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DNA immunization: mechanistic studies
Vaccine 17 (1999) 1612±1619
DNA immunization: mechanistic studies J. Lindsay Whitton *, Fernando Rodriguez, Jie Zhang, Daniel E. Hassett Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
Abstract DNA immunization works, as has been amply demonstrated in a variety of microbial and tumor models. However, the mechanisms which underpin its success remain unclear. Using intramuscular delivery of DNA, we wish to precisely de®ne how DNA-encoded antigens induce CD8+ T-cells (most cytotoxic T-cells; CTL), CD4+ T-cells (mostly helper cells) and antibodies; and to use the accrued knowledge to rationally manipulate DNA vaccines, thus enabling us to optimize each of the above three types of immune response. We consider it likely that dierent mechanisms operate in each case. We have designed a DNA vaccine which induces CTL, but not antibodies. We will present evidence that CTL are induced by endogenously-synthesized protein, not by protein released from cells; and that in the absence of release of intact protein, antibodies are not induced, while CTL induction remains strong. We have used plasmid-encoded minigenes and have found that these short sequences also induce CTL; this, too, argues that CTL are induced by antigens presented following endogenous synthesis. We are attempting to determine how antigens are released from transfected cells, to interact with B-cells and induce antibodies, and are currently evaluating the CD4 responses induced by DNA vaccines. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction An understanding of the immunological response of the host to infection is essential for rational vaccine design. Antigens derived from intracellularly replicating pathogens (i.e. viruses and some bacteria) are processed within the cell, the full-length proteins being reduced to peptides by an intracellular complex named the proteasome; these peptides are moved into the endoplasmic reticulum (ER) by the `TAP' transporters; in the ER the peptides associate with major histocompatibility complex (MHC) class I molecules and a third protein, b2-microglobulin; and this tri-molecular complex is transported to the cell surface for perusal by circulating T-cells. Upon encountering the complex of b2-microglobulin±MHC class I and peptide, antigen-speci®c CD8+ T-cells are activated, acquiring cytotoxic functions. These activated cytotoxic T-lymphocytes (CTL) can lyse infected host cells, thereby limiting the reproduction of the pathogen and controlling the spread of the infection. Extracellular antigen (e.g. virions, bacteria and microbial proteins released * Corresponding author. Tel.: +1-619-784-7090; fax: +1-619-7847380; e-mail: [email protected]. 0264-410X/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 8 ) 0 0 4 1 8 - 6
into the extracellular milieu) is taken up into lysosomes of specialized antigen presenting cells (APC), where it is proteolysed to peptides, which encounter and bind to MHC class II molecules before travelling to the surface of the APCs. In addition, recent studies indicate that some APC can introduce endogenous antigen directly into the MHC class II pathway, (without the protein ®rst having to be released in soluble form); although this may apply only to proteins with natural access to the endoplasmic reticulum (for example, cell membrane proteins) [1±5]. The MHC class II±peptide complex is recognized by CD4+ Thelper cells (Th) which secrete soluble factors (cytokines) that regulate the eector mechanisms of other immune cells. CD4+ cells provide help to B-cells, stimulating their growth and `class-switching' (changing the class of antibody from IgM to, for example, IgG). The role of CD4+ helper in generation of CD8+ CTL is more controversial; however we have recently shown that mice lacking CD4+ T-cells retain largely intact primary CTL responses, but CTL memory is dramatically reduced and vaccination is much less eective [6]. NaõÈ ve B-cells encounter antigen in solution, or displayed on cell surfaces. Upon interacting with the appropriate antigen, clonal expansion occurs and (usually) class switching. The great majority of anti-
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body responses require CD4+ T-cell help for their full development. The ideal vaccine might be characterized as safe, cheap, heat stable, containing protective immunogenic sequences from multiple pathogens and preferably administered as a single oral dose. Although to date no vaccine for human use meets all of these criteria, current vaccines have been remarkably successful in reducing the morbidity and mortality associated with many common infectious diseases and, in the case of smallpox virus, have succeeded in globally eradicating a signi®cant human pathogen [7]. Current vaccines may be divided into two categories, `live' and `dead'. Live vaccines comprise traditional attenuated microbes, viral or bacterial, selected for reduced pathogenicity with maintained immunogenicity; and `recombinant' vaccines, in which foreign antigens are expressed from a replicating viral or bacterial vector. Although a few live recombinant vector vaccines are used in veterinary medicine, none has yet been approved for human use. `Dead' vaccines consist of killed whole pathogens, or soluble pathogen proteins or protein subunits. The nature of the vaccine Ð live or dead Ð is pivotal in determining the type of immune response induced. Both types of vaccine can induce antibodies, but dead vaccines do not eciently enter the MHC class I pathway, and thus are inecient in inducing CTL. Live vaccines may be dangerous to pregnant women or immunocompromized hosts, can revert to pathogenicity within the vaccinee and also may be contaminated by potentially harmful adventitious agents during production. From an economic standpoint, one major drawback of these vaccines is their requirement for refrigeration, increasing the cost, limiting shelf life, and greatly complicating distribution in countries lacking a `cold chain'. Although safer, vaccines composed of inactivated pathogens or immunogenic protein subunits may be less eective in inducing long-term cross-reactive immunity, and the cell-mediated immune responses critical to protection against many diseases caused by intracellularly-replicating organisms. DNA immunization, the inoculation of plasmid DNA encoding microbial proteins, is a recent addition to the vaccine arsenal. It has many apparent advantages, which are reviewed elsewhere [8], and are summarized below. Direct inoculation of plasmid DNA containing open reading frames with appropriate eukaryotic transcription and translation control signals should result in the in vivo synthesis of a protein with conformation and posttranslational modi®cation patterns identical, in most cases, to those which occur during normal infection. Endogenous protein synthesis should mimic viral infection in allowing presentation of the foreign antigen by MHC class I, and uptake of soluble protein by specialized antigen presenting cells
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(APC) allows presentation by MHC class II; both arms of the immune response may therefore be induced. We have shown that DNA vaccines are useful in immunizing infants carrying maternally-derived antibodies, which reduce the eectiveness of live vaccines by neutralizing the microbe and preventing its replication; these antibodies cannot recognize the nucleic acid vector, and therefore synthesis of the encoded protein and induction of an immune response proceeds normally [9]. DNA vaccines, lacking a replicating agent, should be safer than live vaccines for administration to pregnant or immunocompromized individuals. Finally, DNA is relatively cheap to produce, is heat stable and is amenable to genetic manipulation, making it possible to apply our approach of vaccinating with multiple immunologically reactive sequences [10] derived from multiple pathogens. Both humoral and cell-mediated immune responses have been detected following immunization with plasmid DNAs encoding a diverse array of foreign antigens, too numerous to be discussed in detail here. Excellent reviews are available [11, 12] and a collection of papers devoted to this topic has recently been published [12]. DNA immunization `works'; it induces both humoral and cellular immunity (including both CD4+ and CD8+ T-cells), which often are protective against microbial challenge. This has been shown in many model systems and we will not review them here. A frequently-updated list of the animal models, pathogens and antigens studied is available at a World Wide Web site, provided by Dr. R.G. Whalen at address http://www.genweb.com/dnavax/dnavax.html. This paper focuses on understanding the mechanisms which underpin DNA-induced immunity; these mechanisms remain unclear, and are the subject of considerable interest. Our studies exploit the model system of lymphocytic choriomeningitis virus (LCMV) infection of the mouse. LCMV is the prototype of the arenavirus family, and has a bisegmented single stranded ambisense RNA genome [13]. The short genomic segment encodes two proteins, the nucleoprotein (NP) and glycoprotein (GP); the latter undergoes posttranslation modi®cations and cleavage into its mature virus structural proteins GP1 and GP2 [14]. CTL responses are pivotal in the biology of LCMV and are vital to the control of primary infection [15] and to vaccineinduced immunity [16±18]. Our DNA immunization experiments take advantage of our detailed knowledge of LCMV and, together with the large number of reagents already available to us, permit us to investigate the mechanisms which underlie the induction of immune responses by plasmid DNA inoculation.
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2. DNA-mediated induction of CD8+ CTL It has been known for some time that live viral vaccines induce good CD8+ T-cell responses (which for the most part are CTL), because the vaccine organism replicates and synthesizes its proteins intracellularly, and thus its proteins can be eciently introduced into the class I MHC antigen presentation pathway. There is no doubt that the encoded proteins are synthesized within the transfected muscle cells, as has been shown for CAT, b-galactosidase and green ¯uorescent protein (GFP), and this capacity for endogenous protein synthesis led to the prediction that DNA immunization would be rather eective in inducing CTL responses, as has proven to be the case [19±26]. However the mechanism underlying CTL induction remains controversial. The overwhelming expression of encoded antigen by muscle cells provoked suggestions that the muscle cells themselves present antigen and directly induce T-cell responses. How likely is this? T-cells can recognize muscle in certain myopathies [27, 28], and we [29, 30] and others [31] have shown that muscle cells can be recognized by T-cells following DNA immunization, but this does not imply that myocytes can induce T-cell responses. Most workers believe that CTL induction requires at least two signals on the surface of the inducing cell; ®rst, the appropriate class I MHC±peptide complex, which interacts with the T-cell receptor and, second, a co-stimulatory molecule such as B7.1, which interacts with CD28 on the T-cell membrane. Myocytes express only low levels of MHC class I (although this is upregulated in the presence of interferon), and levels of accessory molecules appear to be extremely low; thus the ability of myocytes to present antigen in a manner capable of inducing CTL must be questioned. In addition, recent studies have shown that APCs play a critical role in DNA immunization [32±34]. Chimeric mice expressing two MHC haplotypes on their muscle cells, but only one on their bone marrow derived APCs, mounted CTL responses restricted to the bone marrow haplotype. Thus, it appears unlikely that muscle cells play a direct role in CTL induction. So, are CTL induced by APCs which have taken up soluble protein, perhaps released from muscle cell depots, or are they induced by APCs which have been transfected with DNA? Here the picture becomes unclear. A recent study showed that stably-transfected muscle cells, when transplanted, can induce immune responses (antibody and CTL) [33]. The authors concluded that antigen is released from muscle cells and taken up by APCs, thereby inducing immunity; and others have shown that exogenous proteins can be taken up by specialized APCs (such as macrophages) which can perform the unusual contortion of presenting exogenous antigen via the MHC class I pathway [35±37]. However Robinson's lab has
shown that removal of injected muscle within 10 min of DNA inoculation has no eect on the magnitude of the CTL or antibody responses induced, and the authors concluded that transfected cells in injected muscle do not play a vital role in DNA-initiated antibody and CTL responses [38]. To determine whether APCs take up soluble intact protein or injected DNA, we sought to diminish or prevent release of intact protein following DNA immunization; it is reasonable to suggest that if release of intact protein is required for CTL induction, then preventing this should inhibit CTL responses. We have developed a system to ensure very rapid degradation of an intracellular protein, by stable covalent attachment to ubiquitin, a 76 amino acid cellular protein which `tags' intracellular proteins for proteasomal destruction (reviewed [39]). Space does not permit a thorough review of ubiquitination, nor a detailed account of our strategy to improve delivery to the proteasome, described elsewhere [40]. We covalently linked LCMV NP to a modi®ed ubiquitin, designed to act as a good target for transport to the proteasome. 2.1. Rapid degradation of ubiquitinated NP To determine the intracellular turnover of the ubiquitinated protein, BHK cells were transfected in quadruplicate either with pCMV±NP, or with pCMV± U-NP, and were labeled for 30 min with S35-methionine and S35-cysteine. As a positive control, one plate of cells was infected with LCMV and radio-labeled as described; as a negative control, untreated cells were radio-labeled. After 30 min, the LCMV-infected cells, one of each set of transfected cells and the untreated cells were harvested (tracks labeled L, 0, 0 and U, respectively in Fig. 1a). In the remaining plates the incorporated label was `chased' by incubation of the transfected cells with excess unlabeled amino acids for 30, 60 or 90 min, at which time points cells were harvested. Proteins were immunoprecipitated for 2 h using anti-LCMV±NP antibody, and the products of immunoprecipitation were separated on a polyacrylamide gel and visualized by autoradiography. As shown in Fig. 1a, full length LCMV±NP was readily detected in cells transfected with plasmid pCMV±NP. NP appears relatively stable in these cells since, even after 90 min chase, a band was still easily visible. In contrast, cells transfected with the ubiquitin±NP construct showed very low levels of detectable protein. To con®rm the rapid degradation of the ubiquitinated NP, we used the proteasome inhibitor ALLN [41]; samples with ALLN are indicated with a ` + ' in Fig. 1b. The quantity of NP in cells transfected with pCMV±NP is barely altered (perhaps slightly increased) in the presence of the inhibitor, consistent with NP being stable in the cell. In contrast, there is a marked eect in cells
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LCMV-speci®c responses, but levels were lower. Thus ubiquitination enhances degradation, and improves both recognition by, and induction of, CTL. The CTL responses induced appear more likely to result from uptake by APCs of pCMV±U-NP DNA than uptake of soluble protein, since we show that the ubiquitinated protein is very rapidly degraded within the synthesizing cell, therefore limiting the quantity of intact protein available for release. Thus we favor the hypothesis that CTL induction requires uptake by APCs of plasmid DNA, and that uptake of soluble protein plays a secondary, and expendable, role. 3. DNA-mediated induction of antibodies and CD4+ Tcells
Fig. 1. Ubiquitination of NP enhances intracellular degradation. Cells were infected with LCMV (L), or were transfected with plasmids encoding ubiquitin (U), LCMV NP (NP) or ubiquitinated LCMV NP (U±NP) and were labeled with radioactive amino acids. (a) Cells were `chased' with cold amino acids for 0, 30, 60 or 90 min, at which times the proteins were harvested and precipitated with an antibody speci®c for NP, prior to analysis by SDS-PAGE. (b) Labeling was carried out in the absence (ÿ) or presence (+) of the proteasome inhibitor ALLN.
transfected with pCMV±U-NP; very little material is detected in the absence of inhibitor, but in the presence of ALLN there are readily-detectable levels of protein. Thus the pCMV±U-NP plasmid directs abundant protein synthesis, and the protein is very rapidly degraded by the proteasome, being detectable only in the presence of the proteasome inhibitor. These ®ndings are not cell-speci®c, as similar results were obtained in BALB/c ®broblasts (not shown). Furthermore, we found that the enhanced degradation resulted in improved sensitization of target cells to CTL lysis (not shown). 2.2. Ubiquitinated NP induces CTL in vivo In addition to these tissue culture ®ndings, we have evaluated the ability of pCMV±U-NP to induce CTL in vivo. Similar results were found in C57BL/6 and BALB mice; the latter are shown in Fig. 2. All mice receiving the ubiquitin±NP plasmid were primed for high levels of anti-LCMV CTL. Mice receiving pCMV±NP (nonubiquitinated protein) also mounted
Induction of antibodies usually involves two steps. First, the protein must be accessible to membranebound antibody on naõÈ ve B-lymphocytes, permitting recognition by the appropriate Fab, thus beginning the process of clonal expansion. Second, immunoglobulin class switching (e.g. from IgM to IgG) requires `help' from CD4+ T-cells; and these cells recognize antigen presented by the MHC class II pathway. During a normal virus infection it is easy to see how both of these steps are taken. Virus proteins are displayed on the virion or cell surface, secreted into the extracellular milieu, or released when the cell dies; in these ways they become available to antibodies present on the cell membranes of naõÈ ve B-lymphocytes. Furthermore, the soluble proteins may be engulfed by APCs (including B-lymphocytes themselves [42]), and presented by class II MHC, permitting the provision of cognate help. However, DNA immunization is very dierent from virus infection. First, most proteins are not cytopathic, and so should not directly cause lysis of transfected cells. Many of the encoded proteins (for example, LCMV NP) are neither displayed on the cell surface, nor secreted. Nevertheless many groups Ð including ours Ð have shown that DNA immunization induces antibodies even against such proteins. How, then, are such cytoplasmic, noncytopathic proteins recognized by B-cells? 3.1. Induction of antibody responses is diminished when protein undergoes rapid intracellular degradation To determine plasmid-mediated antibody induction requires release of intact protein, the pCMV±U-NP immunized mice described above were bled six weeks postimmunization, and anti-LCMV antibody titers were evaluated by ELISA using whole LCMV as target antigen. In both BALB/c (Fig. 3) and C57BL/6 mouse strains, the plasmid encoding ubiquitinated NP failed to induce anti-LCMV antibodies; the antibody
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Fig. 2. Ubiquitinated NP induces anti-LCMV CTL. BALB/c mice were immunized with one of the three plasmids shown and six weeks later were challenged with LCMV. Four days later Ð at which time no CTL activity has yet developed in previously-naõÈ ve mice Ð spleens were harvested and an in vitro cytotoxicity assay was carried out. Each bar represents an individual mouse, at eector:target ratios of 50:1 and 25:1. Mice immunized with LCMV rather than with DNA, and rechallenged with LCMV 6 weeks later were included as a positive control for successful in vivo secondary stimulation (LCMV); and mice infected with LCMV 7 days previously were used as a positive control for target cell lysis by CTL (LCMV d7).
titers of mice immunized with this plasmid were indistinguishable from those of mice immunized with a plasmid encoding ubiquitin alone [40]. The failure of the ubiquitin±NP construct to induce antibodies does not re¯ect an absence of gene expression, since other immune responses (CTL induction) were demonstrable in the same mice, as reported above. It therefore appears likely that this phenomenon is the in vivo re¯ection of what we found in tissue culture; the degradation of the ubiquitinated NP is so rapid and complete that intact protein cannot escape the cell in intact form, and so the interaction of intact protein and B-cells cannot take place. Thus, CTL are induced by ubiquitinated protein, but not antibodies. These conclusions are bolstered by another lab's evaluation of ubiquitination in DNA immunization [43]. Our ®nd-
Fig. 3. Ubiquitination of NP abrogates anti-NP antibody induction in BALB/c mice. The DNA-immunized mice described in the legend to Fig. 2 were bled immediately prior to LCMV challenge and their antibody levels were determined by ELISA.
ings suggest that the two arms of the immune response may be induced by dierent mechanisms following DNA immunization. Perhaps release and uptake of intact protein is required for the induction of antibodies, but not for the induction of CTL. 3.2. From what cells might antigen be released? If soluble protein is required for antibody induction, in which cells is the protein synthesized prior to release? As discussed earlier, the ability of myocytes to induce CTL is questionable. However it appears very likely that myocytes can be recognized by CTL. CD8+ cells have been noted in the in®ltrates of several myopathic diseases [28]. So, do myocytes acts as antigen depots, releasing antigen upon CTL lysis? If so, one might expect local in¯ammation. We evaluated the issue in naõÈ ve and in immune mice and found that inoculation of pCMV±NP into a nonimmune mouse induced at most a mild in¯ammatory response. In contrast, the same DNA inoculated into an immune mouse induced a ¯orid myositis; the response is antigen speci®c, since inoculation of pCMV into an immune mouse engenders no such reaction. The in¯ammatory response occurred both in acutely-infected mice, and in mice carrying memory CTL (Fig. 4). The extent and localization of the response, and the destruction of muscle ®bers, suggest that recognition of skeletal muscle by T-cells may be occurring; this has recently been con®rmed [31]. However the role of muscle cells as an important antigen depot is questionable, since excision of the injected muscle bundle (which can be cleanly accomplished when the tibialis
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Fig. 4. Antibody responses are induced in perforin knockout mice. C57BL/6 and PKO mice were immunized with pCMV±NP and six weeks later their anti-LCMV antibody titers were determined by ELISA. (a) Individual mice. (b) Group means. Controls include mice immunized with LCMV and nonimmune mice.
anterior muscle is employed) as soon as 10 min postinjection has little eect on the induction of antibody or CTL responses [38]. It appears unlikely that, in this short time, muscle cells could take up the DNA, express the protein and release sucient material to prime both antibody and CTL responses. Perhaps resident muscle immune cells (macrophages, dendritic cells) take up the DNA and depart the tissue; or perhaps the DNA itself exits the muscle, to transfect other cells. Thus, while i.m. DNA immunization certainly can lead to immune-mediated myocyte destruction, it is unclear whether muscle cells contribute to immunity. While they may act as depots, so too may other cell types which take up the DNA.
(Fig. 5). Thus, we conclude that perforin-mediated lysis is not required for release of antigen expressed following injection of plasmid DNA. Of course it remains possible that myocytes or other cells do act as antigen depots, releasing antigen by an as-yet-unidenti®ed mechanism.
Acknowledgements The authors are grateful to Annette Lord for skilled administrative assistance. These studies were supported
3.3. Perforin-mediated lysis is not required for the induction of antibodies by i.m. DNA immunization Since antigen release is required for antibody induction, an obvious underlying mechanism would be antigen-speci®c CTL-mediated lysis of transfected antigen depots. Therefore we have attempted to assess the role of CTL lysis using mice de®cient in the major CTL cytolytic protein, perforin [44±47]. As shown in Fig. 4, these perforin knockout (PKO) mice mounted readilydetectable antibody responses. Individual (Fig. 4a) and average (Fig. 4b) antibody levels are shown for PKO and C57BL/6 mice. The results show conclusively that perforin-mediated lysis of muscle cells (or indeed of any cells) is not required for induction of antibody responses following DNA immunization. In fact, the results suggest that antibody responses may even be elevated in PKO mice. Our ®ndings are not limited to LCMV NP; responses to a plasmid encoding LCMV glycoprotein are similarly enhanced in PKO mice
Fig. 5. Enhanced response to GP in PKO mice. C57BL/6 and PKO mice were immunized with pCMV-GP, and six weeks later their antiLCMV antibody titers were determined by ELISA. Each set of points represents an individual mouse.
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by NIH grants AI-37186-04 and T32-AI-07354-08. This is manuscript number 11595-NP from the Scripps Research Institute.
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DNA immunization: mechanistic studies J. Lindsay Whitton *, Fernando Rodriguez, Jie Zhang, Daniel E. Hassett Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA
Abstract DNA immunization works, as has been amply demonstrated in a variety of microbial and tumor models. However, the mechanisms which underpin its success remain unclear. Using intramuscular delivery of DNA, we wish to precisely de®ne how DNA-encoded antigens induce CD8+ T-cells (most cytotoxic T-cells; CTL), CD4+ T-cells (mostly helper cells) and antibodies; and to use the accrued knowledge to rationally manipulate DNA vaccines, thus enabling us to optimize each of the above three types of immune response. We consider it likely that dierent mechanisms operate in each case. We have designed a DNA vaccine which induces CTL, but not antibodies. We will present evidence that CTL are induced by endogenously-synthesized protein, not by protein released from cells; and that in the absence of release of intact protein, antibodies are not induced, while CTL induction remains strong. We have used plasmid-encoded minigenes and have found that these short sequences also induce CTL; this, too, argues that CTL are induced by antigens presented following endogenous synthesis. We are attempting to determine how antigens are released from transfected cells, to interact with B-cells and induce antibodies, and are currently evaluating the CD4 responses induced by DNA vaccines. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction An understanding of the immunological response of the host to infection is essential for rational vaccine design. Antigens derived from intracellularly replicating pathogens (i.e. viruses and some bacteria) are processed within the cell, the full-length proteins being reduced to peptides by an intracellular complex named the proteasome; these peptides are moved into the endoplasmic reticulum (ER) by the `TAP' transporters; in the ER the peptides associate with major histocompatibility complex (MHC) class I molecules and a third protein, b2-microglobulin; and this tri-molecular complex is transported to the cell surface for perusal by circulating T-cells. Upon encountering the complex of b2-microglobulin±MHC class I and peptide, antigen-speci®c CD8+ T-cells are activated, acquiring cytotoxic functions. These activated cytotoxic T-lymphocytes (CTL) can lyse infected host cells, thereby limiting the reproduction of the pathogen and controlling the spread of the infection. Extracellular antigen (e.g. virions, bacteria and microbial proteins released * Corresponding author. Tel.: +1-619-784-7090; fax: +1-619-7847380; e-mail: [email protected]. 0264-410X/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 8 ) 0 0 4 1 8 - 6
into the extracellular milieu) is taken up into lysosomes of specialized antigen presenting cells (APC), where it is proteolysed to peptides, which encounter and bind to MHC class II molecules before travelling to the surface of the APCs. In addition, recent studies indicate that some APC can introduce endogenous antigen directly into the MHC class II pathway, (without the protein ®rst having to be released in soluble form); although this may apply only to proteins with natural access to the endoplasmic reticulum (for example, cell membrane proteins) [1±5]. The MHC class II±peptide complex is recognized by CD4+ Thelper cells (Th) which secrete soluble factors (cytokines) that regulate the eector mechanisms of other immune cells. CD4+ cells provide help to B-cells, stimulating their growth and `class-switching' (changing the class of antibody from IgM to, for example, IgG). The role of CD4+ helper in generation of CD8+ CTL is more controversial; however we have recently shown that mice lacking CD4+ T-cells retain largely intact primary CTL responses, but CTL memory is dramatically reduced and vaccination is much less eective [6]. NaõÈ ve B-cells encounter antigen in solution, or displayed on cell surfaces. Upon interacting with the appropriate antigen, clonal expansion occurs and (usually) class switching. The great majority of anti-
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body responses require CD4+ T-cell help for their full development. The ideal vaccine might be characterized as safe, cheap, heat stable, containing protective immunogenic sequences from multiple pathogens and preferably administered as a single oral dose. Although to date no vaccine for human use meets all of these criteria, current vaccines have been remarkably successful in reducing the morbidity and mortality associated with many common infectious diseases and, in the case of smallpox virus, have succeeded in globally eradicating a signi®cant human pathogen [7]. Current vaccines may be divided into two categories, `live' and `dead'. Live vaccines comprise traditional attenuated microbes, viral or bacterial, selected for reduced pathogenicity with maintained immunogenicity; and `recombinant' vaccines, in which foreign antigens are expressed from a replicating viral or bacterial vector. Although a few live recombinant vector vaccines are used in veterinary medicine, none has yet been approved for human use. `Dead' vaccines consist of killed whole pathogens, or soluble pathogen proteins or protein subunits. The nature of the vaccine Ð live or dead Ð is pivotal in determining the type of immune response induced. Both types of vaccine can induce antibodies, but dead vaccines do not eciently enter the MHC class I pathway, and thus are inecient in inducing CTL. Live vaccines may be dangerous to pregnant women or immunocompromized hosts, can revert to pathogenicity within the vaccinee and also may be contaminated by potentially harmful adventitious agents during production. From an economic standpoint, one major drawback of these vaccines is their requirement for refrigeration, increasing the cost, limiting shelf life, and greatly complicating distribution in countries lacking a `cold chain'. Although safer, vaccines composed of inactivated pathogens or immunogenic protein subunits may be less eective in inducing long-term cross-reactive immunity, and the cell-mediated immune responses critical to protection against many diseases caused by intracellularly-replicating organisms. DNA immunization, the inoculation of plasmid DNA encoding microbial proteins, is a recent addition to the vaccine arsenal. It has many apparent advantages, which are reviewed elsewhere [8], and are summarized below. Direct inoculation of plasmid DNA containing open reading frames with appropriate eukaryotic transcription and translation control signals should result in the in vivo synthesis of a protein with conformation and posttranslational modi®cation patterns identical, in most cases, to those which occur during normal infection. Endogenous protein synthesis should mimic viral infection in allowing presentation of the foreign antigen by MHC class I, and uptake of soluble protein by specialized antigen presenting cells
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(APC) allows presentation by MHC class II; both arms of the immune response may therefore be induced. We have shown that DNA vaccines are useful in immunizing infants carrying maternally-derived antibodies, which reduce the eectiveness of live vaccines by neutralizing the microbe and preventing its replication; these antibodies cannot recognize the nucleic acid vector, and therefore synthesis of the encoded protein and induction of an immune response proceeds normally [9]. DNA vaccines, lacking a replicating agent, should be safer than live vaccines for administration to pregnant or immunocompromized individuals. Finally, DNA is relatively cheap to produce, is heat stable and is amenable to genetic manipulation, making it possible to apply our approach of vaccinating with multiple immunologically reactive sequences [10] derived from multiple pathogens. Both humoral and cell-mediated immune responses have been detected following immunization with plasmid DNAs encoding a diverse array of foreign antigens, too numerous to be discussed in detail here. Excellent reviews are available [11, 12] and a collection of papers devoted to this topic has recently been published [12]. DNA immunization `works'; it induces both humoral and cellular immunity (including both CD4+ and CD8+ T-cells), which often are protective against microbial challenge. This has been shown in many model systems and we will not review them here. A frequently-updated list of the animal models, pathogens and antigens studied is available at a World Wide Web site, provided by Dr. R.G. Whalen at address http://www.genweb.com/dnavax/dnavax.html. This paper focuses on understanding the mechanisms which underpin DNA-induced immunity; these mechanisms remain unclear, and are the subject of considerable interest. Our studies exploit the model system of lymphocytic choriomeningitis virus (LCMV) infection of the mouse. LCMV is the prototype of the arenavirus family, and has a bisegmented single stranded ambisense RNA genome [13]. The short genomic segment encodes two proteins, the nucleoprotein (NP) and glycoprotein (GP); the latter undergoes posttranslation modi®cations and cleavage into its mature virus structural proteins GP1 and GP2 [14]. CTL responses are pivotal in the biology of LCMV and are vital to the control of primary infection [15] and to vaccineinduced immunity [16±18]. Our DNA immunization experiments take advantage of our detailed knowledge of LCMV and, together with the large number of reagents already available to us, permit us to investigate the mechanisms which underlie the induction of immune responses by plasmid DNA inoculation.
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2. DNA-mediated induction of CD8+ CTL It has been known for some time that live viral vaccines induce good CD8+ T-cell responses (which for the most part are CTL), because the vaccine organism replicates and synthesizes its proteins intracellularly, and thus its proteins can be eciently introduced into the class I MHC antigen presentation pathway. There is no doubt that the encoded proteins are synthesized within the transfected muscle cells, as has been shown for CAT, b-galactosidase and green ¯uorescent protein (GFP), and this capacity for endogenous protein synthesis led to the prediction that DNA immunization would be rather eective in inducing CTL responses, as has proven to be the case [19±26]. However the mechanism underlying CTL induction remains controversial. The overwhelming expression of encoded antigen by muscle cells provoked suggestions that the muscle cells themselves present antigen and directly induce T-cell responses. How likely is this? T-cells can recognize muscle in certain myopathies [27, 28], and we [29, 30] and others [31] have shown that muscle cells can be recognized by T-cells following DNA immunization, but this does not imply that myocytes can induce T-cell responses. Most workers believe that CTL induction requires at least two signals on the surface of the inducing cell; ®rst, the appropriate class I MHC±peptide complex, which interacts with the T-cell receptor and, second, a co-stimulatory molecule such as B7.1, which interacts with CD28 on the T-cell membrane. Myocytes express only low levels of MHC class I (although this is upregulated in the presence of interferon), and levels of accessory molecules appear to be extremely low; thus the ability of myocytes to present antigen in a manner capable of inducing CTL must be questioned. In addition, recent studies have shown that APCs play a critical role in DNA immunization [32±34]. Chimeric mice expressing two MHC haplotypes on their muscle cells, but only one on their bone marrow derived APCs, mounted CTL responses restricted to the bone marrow haplotype. Thus, it appears unlikely that muscle cells play a direct role in CTL induction. So, are CTL induced by APCs which have taken up soluble protein, perhaps released from muscle cell depots, or are they induced by APCs which have been transfected with DNA? Here the picture becomes unclear. A recent study showed that stably-transfected muscle cells, when transplanted, can induce immune responses (antibody and CTL) [33]. The authors concluded that antigen is released from muscle cells and taken up by APCs, thereby inducing immunity; and others have shown that exogenous proteins can be taken up by specialized APCs (such as macrophages) which can perform the unusual contortion of presenting exogenous antigen via the MHC class I pathway [35±37]. However Robinson's lab has
shown that removal of injected muscle within 10 min of DNA inoculation has no eect on the magnitude of the CTL or antibody responses induced, and the authors concluded that transfected cells in injected muscle do not play a vital role in DNA-initiated antibody and CTL responses [38]. To determine whether APCs take up soluble intact protein or injected DNA, we sought to diminish or prevent release of intact protein following DNA immunization; it is reasonable to suggest that if release of intact protein is required for CTL induction, then preventing this should inhibit CTL responses. We have developed a system to ensure very rapid degradation of an intracellular protein, by stable covalent attachment to ubiquitin, a 76 amino acid cellular protein which `tags' intracellular proteins for proteasomal destruction (reviewed [39]). Space does not permit a thorough review of ubiquitination, nor a detailed account of our strategy to improve delivery to the proteasome, described elsewhere [40]. We covalently linked LCMV NP to a modi®ed ubiquitin, designed to act as a good target for transport to the proteasome. 2.1. Rapid degradation of ubiquitinated NP To determine the intracellular turnover of the ubiquitinated protein, BHK cells were transfected in quadruplicate either with pCMV±NP, or with pCMV± U-NP, and were labeled for 30 min with S35-methionine and S35-cysteine. As a positive control, one plate of cells was infected with LCMV and radio-labeled as described; as a negative control, untreated cells were radio-labeled. After 30 min, the LCMV-infected cells, one of each set of transfected cells and the untreated cells were harvested (tracks labeled L, 0, 0 and U, respectively in Fig. 1a). In the remaining plates the incorporated label was `chased' by incubation of the transfected cells with excess unlabeled amino acids for 30, 60 or 90 min, at which time points cells were harvested. Proteins were immunoprecipitated for 2 h using anti-LCMV±NP antibody, and the products of immunoprecipitation were separated on a polyacrylamide gel and visualized by autoradiography. As shown in Fig. 1a, full length LCMV±NP was readily detected in cells transfected with plasmid pCMV±NP. NP appears relatively stable in these cells since, even after 90 min chase, a band was still easily visible. In contrast, cells transfected with the ubiquitin±NP construct showed very low levels of detectable protein. To con®rm the rapid degradation of the ubiquitinated NP, we used the proteasome inhibitor ALLN [41]; samples with ALLN are indicated with a ` + ' in Fig. 1b. The quantity of NP in cells transfected with pCMV±NP is barely altered (perhaps slightly increased) in the presence of the inhibitor, consistent with NP being stable in the cell. In contrast, there is a marked eect in cells
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LCMV-speci®c responses, but levels were lower. Thus ubiquitination enhances degradation, and improves both recognition by, and induction of, CTL. The CTL responses induced appear more likely to result from uptake by APCs of pCMV±U-NP DNA than uptake of soluble protein, since we show that the ubiquitinated protein is very rapidly degraded within the synthesizing cell, therefore limiting the quantity of intact protein available for release. Thus we favor the hypothesis that CTL induction requires uptake by APCs of plasmid DNA, and that uptake of soluble protein plays a secondary, and expendable, role. 3. DNA-mediated induction of antibodies and CD4+ Tcells
Fig. 1. Ubiquitination of NP enhances intracellular degradation. Cells were infected with LCMV (L), or were transfected with plasmids encoding ubiquitin (U), LCMV NP (NP) or ubiquitinated LCMV NP (U±NP) and were labeled with radioactive amino acids. (a) Cells were `chased' with cold amino acids for 0, 30, 60 or 90 min, at which times the proteins were harvested and precipitated with an antibody speci®c for NP, prior to analysis by SDS-PAGE. (b) Labeling was carried out in the absence (ÿ) or presence (+) of the proteasome inhibitor ALLN.
transfected with pCMV±U-NP; very little material is detected in the absence of inhibitor, but in the presence of ALLN there are readily-detectable levels of protein. Thus the pCMV±U-NP plasmid directs abundant protein synthesis, and the protein is very rapidly degraded by the proteasome, being detectable only in the presence of the proteasome inhibitor. These ®ndings are not cell-speci®c, as similar results were obtained in BALB/c ®broblasts (not shown). Furthermore, we found that the enhanced degradation resulted in improved sensitization of target cells to CTL lysis (not shown). 2.2. Ubiquitinated NP induces CTL in vivo In addition to these tissue culture ®ndings, we have evaluated the ability of pCMV±U-NP to induce CTL in vivo. Similar results were found in C57BL/6 and BALB mice; the latter are shown in Fig. 2. All mice receiving the ubiquitin±NP plasmid were primed for high levels of anti-LCMV CTL. Mice receiving pCMV±NP (nonubiquitinated protein) also mounted
Induction of antibodies usually involves two steps. First, the protein must be accessible to membranebound antibody on naõÈ ve B-lymphocytes, permitting recognition by the appropriate Fab, thus beginning the process of clonal expansion. Second, immunoglobulin class switching (e.g. from IgM to IgG) requires `help' from CD4+ T-cells; and these cells recognize antigen presented by the MHC class II pathway. During a normal virus infection it is easy to see how both of these steps are taken. Virus proteins are displayed on the virion or cell surface, secreted into the extracellular milieu, or released when the cell dies; in these ways they become available to antibodies present on the cell membranes of naõÈ ve B-lymphocytes. Furthermore, the soluble proteins may be engulfed by APCs (including B-lymphocytes themselves [42]), and presented by class II MHC, permitting the provision of cognate help. However, DNA immunization is very dierent from virus infection. First, most proteins are not cytopathic, and so should not directly cause lysis of transfected cells. Many of the encoded proteins (for example, LCMV NP) are neither displayed on the cell surface, nor secreted. Nevertheless many groups Ð including ours Ð have shown that DNA immunization induces antibodies even against such proteins. How, then, are such cytoplasmic, noncytopathic proteins recognized by B-cells? 3.1. Induction of antibody responses is diminished when protein undergoes rapid intracellular degradation To determine plasmid-mediated antibody induction requires release of intact protein, the pCMV±U-NP immunized mice described above were bled six weeks postimmunization, and anti-LCMV antibody titers were evaluated by ELISA using whole LCMV as target antigen. In both BALB/c (Fig. 3) and C57BL/6 mouse strains, the plasmid encoding ubiquitinated NP failed to induce anti-LCMV antibodies; the antibody
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Fig. 2. Ubiquitinated NP induces anti-LCMV CTL. BALB/c mice were immunized with one of the three plasmids shown and six weeks later were challenged with LCMV. Four days later Ð at which time no CTL activity has yet developed in previously-naõÈ ve mice Ð spleens were harvested and an in vitro cytotoxicity assay was carried out. Each bar represents an individual mouse, at eector:target ratios of 50:1 and 25:1. Mice immunized with LCMV rather than with DNA, and rechallenged with LCMV 6 weeks later were included as a positive control for successful in vivo secondary stimulation (LCMV); and mice infected with LCMV 7 days previously were used as a positive control for target cell lysis by CTL (LCMV d7).
titers of mice immunized with this plasmid were indistinguishable from those of mice immunized with a plasmid encoding ubiquitin alone [40]. The failure of the ubiquitin±NP construct to induce antibodies does not re¯ect an absence of gene expression, since other immune responses (CTL induction) were demonstrable in the same mice, as reported above. It therefore appears likely that this phenomenon is the in vivo re¯ection of what we found in tissue culture; the degradation of the ubiquitinated NP is so rapid and complete that intact protein cannot escape the cell in intact form, and so the interaction of intact protein and B-cells cannot take place. Thus, CTL are induced by ubiquitinated protein, but not antibodies. These conclusions are bolstered by another lab's evaluation of ubiquitination in DNA immunization [43]. Our ®nd-
Fig. 3. Ubiquitination of NP abrogates anti-NP antibody induction in BALB/c mice. The DNA-immunized mice described in the legend to Fig. 2 were bled immediately prior to LCMV challenge and their antibody levels were determined by ELISA.
ings suggest that the two arms of the immune response may be induced by dierent mechanisms following DNA immunization. Perhaps release and uptake of intact protein is required for the induction of antibodies, but not for the induction of CTL. 3.2. From what cells might antigen be released? If soluble protein is required for antibody induction, in which cells is the protein synthesized prior to release? As discussed earlier, the ability of myocytes to induce CTL is questionable. However it appears very likely that myocytes can be recognized by CTL. CD8+ cells have been noted in the in®ltrates of several myopathic diseases [28]. So, do myocytes acts as antigen depots, releasing antigen upon CTL lysis? If so, one might expect local in¯ammation. We evaluated the issue in naõÈ ve and in immune mice and found that inoculation of pCMV±NP into a nonimmune mouse induced at most a mild in¯ammatory response. In contrast, the same DNA inoculated into an immune mouse induced a ¯orid myositis; the response is antigen speci®c, since inoculation of pCMV into an immune mouse engenders no such reaction. The in¯ammatory response occurred both in acutely-infected mice, and in mice carrying memory CTL (Fig. 4). The extent and localization of the response, and the destruction of muscle ®bers, suggest that recognition of skeletal muscle by T-cells may be occurring; this has recently been con®rmed [31]. However the role of muscle cells as an important antigen depot is questionable, since excision of the injected muscle bundle (which can be cleanly accomplished when the tibialis
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Fig. 4. Antibody responses are induced in perforin knockout mice. C57BL/6 and PKO mice were immunized with pCMV±NP and six weeks later their anti-LCMV antibody titers were determined by ELISA. (a) Individual mice. (b) Group means. Controls include mice immunized with LCMV and nonimmune mice.
anterior muscle is employed) as soon as 10 min postinjection has little eect on the induction of antibody or CTL responses [38]. It appears unlikely that, in this short time, muscle cells could take up the DNA, express the protein and release sucient material to prime both antibody and CTL responses. Perhaps resident muscle immune cells (macrophages, dendritic cells) take up the DNA and depart the tissue; or perhaps the DNA itself exits the muscle, to transfect other cells. Thus, while i.m. DNA immunization certainly can lead to immune-mediated myocyte destruction, it is unclear whether muscle cells contribute to immunity. While they may act as depots, so too may other cell types which take up the DNA.
(Fig. 5). Thus, we conclude that perforin-mediated lysis is not required for release of antigen expressed following injection of plasmid DNA. Of course it remains possible that myocytes or other cells do act as antigen depots, releasing antigen by an as-yet-unidenti®ed mechanism.
Acknowledgements The authors are grateful to Annette Lord for skilled administrative assistance. These studies were supported
3.3. Perforin-mediated lysis is not required for the induction of antibodies by i.m. DNA immunization Since antigen release is required for antibody induction, an obvious underlying mechanism would be antigen-speci®c CTL-mediated lysis of transfected antigen depots. Therefore we have attempted to assess the role of CTL lysis using mice de®cient in the major CTL cytolytic protein, perforin [44±47]. As shown in Fig. 4, these perforin knockout (PKO) mice mounted readilydetectable antibody responses. Individual (Fig. 4a) and average (Fig. 4b) antibody levels are shown for PKO and C57BL/6 mice. The results show conclusively that perforin-mediated lysis of muscle cells (or indeed of any cells) is not required for induction of antibody responses following DNA immunization. In fact, the results suggest that antibody responses may even be elevated in PKO mice. Our ®ndings are not limited to LCMV NP; responses to a plasmid encoding LCMV glycoprotein are similarly enhanced in PKO mice
Fig. 5. Enhanced response to GP in PKO mice. C57BL/6 and PKO mice were immunized with pCMV-GP, and six weeks later their antiLCMV antibody titers were determined by ELISA. Each set of points represents an individual mouse.
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by NIH grants AI-37186-04 and T32-AI-07354-08. This is manuscript number 11595-NP from the Scripps Research Institute.
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