% survival), whereas the group that received 1.7 x
107 showed an 80% survival rate, and the group that
received 5 x 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 x 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 DNA-primed
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 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.
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To study the association of NP147–155 CTLp
frequency and cross-strain 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
µg 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).
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A, the survival rate was found to inversely correlate
with the reciprocal frequency of the
NP147–155-specific CTLp (Fig. 5B).
The Spearman correlation coefficient between CTLp frequency and
survival rate was -0.81 (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. Protection mediated by immune responses against HA
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 µg of A/PR/8/34 HA DNA or 100
µg 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunizing mice with titrated doses of NP DNA provided a means to quantitatively measure the different aspects of NP-specific immune responses. A reproducible correlation with the injection dose was documented for NP147–155-specific CTLp frequency by LDA as well as anti-NP Ab responses by ELISA. The dose dependence of the Ab response induced by DNA immunization has also been demonstrated with other influenza virus gene constructs, such as HA DNA vaccine (J.B.U., et al., unpublished observations). Surprisingly, no such obvious correlation with the immunizing doses of NP DNA could be demonstrated for T cell proliferative responses. Yet, it is likely that CD4+ T cells were activated even at low doses of DNA immunization, since IgG Ab responses were detected, and IgM to IgG isotype switching in mice generally requires the involvement of Th cells. This may indicate that there is a threshold dose of NP DNA vaccine required for the positive detection of proliferative responses in mice. This is supported by our recent observations that T cell proliferation and cytokine secretion by CD4+ lymphocyte cultures were most vigorous when a high immunization dose of NP DNA of 50–200 µg was applied (30). This may also be a function of the adjuvant effects of bacterial DNA described by Krieg (34) and others (35). In addition, the NP-specific CD4+ T cell responses may not be as monospecific as the CTL responses in BALB/c mice. Potentially, a greater diversity of MHC class II-restricted epitopes, in contrast to the single dominant CD8+ CTL epitope 147–155 (23), might also limit our ability to detect a linear dose-dependent relationship, in contrast to the threshold effect that was seen (36).
In contrast, comparison of
NP147–155-specific reciprocal CTLp frequency
with survival rate strongly indicated that a reciprocal CTLp frequency
of 100,000 or lower, i.e., one or more CTLp in every 100,000
splenocytes, was associated with the highest survival rates after
cross-strain influenza virus challenge (Fig. 5B). Thus,
these experiments indicate that
NP147–155-specific CTLp frequency can serve as a
surrogate marker to assess the cross-strain resistance to lethal
influenza virus challenge in BALB/c mice. The ability of a single
epitope response to exhibit such a relationship may be surprising given
that the potential for recognition of multiple epitopes exists.
However, immune dominance appears to limit the diversity of the
effector CTL response, at least in BALB/c mice. We and others have
described an immunorecessive CTL epitope within NP, residues 218–226.
However, the CTL response against this epitope could only be detected
when we deleted the immunodominant CTL epitope,
NP147– 155, in NP DNA construct (22),
and the CTLp frequency for this epitope appears low even in multiply
immunized mice (T.-M.F., unpublished observations). Furthermore,
when we challenged naive mice that had received activated lymphocytes
from the mice immunized with NP DNA in which the 147–155 epitope had
been modified (lymphocytes were in vitro restimulated with influenza
virus), they were only partially protected (40–50% vs 90–100%)
(T.-M.F., unpublished observations).
The CTLp frequency estimated in this study is lower than that reported in previous studies (29, 37, 38, 39). There are three points to be considered regarding these data: 1) our estimation for the CTLp frequency is a conservative one, since our arbitrary cut-off value used to score a positive microculture in the standard cytotoxicity assay is highly stringent (20% of specific lysis compared with others that generally apply spontaneous lysis counts + 3 SD as cut-offs to score a positive culture well; see Materials and Methods for details); 2) the LDA method for estimating CTLp frequency is far more conservative, as the in vitro culture conditions may fail to expand every in vivo functional CTLp, thus presenting a lower estimate of precursor frequency compared with those assessed by other methods, such as the enzyme-linked immunospot assay and the tetramer-staining assay (40, 41, 42, 43, 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 CD4+ or CD8+ 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 CD4+ T cells
contribute to cross-strain protection in mice immunized with NP DNA,
and T cell subset adoptive transfer experiments have shown that
activated CD4+ T cells can provide cross-strain protective
immunity in mice (30). The roles of CD4+ T cells in
antiviral immunity are complex and may include production of antiviral
cytokines, lysis of virus-infected host cells, amplification of
CD8+ 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 CD4+ 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 CD4+ lymphocyte responses. A
more quantitative method, such as the enzyme-linked immunospot assay,
should be considered in the future to assess CD4+ 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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Footnotes |
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2 Current address: Chiron Corp., 4560 Horton St., Emeryville, CA 94608.
3 Abbreviations used in this paper: NP, nucleoprotein; CTLp, cytotoxic T lymphocyte precursor; HA, hemagglutinin; LDA, limiting dilution analysis.
Received for publication July 30, 1998. Accepted for publication January 4, 1998.
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ß and 
T cell subsets in viral immunity. Annu. Rev. Immunol. 10:123.[Medline]
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