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*
Department of Immunology, St. Jude Children’s Research Hospital, Memphis TN 38105, and
Department of Pathology, University of Tennessee, Memphis, TN 38163
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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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It has been established that CD8+ T cells recognize peptide
Ags in the context of self-MHC class I glycoproteins through the TCR
(5). Recognition of a relevant peptide/MHC complex triggers the
specific lysis of virally infected cells and the secretion of
cytokines, such as IFN-
and TNF-
, involved in the noncytopathic
inactivation of virus (6, 7). The number of epitopes involved in the
effector CTL response to viral infection is small relative to the total
number of epitopes potentially available. This is because effector CTL
responses to dominant epitopes tend to suppress responses to
subdominant epitopes, a phenomenon referred to as immunodominance (8).
For example, the effector CTL response to Sendai virus infection is
predominantly directed against a single immunodominant NP epitope,
NP324–332/Kb (9, 10, 11). However, CTL responses
to subdominant epitopes are able to readily compensate for the loss of
an effector CTL response to the dominant epitope (12, 13, 14, 15). In addition,
potent memory CTLp specific for subdominant epitopes are elicited by
primary virus infection, despite the apparent absence of effector CTL
responses to these epitopes (16, 17, 18, 19, 20, 21).
The observation that CD8+ T cell responses tend to be focused on relatively conserved internal viral components has led to interest in vaccines designed to emphasize cell-mediated immunity (8). In this regard, both peptide- and vaccinia-based vaccines that activate CD8+ T cells have been shown to be protective against respiratory virus infections (9, 22, 23, 24). Recently, it has been established that immunization with naked DNA is an effective way of inducing both cellular and humoral immunity to viruses, parasites, bacteria, and tumor Ags (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). For example, it has been shown that DNA vaccination primes potent MHC class I-restricted CTL activity specific for respiratory virus Ags (38, 39). In the case of influenza nucleoprotein (NP),3 DNA vaccination has been shown to confer protection against infection with other strains of influenza virus (39, 40).
While the efficacy and utility of DNA vaccines have been established, relatively little is known about their mechanism of action or the quality of the CD8+ T cell response elicited. For example, it is not clear whether concomitant Ab responses are necessary for the efficient priming of CTL precursors (CTLp) in vivo. In addition, it is not clear whether DNA vaccination elicits the same breadth of response as natural infection in terms of the specificity of the T cells. In the current study, we analyzed the CD8+ T cell response induced following vaccination with a plasmid construct encoding the Sendai virus NP. Various parameters of the response were investigated including the establishment of memory to both dominant and subdominant epitopes, the trafficking of primed CD8+ T cells to sites of infection, and the role of Ab in establishing CD8+ T cell immunity. The data show that vaccination with DNA encoding Sendai NP can prime strong CTL responses specific for both dominant and subdominant epitopes, and that neither MHC class II+ cells or Ab are required for the priming of these T cells. In addition, primed CD8+ memory T cells are able to migrate to the lung following intranasal (i.n.) Sendai virus infection. Finally, DNA vaccination with Sendai NP confers Ab-independent protection against challenge with a lethal dose of Sendai virus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b), B6CBAF1/J (H-2b/k), and B6.C-H-2bm1/ByJ (H-2bm1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C2- (H-2b) mice, which lack MHC class II molecules (41), and µMT (H-2b) mice, which lack mature B cells (42), were bred under license at St. Jude Children’s Research Hospital (Memphis, TN). All mice were held under specific pathogen-free conditions until infection or immunization.
Cell lines and culture conditions
The L929, L929-Kb, L929-Db, SVBM1, and MC57G cell lines have been described previously (43, 44, 45, 46). SVBM1 cells were a kind gift of Dr. Linda Gooding (Emory University School of Medicine, Atlanta, GA), and Cos7 cells were provided by Dr. Mary Ellen Conley (St. Jude Children’s Research Hospital). All cell lines were maintained in complete tumor medium (CTM) (47) at 37°C in the presence of 10% CO2. L929-Db and L929-Kb were cultured in CTM supplemented with 0.5 g/ml G418 (Life Technologies, Grand Island, NY) to maintain Db and Kb expression.
Synthetic peptide
Sendai virus NP peptide, NP324–332 (FAPGNYPAL), was synthesized at St. Jude Children’s Research Hospital Center for Biotechnology on an Applied Biosystems model 433A peptide synthesizer (Berkeley, CA). Peptide purity was monitored by reverse phase HPLC analysis. Peptide stock solutions were prepared in PBS and diluted in CTM to the appropriate concentration.
Generation of polyclonal CTL lines and limiting dilution analysis
Short-term CTL lines were generated from the spleens of immunized mice as described previously (16). Briefly, spleen cells were teased through nylon meshes to generate single-cell suspensions and then depleted of erythrocytes by suspension in Gey’s solution for 5 min. Spleen cells (6 x 106) from immunized mice were incubated with 6 x 106 irradiated syngeneic splenocytes in 2 ml CTM in the presence of 0.5 µg/ml NP324–332 and 10 U human rIL-2 in 24-well plates for 5 to 6 days. It should be noted that the NP324–332 peptide binds to Kb and Db with approximately equal affinity and stimulates both NP324–332/Kb- and NP324–332/Db-specific T cells in this system. Limiting dilution analysis was used to determine CTLp frequencies as described previously (16, 48, 49). Briefly, titered numbers of spleen cells from vaccinated mice were cocultured with 5 x 104 irradiated C57BL/6 splenocytes in the presence of 0.5 µg/ml NP324–332 peptide and 10 U/ml human rIL-2 in a final volume of 200 µl in round-bottom microtiter wells. After 7 days, the contents of individual wells were split and tested in a 6-h cytotoxicity assay on 2 x 103 51Cr-labeled target cells. Minimal estimates of CTLp frequency were obtained according to the Poisson equation of the slope of the line (cell number vs log fraction of nonresponding wells) using x2 analysis. This yields the minimal frequency estimate and 95% confidence intervals.
Virus infection, virus challenge
The Enders strain of Sendai virus was prepared and stored as described previously (4, 50). Mice were anesthetized by i.p. injection with Avertin (2,2,2-tribromoethanol) and infected i.n. with either 1 x 103 50% egg infectious doses (EID50) of Sendai virus for the generation of CTL responses, or lethal doses of Sendai virus for the protection studies (1.32 x 105 EID50 for C2- and C57BL/6 mice and 6.6 x 104 EID50 for µMT mice). Lymphocytes responding in the lungs of infected mice were collected 7 or 8 days after infection by bronchoalveolar lavage (BAL), as described previously (49). The cells were allowed to adhere to a tissue culture flask for 1 h at room temperature to remove the adherent cells. The nonadherent BAL cells were depleted of erythrocytes with Gey’s solution and then used directly in the cytotoxicity assay. For the protection studies, mice were observed for 21 days following challenge with a lethal dose of Sendai virus.
Cytotoxicity assay
Cytotoxic activity was determined as described previously (16). Target cells (L929, L929-Kb, L929-Db, MC57G, or SVBM1) were labeled with Na51CrO4 (Amersham Life Science, Arlington Heights, IL) and either infected with Sendai virus or pulsed with NP324–332 peptide. Various numbers of effector CTL were incubated with 2 x 103 target cells in 96-well plates at 37°C in the presence of 10% CO2 for 5 h. The percent of specific release was calculated by the formula: % of specific release = ([experimental release - spontaneous release]/[maximum release - spontaneous release]) x 100 cpm. Spontaneous release was typically <20% of the total release induced by 1% Triton X-100.
Construction and purification of DNA vaccine
The pJW4303 vector has been described previously (51). It contains a CMV immediate/early gene promoter that drives the transcription of the encoded gene. The vector was linearized with BamH1 and HindIII, and the ends were filled in with T4 DNA polymerase. An EcoR1/BamH1 fragment encoding the entire cDNA of Sendai virus NP was isolated from pTF1-SNP, kindly provided by Drs. A. Portner and T. Takimoto (St. Jude Children’s Research Hospital). The ends of the NP gene were filled in with T4 DNA polymerase and the fragment was blunt-ligated to pJW4303 with a DNA Ligation Kit (Takara Shuzo Co. Ltd., Kyoto, Japan) to generate pJW-SNP. pJW-SNP was shown to contain the full length Sendai NP gene in the correct orientation. Both pJW4303 and pJW-SNP were transformed into C600 bacteria, cultured in Luria Broth, and purified with a Qiagen Maxiprep Kit (Chatsworth, CA).
Transfection
Cos7 (106) cells were transfected with 10 µg
of the pJW-SNP plasmid by electroporation with a Gene Pulsar II
apparatus (Bio-Rad, Hercules, CA). Briefly, cells were trypsinized,
washed, and resuspended in 0.7 ml of PBS in the presence of 10 µg
pJW-SNP. Cells were then transferred to a 0.4-cm Gene Pulsar Cuvette
(Bio-Rad) and pulsed twice using a voltage of 850 V, a capacitor
setting of 25 microfarad, and a time constant after pulse of about 0.5
milliseconds. After electroporation, the cells were rested on ice for 5
min and then transferred to 12-well plates containing 4 ml CTM. The
electroporated cells were cultured for 2 days and then tested for NP
gene expression using an immunobrown assay. Briefly, transfected cells
were permeabilized with pure methanol for 15 min prior to the addition
of a mouse mAb specific for Sendai NP, A-SVNP (kindly provided by Dr.
A. Portner). Then biotinylated anti-mouse Ig Ab (Vector
Laboratories, Burlingame, CA) was added and incubated for an additional
30 min. The cells were then washed, and strepavidin-coupled horseradish
peroxidase (Vector Laboratories) was added followed by
3,3-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis,
MO). Cells expressing Sendai NP turned brown and could be identified by
standard light microscopy. Transfection efficiency was 
30%.
Immunization of mice
The Accell helium-driven particle delivery device (gene gun) (Auragen Inc., Middleton, WI) was used in the experiments. 40 µg of pJW-SNP or pJW4303 was coated onto 25 mg of 2.1-diameter gold beads (Auragen Inc.) in the presence of 100 µl of 1 M CaCl2 and 100 µl of 0.05 M spermidine. Following precipitation of DNA onto the gold beads, the DNA was washed three times with ethanol and then resuspended in 3.6 ml ethanol in the presence of 0.1 mg/ml polyvinyl pyrrolidone. The DNA/gold was loaded onto the interior surface of Tefzel tubing (McMaster-Carr, Chicago, IL) using a tube loader. The tubing was cut into 0.5-inch segments, each of which contained about 0.4 µg to 0.6 µg of DNA. In general, 8- to 12-wk-old mice were vaccinated twice (at a 3-wk interval) on a shaved region of the abdomen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that infection of C57BL/6 mice with
Sendai virus, a murine parainfluenza-1 virus (52), elicits a potent
effector CTL response in the lung that appears to be exclusively
directed to an immunodominant NP epitope,
NP324–332/Kb (9, 10, 11). However, the pool
of memory CTLp that are established following viral clearance include
populations specific for both the dominant
NP324–332/Kb and subdominant
NP324–332/Db epitopes (16). Importantly, CTLp
specific for each of these epitopes are present at similar frequencies,
indicating that potent memory CTLp specific for subdominant epitopes
can be induced in the absence of a detectable effector response. To
investigate whether a similar spectrum of CTLp is elicited by DNA
immunization, C57BL/6 mice were immunized twice (at 3-wk intervals)
with a plasmid containing the Sendai virus NP gene, pJW-SNP. Control
animals were immunized with the empty vector, pJW4303. Two weeks after
the second immunization, splenocytes were restimulated in vitro with
the NP324–332 peptide and tested for their ability to lyse
L929-Kb and L929-Db transfectants pulsed with
the NP324–332 peptide. As shown in Figure 1
, CTL isolated from pJW-SNP-primed but
not pJW4303-primed animals were specific for both the immunodominant
NP324–332/Kb and subdominant
NP324–332/Db epitopes. In addition, these CTL
were able to lyse Sendai virus-infected MC57G (H-2b)
cells.
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A and Table I, the frequency of CTLp specific for the
dominant NP324–332/Kb epitope was 1:9000
spleen cells (equivalent to 1:1500 CD8+ T cells). This is
similar to the frequency of memory
NP324–332/Kb-specific CTLp elicited by Sendai
virus infection (1:1000 CD8+ T cells) (16). The
frequency of CTLp specific for the subdominant
NP324–332/Db epitope was 1:24,000 spleen
cells (equivalent to 1:3500 CD8+ T cells). Again, this is
very similar to the frequency of
NP324–332/Db-specific CTLp elicited by Sendai
virus infection (1:2000 CD8+ T cells) (16). Thus, these
are the first data to clearly demonstrate that DNA vaccination can
induce high frequency T cell memory to subdominant epitopes in the face
of a competing response to a dominant epitope. Notably, memory CTLp
populations specific for both dominant and subdominant epitopes were
long-lived and could be readily recovered 5 mo after the initial
vaccination (Table I). In addition, similarly high frequencies of
memory CTLp were also generated following only a single immunization
with the appropriate DNA construct (data not shown).
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Although the data demonstrated that DNA vaccination was able to
prime CTLp specific for both dominant and subdominant T cell epitopes,
it was not clear that such cells had the appropriate avidity,
longevity, or homing properties to effectively participate in a
subsequent immune response. To investigate this, we took advantage of a
system that allowed us to track primed T cells to the site of
infection. Previous studies have shown that
H-2k-restricted T cells dominate the effector
CD8+ T cell response to Sendai virus infection in the lungs
of B6CBAF1 mice (H-2b/k) (16). Despite the presence of the
Kb MHC molecule, there is little to no detectable effector
response to the NP324–332/Kb epitope, which
has been rendered subdominant in this strain of mice. However, priming
of NP324–332/Kb-specific T cells prior to
infection (for example, by infection with a recombinant vaccinia virus
that expresses NP) results in a switch in the specificity of effector
cells in the lung induced by subsequent Sendai virus infection (16).
Thus, to demonstrate that CTLp primed by DNA vaccination could be
recruited to the site of infection, B6CBAF1 mice were vaccinated with
either pJW-SNP or pJW4303. Two weeks after the second vaccination, the
mice were infected i.n. with Sendai virus, and the specificity of the
BAL was assessed 8 days later. As shown in Figure 3A, prior vaccination with the
control vector (pJW4303) resulted in the expected dominance of
H-2k-restricted effector CTL in the lung following Sendai
virus infection. However, prior vaccination with pJW-SNP resulted in a
complete switch in specificity such that the response was now dominated
by NP324–332/Kb-specific effector CTL (Fig. 3B). These data demonstrate that memory CTLp induced
by DNA vaccination are able to traffic to and function at the site of
infection.
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C). Sendai virus infection of
B6.C-H-2bm1 mice that had been vaccinated with pJW-SNP
elicited a potent effector CTL response to the subdominant
NP324–332/Db-epitope (Fig. 3D). It should be noted in this case that the SVBM1
target cells coexpress Kbm1 and Db so that the
true level of Kbm1-restricted lysis cannot be determined in
Figure 3D. Again, the data demonstrate that memory CTLp
primed by DNA vaccination are fully functional in the context of a
subsequent live virus infection. As a control for these experiments, we
also vaccinated C57BL/6 mice with pJW4303 and pJW-SNP (Fig. 3, E and F). Vaccination with pJW-SNP in this
case will prime CTLp specific for both the dominant
NP324–332/Kb and subdominant
NP324–332/Db epitopes (Fig. 1). As expected,
prior vaccination did not alter the specificity of the effector
response in the lung, except to allow detection of a minor
NP324–332/Db-specific effector response (Fig. 3F) that is never seen upon primary virus challenge.
Thus, the normal pattern of immunodominance between
NP324–332/Kb and
NP324–332/Db is maintained. Immunization with DNA encoding NP confers protection against viral challenge
Having shown that memory CTLp primed by DNA vaccination could
participate in an effector CTL response to subsequent Sendai virus
infection, we next asked whether this vaccination protocol induced any
degree of protection against virus challenge. Cohorts of 10 C57BL/6
mice were vaccinated twice with either pJW4303 or pJW-SNP. An
additional cohort of 10 control mice was left unimmunized. Two weeks
later, all of the mice were challenged i.n. with a lethal dose of live
Sendai virus (1.32 x 105 EID50,
equivalent to 41 LD50). As shown in Figure 4, all of the mice that were either left
untreated or vaccinated with the blank pJW4303 vector died between days
7 and 9. In contrast, the pJW-SNP-vaccinated mice showed substantial
protection, with 6 of 10 mice surviving for 21 days. This is a
substantial degree of protection for a CD8+-mediated
response and is consistent with other studies on CD8+ T
cell protection against respiratory virus infection following
vaccination with either peptides or vaccinia-recombinants (9, 22, 23, 24).
These data are also consistent with studies in mice demonstrating
protection from a lethal dose of influenza virus following DNA
vaccination with an influenza NP construct (39).
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The experiments described above as well as other published reports
have demonstrated that DNA immunization with genes encoding internal
viral components can confer protective immunity (39, 40). While this
protection appears to be mediated by CD8+ T cells, the
role of humoral responses in protection has not been completely
excluded. To determine whether Ab is required for this protective
response, we took advantage of B cell-deficient (µMT,
H-2b)(42) and MHC class II- (C2-,
H-2b) (41) mice that are both profoundly deficient in
generating Ab responses. In initial experiments, we assessed whether
DNA vaccination was able to induce CD8+ T cell responses in
these mouse strains. µMT and C2- mice were vaccinated
with pJW-SNP, and splenocytes were subsequently restimulated in vitro
with the NP324–332 peptide. As shown in Figure 5, CTLp were primed against both dominant
and subdominant epitopes, although this priming appeared to be less
efficient compared with intact C57BL/6 mice in terms of lytic activity.
In the case of µMT mice, there was a specific reduction in the lytic
activity of CTL specific for the subdominant
NP324–332/Db epitope (Fig. 5A). Nonetheless, these data demonstrate that neither
class II+ cells nor functional B cell responses are
essential for the priming of CD8+ memory CTLp by DNA
vaccination. This is consistent with the observation that these cells
are not essential for the priming of CD8+ responses to live
virus infection and further demonstrates that DNA vaccination induces
responses similar to those induced by live virus (4, 53, 54).
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, vaccination with
pJW-SNP induced significant protection in both MHC class II-deficient
and B cell-deficient mice. This level of protection was similar to that
observed following pJW-SNP immunization of intact mice. Thus, these
data strongly suggest that humoral responses are not required for the
induction of protective responses by DNA vaccination with pJW-SNP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been known for some time that effector CD8+ T cell
responses to infection are restricted to a relatively small number of
epitopes compared with the total number of epitopes available (8).
Importantly, the response to the dominant epitopes suppresses, or
masks, the response to subdominant epitopes (12, 13, 14, 15, 59). Despite the
immunodominance of the effector CTL response, several recent studies
have shown that the specificity of the memory CTLp induced by infection
is substantially broader (17, 18, 19, 20, 21). For example, in the case of Sendai
virus infection, similar frequencies of memory CTLp are induced to both
dominant (NP324–332/Kb) and subdominant
(NP324–332/Db) epitopes, despite the fact that
the effector response is almost exclusively directed at
NP324–332/Kb (16). The observation that DNA
vaccination induces memory CTLp specific for subdominant epitopes at a
high frequency indicates that this mode of vaccination induces a
similar quality of memory to that which is normally induced by viral
infection. Another study has also shown that DNA vaccination can be
used to elicit CD8+ T cells specific for a subdominant T
cell epitopes (40). However, in this case the subdominant response was
studied using a DNA construct in which the competitive dominant epitope
had been specifically eliminated. This effectively rendered the
remaining epitope immunodominant in the context of this vaccine. The
current studies demonstrate that memory CTLp populations specific for
subdominant epitopes can be efficiently primed despite the presence of
a strongly immunodominant epitope. Moreover, the frequency of memory
CTLp specific for the subdominant epitope approaches that of the
dominant epitope (Table I). The capacity to induce relevant memory to
subdominant epitopes may be very important in the context of viruses
that induce persistent or latent infection in the host, such as HIV or
EBV. In these situations, the dominant effector CTL response may be
rapidly exhausted, or the virus may be able to eliminate the dominant
epitope, forcing the response to the subdominant epitope. A DNA vaccine
that induces broad memory to multiple epitopes is likely to be more
effective against persistent infection.
The induction of memory CTLp by natural virus infection is poorly understood. It is not clear whether memory cells represent a subset of activated effector cells, or whether there is a separate pathway of differentiation. It is known that the induction of CD8+ T cell responses and memory CTLp does not require help from CD4+ T cells. For example, potent CTL responses are generated in MHC class II- mice that lack CD4+ T cells and helper pathways following virus infection (4, 53, 54). However, it should be noted that some studies have suggested that the absence of CD4+ T cells results in a reduced formation of memory CTLp (60, 61). Vaccination of CD4-deficient mice with pJW-SNP resulted in potent priming of CTLp specific for the dominant NP324–332/Kb epitope, although the priming of CTLp to the subdominant NP324–332/Db epitope was less efficient. The current studies also suggest that Ab responses are not absolutely required for the induction of CTLp by DNA vaccines. This indicates that the priming of CTLp by DNA vaccination does not require Ab-mediated uptake of free Ag by FcR+ APCs using alternative processing pathways (62, 63, 64, 65, 66, 67). Thus, it seems likely that direct transfection of the APCs with the DNA vaccine is the primary mechanism for CTLp induction.
Several studies have shown that primed CD8+ T cells are able to provide a moderate degree of protection from challenge with a lethal dose of virus. For example, immunization with the Sendai NP324–332/Kb peptide provides protection from Sendai virus infection (9). In general, this protection is substantially weaker than the protection mediated by Ab, presumably because CD8+-mediated responses are only activated following the establishment of an infection. Nonetheless, the CTL response plays an important role in control of the degree of infection by reducing the viral load to be cleared by other mechanisms. Importantly, the CTL response has the advantage in that it tends to be directed against relatively conserved viral gene products allowing heterologous protection. Elegant studies by other laboratories using the influenza system have shown that DNA vaccination with genes for internal viral proteins can induce protective memory responses (39). However, while a role for NP-specific Ab was ruled out, it was not clear from these studies that the response was actually mediated by CD8+ T cells. This issue was addressed in part by demonstrating that CTL cultures generated from DNA-vaccinated mice could protect animals from subsequent viral infection. But it is possible that the extensive in vitro amplification of Ag-specific CTL was crucial for this effect. The current studies demonstrate that DNA vaccine-primed CTLp are able to traffic to the site of inflammation, in this case the lung, following Sendai virus infection. Moreover, CTLp primed against subdominant epitopes effectively out-compete unprimed T cells specific for the dominant epitope. The degree of protection induced by this type of vaccination is equivalent to that seen by other vaccination strategies, such as vaccination with vaccinia-NP (22). In addition, the data show that neither conventional CD4+ T cells nor Ab is required for this type of protection.
Collectively, the current data demonstrate that the memory CTLp pool induced by DNA vaccination is very similar to that induced by natural viral infection. Thus, the breadth of specificity, the longevity, the ability to participate in a subsequent effector response, and the requirements for T cell help are all indistinguishable from responses induced by normal viral infection. These data suggest that the underlying mechanisms of priming of memory CTLp by viral infection and DNA vaccination are likely to be the same. Importantly, DNA vaccines are likely to induce CD8+ T cell memory, which produces appropriate responses to subsequent viral infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David L. Woodland, Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105.
3 Abbreviations used in this paper: NP, nucleoprotein; BAL, bronchoalveolar lavage; CTLp, CTL precursors; CTM, complete tumor medium; EID50, 50% egg infectious dose; i.n., intranasal.
Received for publication September 23, 1997. Accepted for publication November 11, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. Liu, E. J. Usherwood, M. A. Blackman, and D. L. Woodland T-Cell Vaccination Alters the Course of Murine Herpesvirus 68 Infection and the Establishment of Viral Latency in Mice J. Virol., December 1, 1999; 73(12): 9849 - 9857. [Abstract] [Full Text]
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 Y Chen, E. Usherwood, S. Surman, T. Hogg, and D. Woodland Long-term CD8+ T cell memory to Sendai virus elicited by DNA vaccination J. Gen. Virol., June 1, 1999; 80(6): 1393 - 1399. [Abstract]
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)
and L929-Kb (•) or Sendai virus-infected MC57G (
)
cells. The controls are unpulsed L929-Db (
) and
L929-Kb (
) and uninfected MC57G (
) cells. The data
are a representative example of four independent experiments.
) and L cells
(Kk/Dk) (
), and L929 (
