Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
VIRAL PATHOGENESIS AND IMMUNITY

Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

Jeffrey B. Ulmer, Tong-Ming Fu, R. Randall Deck, Arthur Friedman, Liming Guan, Corrille DeWitt, Xu Liu, Su Wang, Margaret A. Liu, John J. Donnelly, Michael J. Caulfield
Jeffrey B. Ulmer
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tong-Ming Fu
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
R. Randall Deck
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Arthur Friedman
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Liming Guan
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Corrille DeWitt
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Xu Liu
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Su Wang
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Margaret A. Liu
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
John J. Donnelly
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael J. Caulfield
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.72.7.5648-5653.1998
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.

Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g.,Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4+ and CD8+ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4+ T-cell responses but not CD8+ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4+ T cells secreting Th1-type cytokines and CD8+ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.

MATERIALS AND METHODS

Vaccination of animals.Female BALB/c mice (4 to 6 weeks old) were purchased from Charles River Laboratories (Raleigh, N.C.). Animals were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited facility and cared for in accordance with the “Guide for the Care and Use of Laboratory Animals.” The plasmid DNA expression vector containing the NP gene cloned from the A/PR/8/34 influenza virus strain was prepared as previously described (17, 29).

Measurement of lymphoproliferation and cytokines.Single-cell suspensions of spleen cells from DNA-vaccinated animals were depleted of erythrocytes in ACK lysis buffer (Gibco) and stimulated with recombinant NP (10 μg/ml) in vitro in round-bottom microwell plates at 5 × 105 cells/ml in RPMI 1640 medium supplemented with HEPES, glutamine, 10% fetal calf serum, and 50 μM 2-mercaptoethanol. Cells were cultured for 5 days, and [3H]thymidine was added at 1 μCi/well during the last 24 h. Cells were harvested onto glass fiber filter mats by using a Tomtek cell harvester, and radioactivity was measured in a liquid scintillation counter (Betaplate; Wallac).

For analysis of cytokine secretion, culture supernatants from restimulated spleen cells (see above) were harvested on day 4. Interleukin-2 (IL-2), IL-4, IL-10, granulocyte-macrophage colony-stimulating factor, and gamma interferon (IFN-γ) levels were measured by an enzyme-linked immunosorbent assay (ELISA) according to kit instructions (Endogen and Genzyme).

Measurement of antibody responses.For measurement of anti-NP antibodies, an ELISA was used as previously described (29). To detect specific immunoglobulin isotypes, peroxidase-conjugated rabbit anti-mouse immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 (Zymed) were used as detailed elsewhere (5). For determination of geometric mean titer, samples below the limit of detection were assigned a value of 50, since the serum samples were diluted in 10-fold increments.

In vitro depletion of T-cell subsets.Spleen cells were depleted of specific T-cell subsets by using two methods. First, R&D Systems murine CD4 or CD8 Subset Column kits were used according to the instructions provided. Briefly, 2.0 × 108 cells in 1 ml of sterile 1× column buffer were gently mixed with the contents of 1 ml of monoclonal anti-CD4 or anti-CD8 cocktail and incubated at room temperature for 15 min. The cells were washed and sedimented twice with 10 ml of 1× column buffer. The columns were washed with 10 ml of 1× column buffer, and the antibody-treated cells were applied to a column, allowed to enter into the column, and then incubated at room temperature for 10 min. The cells were eluted with column buffer and then sedimented prior to resuspension in culture medium for antigen restimulation.

Second, T-subset purification columns (Biotex Laboratories, Inc., Edmonton, Alberta, Canada) were used as instructed by the manufacturer. Briefly, splenocyte suspensions from mice immunized with DNA were washed and incubated with the monoclonal antibody (MAb) cocktails. These cocktail preparations consist of MAbs directed against surface marker antigens of B cells and of the T-cell subset which is intended to be depleted. The cells were then passed through a column of glass beads coated with anti-mouse IgG which bound the cells coated with MAbs. The unbound cells, the majority of which were the desired T-cell subset, were eluted from the column and collected. These enriched subsets of CD4+ or CD8+ lymphocytes contained <5% of the depleted cell population, as estimated by fluorescence-activated cell sorting (FACS) analysis. The lymphocytes were cultured and restimulated for 7 days with syngeneic cells that had been infected with A/PR/8/34 virus or pulsed with human immunodeficiency virus (HIV) Gag synthetic peptide 193-212. IL-2 (10 U/ml; Cellular Products Inc., Buffalo, N.Y.) was added on the second day of culture. Lymphocytes were washed three times with, and resuspended at desired concentrations in, phosphate-buffered saline.

To assess the purity of cell populations, cells were stained with fluorescein isothiocyanate-labeled anti-CD4 clone RM4-4 (Pharmingen) and phycoerythrin-labeled anti-CD8b clone 53-5.8 (Pharmingen), and flow cytometry analysis was performed with a FACScan (Becton Dickinson).

In vivo depletion of T-cell subsets.The regimen described previously by Wofsy and Seaman (34) was used to deplete T-cell subsets in vivo. Two separate experiments involving groups of 10 female BALB/c mice were injected with NP DNA (200 μg) on weeks 0, 3 and 6 and then challenged with live influenza virus on week 9. Starting 3 days before viral challenge, mice were given daily injections (100 μg each) of either normal rat IgG (Sigma), rat anti-mouse CD4 MAb (clone RM4-5; Pharmingen), or rat anti-mouse-CD8a MAb (clone 53-6.7; Pharmingen). Cell depletion was monitored by staining peripheral blood lymphocytes with antibodies specific for different epitopes on CD4 and CD8. Briefly, peripheral blood was collected from the tail veins of individual mice into tubes containing 10 ml of 0.85% saline. Cells were pelleted at 1,200 rpm for 10 min and then washed twice with Tris-ammonium chloride buffer (2) to lyse erythrocytes. Cells were stained and analyzed by flow cytometry as described above.

Adoptive transfer of T cells.The adoptive transfer protocol was modified from a method described previously (9, 33). The recipient mice, 4 h after challenge infection with influenza virus A/HK/68, received 0.2-ml volumes of lymphocytes through the tail veins.

Influenza virus challenge model.Challenge with live influenza virus A/HK/68 was performed essentially as previously described (29). Briefly, virus was administered by intranasal instillation of 20 μl containing 103 50% tissue culture infective doses (TCID50) onto the nares of anesthetized mice, which in this study led to a rapid lung infection that was lethal to approximately 50% of nonimmunized mice. Individual mice were monitored daily for weight loss and survival. Data were calculated as average individual weight in a group, or as a percentage of group prechallenge weight, versus days after challenge. Statistical analyses were performed by using the t test for independent samples.

RESULTS

Induction of cellular immune responses.Previous studies have demonstrated that injection of NP DNA into mice resulted in the induction of IgG anti-NP antibodies and CD8-restricted CTL (29), the latter of which were detected up to 1 to 2 years after injection (30, 37). These data suggest that a helper T-cell response against NP was also induced, resulting in a source of cytokines that facilitated switching of the immunoglobulin isotype and priming of a memory CTL response. Indeed, spleen cells from mice that were injected with NP DNA showed robust lymphoproliferative responses upon restimulation (Fig. 1). The magnitude of these responses from NP DNA-injected mice was greater than that induced by live influenza virus infection or vaccination with formalin-inactivated virus, possibly due to potential immunostimulatory effects of DNA or longevity of NP expression after DNA vaccination (6). Lymphoproliferative responses have been detected in spleen cells from mice as soon as 2 weeks and as late as 1 year after injection with NP DNA (not shown). Certain cytokines also were secreted from these spleen cells during antigen restimulation in vitro. The profile of cytokines secreted was indicative of a Th1 type of helper T-cell response, with high levels of IFN-γ and IL-2 (Fig.2), but little or no IL-4 or IL-10 secreted into the culture supernatants of restimulated cells (not shown). In addition, granulocyte-macrophage colony-stimulating factor was detectable in the culture supernatants, but at modest levels (not shown). As might be expected from this Th1 type of response, the immunoglobulin subtype profile of anti-NP antibodies was predominated by IgG2a and IgG2b, with lesser amounts of IgG1 (Fig.3).

Fig. 1.
  • Open in new tab
  • Download powerpoint
Fig. 1.

Lymphoproliferative responses after NP DNA vaccination. Female BALB/c mice were uninjected or injected with NP DNA (50 μg), control DNA (50 μg), or inactivated influenza virus (A/PR/8/34) (flu; 15 μg) on weeks 0 and 3 or were infected awake with 1,000 TCID50 of influenza virus (A/PR/8/34) on week 0. Spleens were collected and pooled from three mice per group on week 7 and restimulated in vitro with NP. Lymphoproliferation data are presented as a stimulation index.

Fig. 2.
  • Open in new tab
  • Download powerpoint
Fig. 2.

Cytokine secretion from restimulated spleen cells. Female BALB/c mice were injected with NP DNA (50 μg) or control DNA (50 μg) on weeks 0 and 3, and spleens were collected and pooled from three mice per group on week 7. Cells from DNA-injected mice were restimulated in vitro specifically with recombinant NP protein, and cells from NP DNA-injected mice were nonspecifically activated with the mitogen concanavalin A (Con A). Cytokines secreted into the culture supernatant were detected by ELISA and are presented as picograms/milliliter of culture supernatant.

Fig. 3.
  • Open in new tab
  • Download powerpoint
Fig. 3.

Anti-NP immunoglobulin subtype profile. Female BALB/c mice were injected with NP DNA (50 μg) or NP protein (10 μg) on weeks 0 and 3, and sera were collected on week 5. Anti-NP antibody subtypes were measured by ELISA as described in Materials and Methods. Data are presented as geometric mean ELISA titers ± standard errors of the means for groups of five mice.

Analysis of T-cell subsets in vitro.To ascertain the type of T cells responsible for lymphoproliferation and cytokine secretion in vitro, T cells were depleted of either CD4+ or CD8+ T cells prior to restimulation with antigen. In three separate experiments, depletion of CD4+ T cells resulted in preparations containing 0.3 to 0.6% CD4+ and 63 to 82% CD8+ cells, while depletion of CD8+ T cells resulted in preparations containing 80 to 85% CD4+ and 0.05 to 0.3% CD8+ cells, as quantified by FACS analysis. Unseparated populations consisted of 20 to 22% CD4+ and 8 to 10% CD8+ cells. The relative proportion of cells did not change appreciably during the 5-day restimulation period. Measurement of proliferation in these separated T-cell populations indicated that under these conditions most, if not all, lymphoproliferation was due to CD4+ T cells (Fig.4A). The higher level of proliferation in the CD8-depleted population, compared to unseparated spleen cells, was likely due to the three- to fourfold enrichment in CD4+cells. Similarly, detectable cytokine (IFN-γ and IL-2) secretion upon restimulation was mediated solely by CD4+ T cells (Fig. 4B and C). However, it is possible that the NP-specific CD8+ T cells can undergo lymphoproliferation and cytokine secretion after restimulation of an unseparated spleen cell population. Regardless, the data show that, in addition to CD8+ CTL (26), Th1-type cytokine-secreting helper T cells were induced by vaccination with NP DNA.

Fig. 4.
  • Open in new tab
  • Download powerpoint
Fig. 4.

In vitro depletion of T-cell subsets. Female BALB/c mice were injected with NP DNA (200 μg) on weeks 0, 3, and 6, and spleens were collected and pooled from groups of three mice on week 23. T-cell subsets were prepared and restimulated as described in Materials and Methods. Cells from NP DNA-injected and uninjected mice were restimulated with NP protein and analyzed for proliferation plotted as a stimulation index (A) and secretion of IFN-γ (B) or IL-2 (C), as measured by ELISA and plotted as picograms/milliliter of culture supernatant.

Determination of effector cells.Elucidation of the NP-specific effector cells responsible for protection from lethal influenza virus challenge of NP DNA-vaccinated mice was accomplished in two complementary ways. First, specific T-cell subsets were depleted in vivo in mice that had been inoculated with NP DNA. Mice were given injections of anti-CD4 or anti-CD8 antibodies on 3 successive days prior to challenge with influenza virus. Based on FACS analysis of cells from blood drawn on day 0 or 7 after challenge, these mice were substantially depleted of CD4 (<4.2%) or CD8 (<0.6%) T cells. Similar treatment with isotype control antibodies did not affect the levels of CD4 or CD8 cells. To ensure that such antibody treatment did not have an effect on the influenza virus challenge model, unimmunized mice were treated with anti-CD4, anti-CD8, or control antibodies, then challenged with virus, and monitored for survival and weight loss. Neither survival nor weight loss was discernibly affected after challenge with ∼1 50% lethal dose of virus (data not shown). Similarly, tail vein bleeding of the mice on the day of challenge had no effect on survival or weight loss. This latter issue was important, since every mouse was bled on the day of virus challenge for determination of levels of circulating CD4+ and CD8+ T cells. Groups of mice were vaccinated with NP DNA or control DNA not encoding a protein and then challenged with virus. Mice that had received NP DNA and were untreated prior to challenge were completely protected from death (Fig. 5A) and showed minimal weight loss after challenge (Fig. 5B), as did NP DNA-vaccinated mice that were treated with control antibody. However, protection was abrogated in NP DNA-vaccinated mice that were depleted of CD8+ T cells prior to challenge, as measured by survival (P < 0.0001) and weight loss (P < 0.05). Depletion of CD4+ T cells also decreased the level of protection in NP DNA-vaccinated mice but not to the same degree as that seen with CD8 depletion, as reduction was significant when measured by survival (P < 0.001) but not when measured by weight loss (P > 0.05). Therefore, both CD4+ and CD8+ T cells appear to play a role in protection induced by NP DNA.

Fig. 5.
  • Open in new tab
  • Download powerpoint
Fig. 5.

In vivo depletion of T-cell subsets. Groups of 10 female BALB/c mice were injected with NP DNA (200 μg) on weeks 0, 3, and 6 and then were untreated or treated with anti-CD4, anti-CD8, or control (rat IgG) antibody on week 9. As a negative control, mice were injected three times with control DNA (200 μg). All groups were challenged under anesthesia with 1,000 TCID50 of influenza virus A/HK/68 and monitored for survival (A) and weight loss (B). The results of two experiments were similar, and the data were combined in Fig. 5to achieve an n of 20 per data point.

The ability of the mice depleted of T cell subsets to mount antibody responses was also investigated. Mice were vaccinated with hepatitis B surface antigen (HBsAg) after the 3-day antibody treatment and then monitored for the development of anti-HBsAg antibodies. As expected, mice depleted of CD4 cells were severely limited in the ability to generate anti- HBsAg antibodies, while no such impairment was seen in mice depleted of CD8 cells (data not shown). In NP DNA-immunized mice subsequently challenged with influenza virus A/HK/68, antibody responses to the challenge virus were assessed, as measured by hemagglutination-inhibiting antibodies. In mice depleted of CD4 cells, the postchallenge hemagglutination inhibition titers were lower than in undepleted mice or in mice depleted of CD8 cells (data not shown), indicating that the absence of CD4 cells impaired the development of an antibody response against the challenge virus.

The second approach to assessing the nature of the effector cells after NP DNA vaccination was adoptive transfer of T-cell subsets. Spleen cells from groups of DNA-vaccinated or influenza virus-infected mice were enriched in CD4 or CD8 cells in vitro and then inoculated into naive mice. At 4 h after transfer, mice were challenged with ∼1 50% lethal dose of virus and monitored for survival and weight loss. Recipients of unseparated spleen cells from influenza virus-infected mice were completely protected from death (Fig.6A) and showed minimal weight loss (Fig.6B). Similarly, transfer of either CD4+ or CD8+T cells from NP DNA-vaccinated mice resulted in complete protection from death (P < 0.003 compared to HIV Gag DNA-injected mice) and substantial protection from weight loss (P < 0.01 compared to HIV Gag DNA-injected mice). In contrast, mice that received cells from mice injected with HIV Gag DNA, which contained Gag-specific CD4+ and CD8+ T cells (unpublished observations), were not protected. Therefore, these adoptive transfer studies demonstrate that NP-specific CD4+ and CD8+ T cells can both independently act as effectors for protection from influenza virus challenge, thereby corroborating the results of the in vivo depletion studies. Further, vaccination of mice with NP DNA appears to prime T cells with approximately the same effectiveness as infection of mice with influenza virus.

Fig. 6.
  • Open in new tab
  • Download powerpoint
Fig. 6.

Adoptive transfer of T-cell subsets. Spleen cells from uninjected mice (solid triangles) or mice primed with influenza virus A/PR/8/34 (flu-infected; solid squares), immunized with NP DNA, or injected with HIV Gag DNA (open squares) were harvested. The NP DNA-primed spleen cells were enriched for CD4+ (open circles) or CD8+ T lymphocytes (open triangles). Spleen cells from mice immunized with HIV Gag DNA were restimulated with syngeneic cells pulsed with Gag peptide 193-212, while cells from the remaining groups were restimulated in vitro for 7 days with syngeneic cells infected with A/PR/8/34. These lymphocytes were adoptively transferred into age-matched naive mice (2.5 × 107cells/mouse for the groups denoted by open and solid squares; 107 for groups denoted by open circles and triangles) that had been intranasally challenged with A/HK/68 (H3N2) 4 h previously. Data are plotted as percent survival (A) and weight loss (B) versus days after challenge for groups of 10 mice.

DISCUSSION

Induction of immune responses against influenza virus NP in mice can be accomplished by several means, including inoculation of recombinant protein (together with adjuvant) (26), live influenza virus (8, 15), recombinant live vectors such as vaccinia virus (14) and Salmonella(25) expressing NP, myoblasts expressing NP (31), and DNA vaccines (9, 19, 29, 37). Most of these modes of vaccination can confer heterosubtypic protection against influenza virus challenge (i.e., challenge with a different subtype of virus than that from which the vaccine was prepared). Such protection has long been thought to involve, at least in part, major histocompatibility complex (MHC) class I-restricted CTL (23). However, several lines of evidence suggest that other cells may also be involved in protection. For example, recombinant NP protein plus adjuvant (26) and NP-expressing Salmonella (25) protected mice from challenge despite the apparent lack of induction of MHC class I-restricted CTL. Also, beta-2-microglobulin (B2M) −/− mice, which are deficient in the ability to induce MHC class I-restricted CTL, can be protected when vaccinated with recombinant NP-expressing vaccinia virus or live influenza virus (1, 24). Finally, adoptive transfer of MHC class II-restricted, cytokine-secreting helper T cells can confer protection in normal (11, 27) and nu/nu (20) mice. These results strongly implicate cells other than MHC class I-restricted CTL as effector cells in protection from influenza virus challenge. However, analyses of the T-cell subsets that can act as effectors in protection from influenza virus challenge in immune mice have yielded conflicting results. In vivo depletion of CD4+or CD8+ T cells was shown by Liang et al. (15) to result in partial abrogation of protection, as measured by virus shedding into the nasal cavity, while depletion of CD8+ but not CD4+ T cells led to a diminution of protection, as measured by virus titers in the lungs. Further, in the absence of B cells, CD4+ T cells are inefficient in controlling an influenza virus infection in mice (18, 28). In contrast, Epstein et al. (8) demonstrated that neither depletion of CD4+ T cells nor depletion of CD8+ T cells had any effect on protection from virus challenge, as measured by lung virus titers after a sublethal dose of virus or survival after a lethal challenge. They did, however, find that CD4+ T cells were necessary for protection in B2M −/− mice.

Previously, we demonstrated that vaccination of mice with NP DNA induced robust MHC class I-restricted CTL and heterosubtypic protection and that this protection was not due to antibody responses against NP (29). In this study we sought to investigate the spectrum of cellular immune responses induced by NP DNA and to delineate which of these responses mediate heterosubtypic protection. Here we show that, in addition to MHC class I-restricted CTL, NP DNA induces helper T-cell responses, as measured by lymphoproliferation of CD4+ T cells, with concomitant secretion of Th1-type cytokines. Furthermore, using the two separate approaches of in vivo depletion and adoptive transfer of T-cell subsets, both CD4+ and CD8+T cells were demonstrated to be capable of effector cell function in protection from influenza virus challenge. Based on the depletion studies, CD8+ T cells generated by NP DNA vaccination are necessary for protection, while CD4+ T cells may not be as critical (although they do appear to play a role, as evidenced by the partial abrogation of protection in their absence). However, based on the adoptive transfer experiments, either CD4+ or CD8+ T cells alone are sufficient to confer protection. This apparent contradiction in the necessity of CD4+ or CD8+ T cells for protection conferred by NP DNA could be a result of different levels of these cells present in NP DNA-vaccinated mice versus those levels in mice receiving a bolus inoculation of NP-specific T cells activated in vitro. For example, there may be higher levels of activated CD4+ cells in naive recipient mice than in NP DNA-vaccinated mice that could overcome the necessity for CD8+ T cells seen in vaccinated mice. This argument has been suggested by Epstein et al. (8) to account for differences in previous adoptive transfer (23) and depletion studies (8, 15). Regardless of the relative importance of CD4+ and CD8+ T cells, though, the data presented here are consistent with the hypothesis that both cell types are involved in protection conferred by NP DNA.

The precise nature of the NP-specific effector CD4+ T cells induced by NP DNA is not known. Studies using adoptive transfer of T cells from influenza virus-infected mice into naive mice have demonstrated or implicated cytokine-secreting helper T cells as having an effector function (11, 20, 27), while studies of B2M −/− mice suggest that cytolytic CD4+ T cells can also confer protection (1, 24). The CD4+ T cells induced by NP DNA in our work were clearly of the Th1-type helper T-cell phenotype, as indicated by the profile of immunoglobulin subtypes of anti-NP antibodies and the cytokines secreted from CD4+ T cells upon antigen restimulation in vitro. Several attempts to detect CD4-mediated cytolytic activity were unsuccessful (not shown), even in spleen cells of highly vaccinated mice and using A20-1.11 target cells that express high levels of MHC class II (13). Therefore, while the presence of cytolytic CD4+ T cells in NP DNA-vaccinated mice cannot be ruled out, it is likely that the CD4+ T-cell effectors induced by NP DNA mediated protection through secretion of Th1-type cytokines.

In conclusion, NP DNA vaccines are effective at inducing a broad spectrum of cellular immune responses, including MHC class I-restricted CTL and Th1-type cytokine-secreting helper T cells. Both of these types of cells appear to be important as effector cells for protection against challenge with influenza virus. Taken together with previously published reports, these results indicate that there may be overlapping levels of protection against influenza virus infection involving several types of immune mediators, including MHC class I-restricted CTL, cytokine-secreting helper T cells, and possibly other types of cells. Since the current inactivated virus vaccines are not thought to be efficient at inducing broad-based cellular immune responses, these results have implications for development of human vaccines against influenza virus infection.

FOOTNOTES

    • Received 14 November 1997.
    • Accepted 2 April 1998.
  • Copyright © 1998 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Bender B. S.,
    2. Bell W. E.,
    3. Taylor S.,
    4. Small P. A. Jr.
    Class I major histocompatibility complex-restricted cytotoxic T lymphocytes are not necessary for heterotypic immunity to influenza.J. Infect. Dis. 170 1994 1195 1200
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Boyle W.
    An extension of the 51Cr-release assay for the estimation of mouse cytotoxins.Transplantation 6 1968 761 764
    OpenUrlCrossRefPubMedWeb of Science
  3. 3.↵
    1. Cox G.,
    2. Zamb T. J.,
    3. Babiuk L. A.
    Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA.J. Virol. 67 1993 5664 5667
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Davis H. L.,
    2. Michel M. L.,
    3. Whalen R. G.
    DNA-based immunization induces continuous secretion of hepatitis-b surface antigen and high levels of circulating antibody.Hum. Mol. Genet. 2 1993 1847 1851
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Deck R. R.,
    2. DeWitt C. M.,
    3. Donnelly J. J.,
    4. Liu M. A.,
    5. Ulmer J. B.
    Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine.Vaccine 15 1997 71 78
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    1. Donnelly J. J.,
    2. Ulmer J. B.,
    3. Shiver J. W.,
    4. Liu M. A.
    DNA vaccines.Annu. Rev. Immunol. 15 1997 617 648
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    1. Doolan D. L.,
    2. Sedegah M.,
    3. Hedstrom R. C.,
    4. Hobart P.,
    5. Charoenvit Y.,
    6. Hoffman S. L.
    Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+ T cell-, interferon γ-, and nitric oxide-dependent immunity.J. Exp. Med. 183 1996 1739 1746
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Epstein S. L.,
    2. Lo C.-Y.,
    3. Misplon J. A.,
    4. Lawson C. M.,
    5. Hendrickson B. A.,
    6. Max E. E.,
    7. Subbarao K.
    Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, B2-microglobulin-deficient, and J chain-deficient mice.J. Immunol. 158 1997 1222 1230
    OpenUrlAbstract
  9. 9.↵
    1. Fu T.-M.,
    2. Friedman A.,
    3. Ulmer J. B.,
    4. Liu M. A.,
    5. Donnelly J. J.
    Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA vaccination.J. Virol. 71 1997 2715 2721
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Fynan E. F.,
    2. Webster R. G.,
    3. Fuller D. H.,
    4. Haynes J. R.,
    5. Santoro J. C.,
    6. Robinson H. L.
    DNA vaccines—protective immunizations by parenteral, mucosal, and gene-gun inoculations.Proc. Natl. Acad. Sci. USA 90 1993 11478 11482
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Graham M. B.,
    2. Braciale V. L.,
    3. Braciale T. J.
    Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection.J. Exp. Med. 180 1994 1273 1282
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Huygen K.,
    2. Content J.,
    3. Denis O.,
    4. Montgomery D. L.,
    5. Yawman A. M.,
    6. Deck R. R.,
    7. DeWitt C. M.,
    8. Orme I. M.,
    9. Baldwin S.,
    10. D’Souza C. S.,
    11. Drowart A.,
    12. Lozes E.,
    13. Vandenbussche P.,
    14. Mooren J.-P.,
    15. Liu M. A.,
    16. Ulmer J. B.
    Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.Nat. Med. 2 1996 893 898
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. Kim K. J.,
    2. Kanellopoulos-Langevin C.,
    3. Merwin R. M.,
    4. Sachs D. H.,
    5. Asofsky R.
    Establishment and characterization of BALB/c lymphoma lines with B cell properties.J. Immunol. 122 1979 549
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Lawson C. M.,
    2. Bennink J. R.,
    3. Restifo N. P.,
    4. Yewdell J. W.,
    5. Murphy B. R.
    Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.J. Virol. 68 1994 3505 3511
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    1. Liang S.,
    2. Mozdzanowska K.,
    3. Palladino G.,
    4. Gerhard W.
    Heterosubtypic immunity to influenza type A virus in mice: effector mechanisms and their longevity.J. Immunol. 152 1994 1653 1661
    OpenUrlAbstract
  16. 16.↵
    1. Manickan E.,
    2. Rouse R.,
    3. Yu Z. Y.,
    4. Wire W. S.,
    5. Rouse B. T.
    Genetic immunization against herpes-simplex-virus protection is mediated by CD4+ T-lymphocytes.J. Immunol. 155 1995 259 265
    OpenUrlAbstract
  17. 17.↵
    1. Montgomery D. L.,
    2. Shiver J. W.,
    3. Leander K. R.,
    4. Perry H. C.,
    5. Friedman A.,
    6. Martinez D.,
    7. Ulmer J. B.,
    8. Donnelly J. J.,
    9. Liu M. A.
    Heterologous and homologous protection against influenza-A by DNA vaccination—optimization of DNA vectors.DNA Cell Biol. 12 1993 777 783
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    1. Mozdzanowska K.,
    2. Furchner M.,
    3. Maiese K.,
    4. Gerhard W.
    CD4+ T cells are ineffective in clearing a pulmonary infection with influenza type A virus in the absence of B cells.Virology 239 1997 217 225
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Pertmer T. M.,
    2. Eisenbraun M. D.,
    3. McCabe D.,
    4. Prayaga S. K.,
    5. Fuller D. F.,
    6. Haynes J. R.
    Gene gun-based nucleic-acid immunization—elicitation of humoral and cytotoxic T-lymphocyte responses following epidermal delivery of nanogram quantities of DNA.Vaccine 13 1995 1427 1430
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Scherle P. A.,
    2. Palladino G.,
    3. Gerhard W.
    Mice can recover from pulmonary influenza virus infection in the absence of class I-restricted cytotoxic T lymphocytes.J. Immunol. 148 1992 212 217
    OpenUrlAbstract
  21. 21.↵
    1. Sedegah M.,
    2. Hedstrom R.,
    3. Hobart P.,
    4. Hoffman S. L.
    Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein.Proc. Natl. Acad. Sci. USA 91 1994 9866 9870
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Tang D. C.,
    2. Devit M.,
    3. Johnston S. A.
    Genetic immunization is a simple method for eliciting an immune response.Nature 356 1992 152 154
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    1. Taylor P. M.,
    2. Askonas B. A.
    Influenza nucleoprotein-specific cytotoxic T cell clones are protective in vivo.Immunology 58 1986 417 420
    OpenUrlPubMedWeb of Science
  24. 24.↵
    1. Taylor S. F.,
    2. Bender B. S.
    B2-microglobulin-deficient mice demonstrate class II MHC restricted anti-viral CD4+ but not CD8+ CTL against influenza-sensitized autologous splenocytes.Immunol. Lett. 46 1995 67 73
    OpenUrlCrossRefPubMedWeb of Science
  25. 25.↵
    1. Tite J. P.,
    2. Gao X.-M.,
    3. Hughes-Jenkins C. M.,
    4. Lipscombe M.,
    5. O’Callaghan D.,
    6. Dougan G.
    Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier.Immunology 70 1990 540 546
    OpenUrlPubMedWeb of Science
  26. 26.↵
    1. Tite J. P.,
    2. Hughes-Jenkins C.,
    3. O’Callaghan D.,
    4. Dougan G.,
    5. Russel S. M.,
    6. Gao X.-M.,
    7. Liew F. Y.
    Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. II. Protection from influenza infection and mechanism of protection.Immunology 71 1990 202 207
    OpenUrlPubMedWeb of Science
  27. 27.↵
    1. Topham D. J.,
    2. Tripp R. A.,
    3. Sarawar S. R.,
    4. Sangster M. Y.,
    5. Doherty P. C.
    Immune CD4+ T cells promote the clearance of influenza virus from major histocompatibility complex class II −/− respiratory epithelium.J. Virol. 70 1996 1288 1291
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Topham D. J.,
    2. Doherty P. C.
    Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells.J. Virol. 72 1998 882 885
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Ulmer J. B.,
    2. Donnelly J. J.,
    3. Parker S. E.,
    4. Rhodes G. H.,
    5. Felgner P. L.,
    6. Dwarki V. J.,
    7. Gromkowski S. H.,
    8. Deck R. R.,
    9. Dewitt C. M.,
    10. Friedman A.,
    11. Hawe L. A.,
    12. Leander K. R.,
    13. Martinez D.,
    14. Perry H. C.,
    15. Shiver J. W.,
    16. Montgomery D. L.,
    17. Liu M. A.
    Heterologous protection against influenza by injection of DNA encoding a viral protein.Science 259 1993 1745 1749
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Ulmer J. B.,
    2. Deck R. R.,
    3. Yawman A. M.,
    4. Friedman A.,
    5. DeWitt C. M.,
    6. Martinez D.,
    7. Donnelly J. J.,
    8. Liu M. A.
    DNA vaccines for bacteria and viruses.Adv. Exp. Med. Biol. 397 1996 49 53
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Ulmer J. B.,
    2. Deck R. R.,
    3. DeWitt C. M.,
    4. Donnelly J. J.,
    5. Liu M. A.
    Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells.Immunology 89 1996 59 67
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    1. Wang B.,
    2. Boyer J.,
    3. Srikantan V.,
    4. Coney L.,
    5. Carrano R.,
    6. Phan C.,
    7. Merva M.,
    8. Dang K.,
    9. Agadjanyan M.,
    10. Gilbert L.,
    11. Ugen K. E.,
    12. Williams W. V.,
    13. Weiner D. B.
    DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and nonhuman primates.DNA Cell Biol. 12 1993 799 805
    OpenUrlCrossRefPubMedWeb of Science
  33. 33.↵
    1. Wells M. A.,
    2. Ennis F. A.,
    3. Albrecht P.
    Recovery from a viral respiratory infection. II. Passive transfer of immune spleen cells to mice with influenza pneumonia.J. Immunol. 126 1981 1042 1046
    OpenUrlPubMed
  34. 34.↵
    1. Wofsy D.,
    2. Seaman W. E.
    Successful treatment of autoimmunity in NZB/NZW F1 mice with monoclonal antibody to L3T4.J. Exp. Med. 161 1985 378 391
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Wolff J. A.,
    2. Malone R. W.,
    3. Williams P.,
    4. Chong W.,
    5. Acsadi G.,
    6. Jani A.,
    7. Felgner P. L.
    Direct gene transfer into mouse muscle in vivo.Science 247 1990 1465 1468
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Xiang Z. Q.,
    2. Spitalnik S.,
    3. Cheng J.,
    4. Erikson J.,
    5. Wojczyk B.,
    6. Ertl H.
    Immune responses to nucleic acid vaccines to rabies virus.Virology 209 1995 569 579
    OpenUrlCrossRefPubMedWeb of Science
  37. 37.↵
    1. Yankauckas M. A.,
    2. Morrow J. E.,
    3. Parker S. E.,
    4. Abai A.,
    5. Rhodes G. H.,
    6. Dwarki V. J.,
    7. Gromkowski S. H.
    Long-term antinucleoprotein cellular and humoral immunity is induced by intramuscular injection of plasmid DNA containing NP gene.DNA Cell Biol. 12 1993 771 776
    OpenUrlCrossRefPubMedWeb of Science
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA
Jeffrey B. Ulmer, Tong-Ming Fu, R. Randall Deck, Arthur Friedman, Liming Guan, Corrille DeWitt, Xu Liu, Su Wang, Margaret A. Liu, John J. Donnelly, Michael J. Caulfield
Journal of Virology Jul 1998, 72 (7) 5648-5653; DOI: 10.1128/JVI.72.7.5648-5653.1998

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA
Jeffrey B. Ulmer, Tong-Ming Fu, R. Randall Deck, Arthur Friedman, Liming Guan, Corrille DeWitt, Xu Liu, Su Wang, Margaret A. Liu, John J. Donnelly, Michael J. Caulfield
Journal of Virology Jul 1998, 72 (7) 5648-5653; DOI: 10.1128/JVI.72.7.5648-5653.1998
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

CD4-Positive T-Lymphocytes
CD8-Positive T-Lymphocytes
influenza vaccines
Nucleoproteins
Vaccines, DNA
Viral Core Proteins

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514