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Immune Responses to DNA Vaccines: Induction of CD8 T Cells

and .

CD8 T Cells Are Important in Controlling Most Virus Infections

The importance of CD8 T cells in the control and eradication of viruses has been demonstrated in mice and men. In the mouse, they are critical in combating infection with lymphocytic choriomeningitis virus (LCMV; ref. 1), and in humans, “experiments of nature” strongly suggest that T cells play a vital role in controlling many virus infections.2 For example, children born with hereditary agammaglobulinemia are much more susceptible to suppurative bacterial infections,3 and people with defects in the complement cascade show increased susceptibility to Neisserial diseases;46 however, in contrast to their greatly enhanced vulnerability to bacterial infections, these individuals show only mildly elevated susceptibility to most viral diseases, with the exception of rare enteroviral meningitides, caused most often by picornaviruses such as coxsackievirus7,8 and echovirus type 9 or 11.9,10 For most virus infections, the incidence of disease, and disease severity, are similar in antibodydeficient and in immunocompetent individuals. These observations suggest that other factorsperhaps CD8 T cellsare capable of resolving (most) virus infections in humans. This suggestion is supported by the finding that the frequency and severity of virus infections are markedly increased in humans with impaired Tcell responses [for example, in patients with Di George's syndrome (congenital thymic aplasia), acquired immunodeficiency syndrome (AIDS), leukemia, or recipients of immunosuppressive therapy].11 In HIV infection, CD8 Tcell activity correlates with clearance of initial viral load, and their absence heralds a return to high viral titers, and eventual AIDS.12,14 The importance of T cells in controlling human virus infections is further highlighted by our responses to measles virus. In immunocompetent individuals, the infection is typified by the characteristic (& diagnostic) rash, and complete recovery is the norm. In contrast, in Tcell deficiency, the disease is often fatal.15,17 The rash itself is Tcellmediated and does not develop in severely immunosuppressed individuals; indeed, the presence of a rash in an immunosuppressed victim (e.g., in a leukemic child with measles) is considered a positive prognostic indicator.18 In agammaglobulinemic children, the rash develops normally, and the infection is cleared. Furthermore, these children are subsequently immune to measles,2 suggesting that T cells can play an important role not only in controlling a primary infection, but also in preventing disease following secondary exposure; this observation was an early (and often overlooked) indication that CD8 memory T cells might be important in vaccineinduced antiviral immunity (see below). In the next section, we shall provide a molecular explanation of why CD8 T cells are important in controlling most virus infections; and why bacterial infections, rather than virus infections, are more severe in the absence of antibodies.

Antigen Presentation Pathways Determine the Type of Immune Response Mounted by the Host

In this section, we wish to make two broad points: (i) the host mounts the type of immune response best suited to eradicating the particular type of microbe which it faces; and (ii) the type of immune response mounted is dictated by the interaction of the infectious agent with the host's antigen presentation pathways. These ideas, which are relevant to our subsequent discussion of how DNA vaccination induces CD8 T cells, have been reviewed elsewhere,19 and detailed molecular, immunological, and biological perspectives are available.20 The underlying mechanisms are cartooned in Figure 1 (the molecular details of the antigen presentation pathways are described in other chapters in this volume). Intracellular organisms (usually viruses) will synthesize proteins inside the infected cells, and these antigens therefore gain ready access to the MHC class I pathway; as a result, they often induce strong MHC class I restricted CD8 T cell responses (these cells are usually cytotoxic, although they also secrete a variety of cytokines in response to antigen). Furthermore, the antigens synthesized during intracellular infections usually will be released from the infected cells into the extracellular milieu, and these soluble materials are taken up by specialized antigen presenting cells (APC) which express both classes of MHC molecule. Inside these APC, the antigens enter the MHC class II pathway, permitting them to induce MHC class II restricted CD4 T cells, which usually provide “help” to B cells and, often to CD8 T cells. The soluble antigens will also encounter B lymphocytes, and therefore should (in concert with CD4 T cell “help”) induce antibody responses. Therefore, we would expect most intracellular organisms to induce strong CD4 and CD8 cell responses, and decent antibody responses; and, broadly speaking, this is the case. In contrast, the antigens of extracellular organisms (most bacteria) cannot efficiently enter the MHC class I pathway [crosspriming notwithstanding] and, therefore, most bacteria fail to induce strong CD8 immunity. However, many of the proteins encoded by extracellular microbes will be taken up by APCs and introduced into the MHC class II pathway, and so extracellular organisms induce strong CD4 T cell and antibody responses.

Figure 1. Antigen processing pathways ensure that the immune response which is mounted is that which is best suited to combat the invading microbe.

Figure 1

Antigen processing pathways ensure that the immune response which is mounted is that which is best suited to combat the invading microbe. The MHC class I & II antigen presentation pathways determine the types of adaptive immune response mounted (more...)

These considerations explain how the host “knows” what kind of response to produce against a given microbeit is dictated by the intracellular or extracellular nature of the invader. Furthermore, as also shown in Figure 1, the response induced is that which is most appropriate to deal with the infection. An intracellular organism will induce CD8 T cells (which recognize and eradicate infected cells) as well as antibodies (which mop up the organisms when they are in their extracellular state) & CD4 T cells (which provide help). In contrast, the host will not “waste its time” producing CD8 T cells against an organism whose entire lifecycle is extracellular; instead, strong CD4 T cell & antibody responses predominate. Figure 1 also facilitates understanding of the biological consequences of immune dysfunction, which were described above. Individuals with agammaglobulinemia show increased susceptibility to bacterial diseases because they have no other adaptive immune response which can combat extracellular organisms; CD8 T cells have little effect on our ability to combat most bacterial infections. Therefore, antibodies are absolutely critical for protection against many bacterial infections. In contrast, the absence of antibodies is often not as devastating when the microbe is intracellular, because the host can rely, to a significant extent, on CD8 T cell responses; hence, agammaglobulinemic people can resist most3 [although not all]7,10,21 virus infections. Finally, these ideas have implications for vaccine design.

Designing Vaccines Against Viruses and Bacteria

For many years, antibodies were thought to be the only important facet of vaccineinduced immunity. This misunderstanding had two causes. The first was practical; antibodies were much easier to detect than T cells. The second was conceptual; the observation that protective immunity usually coincided with strong antibody responses led many to concludeillogicallythat protection must be conferred by antibodies. As stated above, measles infection of agammaglobulinemic individuals suggested that other facets of adaptive immunity could protect against disease following a secondary exposure to virus. More than a decade ago, we and others, working in viral model systems, asked if a vaccine which induced CD8 T cells alone could solidly protect the host against subsequent viral challenge. In LCMV infection22,26 and in murine cytomegalovirus (MCMV) infection,27,28 it proved possible to fully protect a naive animal from virus challenge by immunization with a recombinant vaccinia virus (VV) expressing a single viral internal (i.e., nonmembrane) protein. Indeed, recombinant vaccines containing “minigenes” encoding isolated CD8 T cell epitopes as short as 11 residues could confer protection against normallylethal doses of challenge virus, and different epitopes could be linked in a “string of beads” to protect on several MHC backgrounds.29,30 No virusspecific antibody responses are induced by these vaccines, proving that the outcome of infection (death or survival) is determined by vaccineinduced CD8 T cells. In the case of MCMV, a herpesvirus, protection is conferred by CD8 T cells specific for a protein expressed very early in the virus lifecycle. Presumably, it is to the host's benefit to recognize such early proteins, because it gives the host a chance to destroy infected cells before the virus can produce infectious progeny. In human herpesvirus infections, it is clear that CD8 T cell responses are generated against similar classes of proteins. For example, the major CTL response to human cytomegalovirus (HCMV) is to a protein expressed immediately upon infection;31 a similar situation exists for varicellazoster virus32 and herpes simplex virus (HSV).33 Of course, the immediateearly, early & late proteins, seen in herpesviruses, are not found in all virus infections; many viruses express their full complement of proteins more or less synchronously, and in these viruses, many proteins can be targets for biologicallyrelevant CD8 T cell responses. Influenza virus infection induces CTLs directed against most viral components, although a major group is specific for the virus nucleoprotein (NP). These NPspecific T cells, unlike antiinfluenza antibodies, are crossreactive; that is, they lyse HLAmatched target cells infected with a serologically distinct strain of influenza virus. However their presence fails to confer absolute immunity to infection and disease caused by a serotypically distinct influenza virus. These results are sometimes used to challenge the hypothesis that the presence of virusspecific CD8 cells confers protection against virusinduced disease. However, it is important to understand that CD8 T cells cannot prevent infection; on the contrary, recognition by these cells requires that the target cell be infected. Antibodies prevent infection, and CD8 T cells limit virus production and dissemination; together they protect against disease. Thus, the preexisting antiinfluenza CTLs, while failing to prevent infection, may diminish the ensuing morbidity (disease) and mortality (death).

We do not argue that induction of antibodies is unimportant for antibodies unimportant antiviral vaccines; on the contrary, a role for antiviral antibodies in vaccination is unquestionable. Passive antibody therapy can protect against or modify the course of several human virus infections. For example, infusion of antibodies specific for the Junin arenavirus is beneficial in Argentinian hemorrhagic fever,34,35 whereas postexposure rabies prophylaxis relies on vaccination and concurrent administration of virusspecific Igs.36 Furthermore, in experimental models, antibodies can lower viral titers and modulate disease. For instance, recovery from ocular herpesvirus infection is hastened by administration of antiHSV antibody.37 Nevertheless, for the reasons discussed above, we conclude that an antiviral vaccine should not rely solely on antibodies, and instead should induce both arms of the antigenspecific response; T cells (CD8 & CD4) and antibodies. When viewed in the light of Figure 1, this conclusion makes good sense. What of antibacterial vaccines? For bacteria with a largely intracellular life cycle (e.g., Listeria monocytogenes), CD8 T cells, as well as CD4 T cells and antibodies, should be induced. The importance of CD8 T cells in controlling Listeria was demonstrated years ago,38,39 and a recombinant vaccinia vaccine encoding a single CD8 T cell epitope from Listeria could confer some protection against bacterial challenge.40 However, for a strictly extracellular organism, CD8 T cells are much less likely to play a protective role. As stated above, antibodies are vital to the control of most bacterial infections, and therefore antibody induction should be an invariable goal of most bacterial vaccines.

It is appropriate, at this point, to make a general comment on the selection of vaccine target proteins. CD8 T cell responses may be directed against essentially any intracellular protein, since most proteins made inside an infected cell can, potentially, contain epitopes presented by the MHC class I pathway. In many cases, the most important targets for CD8 T cells are defined temporally; the most effective CD8 T cells are often those specific for proteins made early in the microbial life cycle, and these proteins are appropriate vaccine candidates. Similarly, CD4 T cell responses can be mounted against almost any protein, internal or external, since most proteins should be taken up by APCs, where their lysosomal degradation products become available for presentation by MHC class II molecules. In contrast to the large number of antigens open to T cell perusal, biologicallyimportant antibody responses are most likely to be directed against “external” proteins, at the surface of the microbe (be it a virus or a bacterium), since those proteins will be most accessible. So, for most bacteria, the most promising vaccine candidates are likely to be cell surface proteins, or other cell surface structural components, which are accessible to antibodies. There is at least one additional consideration. Many bacterial diseases result not from the bacterial infection per se, but rather from the actions of bacteriallyderived toxins; examples include tetanus and cholera. [In contrast, viruses generally do not produce toxins; although there are exceptions.]41 Vaccines against these diseases may, therefore, be targeted not against the bacterium, but against the toxin. In essence, such a vaccine does not necessarily allow the host to improve its control of infection; instead, it readies the host to neutralize the pathogenic capacity of the organism, and may leave the naturally developing immune response to take care of the infection.

The Effector Functions Through Which CD8 T Cells Exert Their Biological Activities

CD8 T cells have two general effector functions: they can induce lysis of cells expressing the cognate antigen, or they can secrete cytokines in response to antigen contact. Lytic activity was the first criterion by which these cells were identified and, as a result, they often are termed cytotoxic T lymphocytes (CTL). However, it is becoming clear that CD8 T cells can regulate their effector functions in a subtle manner, and that some antigenstimulated cells can produce cytokines, but show low lytic activity.42,43 Furthermore, as discussed below, CD8 T cells can control certain infections by cytokine release alone. Therefore, the term CTL should be used only when lytic activity is proven, and should not be used as a synonym for all antigenresponsive CD8 T cells.

CD8 T Cells Usually Contain Perforin, a Poreforming Protein

Most CD8 T cells contain granules, which align with the target cell upon recognition, and whose contents are released in a calciumdependent manner onto the target cell membrane. These granules include a protein called perforin,44 which undergoes assembly into transmembrane pores and thus punches holes in the cytoplasmic membrane of the target cell. Perforin shares immunologic crossreactivity with the C9 component of complement, and cloning of the murine perforin gene has allowed identification of a short stretch of amino acid homology between the two proteins. The membrane lesions caused by perforin are similar to those induced by the complement C9 complex. Thus, CTLs and complementmediated lysis seem to share one common mechanism of action. The importance of perforin in CTL activity in vivo has been demonstrated.45,47 Transgenic mice with a dysfunctional perforin gene are less effective at controlling infection by some (though not all) viruses; it has been argued that perforin is important for the clearance of “persistent” viruses, but is not required to counter acute virus infections which may be cleared by a combination of cytokines and antibodies.48 This hypothesis, although intriguing, remains unproven.

CD8 T Cells Can Induce Apoptosis in Target Cells

Apoptosis, or programmed cell death, is a well recognized phenomenon responsible for several developmental processes, including the clonal deletion of T cells in the thymus. Its most characteristic features are nuclear blebbing and disintegration, resulting in a nucleosome stepladder of fragmented DNA. The perforin channels inserted into the cell membrane by CTL permit the entry of other CTLproduced proteins (granzymes) which induce apoptosis in the target cell. Furthermore, CD8 (and sometimes CD4) T cells can induce apoptosis when the fasL protein, expressed on the T cell membrane,49 interacts with Fas protein on the target cell, initiating a signaling cascade which ends in target cell apoptosis. Virusspecific T cells may induce this process in infected target cells.50,52

CD8 T Cells Release Antiviral Cytokines

Many CD8 T cells release high levels of cytokines, for example interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Mice lacking the IFNγ receptor have increased susceptibility to several infections, despite apparently normal CTL and Th responses.53 It has been cogently argued that a major role of the TcR/MHC/peptide interaction is to ensure that cytokine production by CD8 T cells is limited to the immediate proximity of virusinfected cells,54,55 and convincing data from mice persistently infected with LCMV,56,57 from hepatitis B virus (HBV) transgenic mice58,59 and from HBVinfected primates60 has shown that viral materials can be eradicated in vivo from neurons56,57 and hepatocytes58,59 in the absence of cytolysis.

Antigenic Control of CD8 T Cell Activation, and Effector Function

Antigenspecific recognition by the TcR, and signal transduction across the cell membrane, initiate a series of events in naïve CD8 T cells, including cell division and the activation of effector functions.

A Single Short Antigenic Pulse is Sufficient to Drive Naïve CD8 T Cells to Become Memory Cells

Recent data strongly suggest that the entire program of CD8 T cell maturation can be initiated by a single, shortterm, exposure to antigen. After antigen contact for as few as 2 hours, naïve CD8 T cells bearing the appropriate TcR are irrevocably committed to divide, expand, develop their effector functions, and pass into the memory phase; it is important to note that no further antigen contact is required.61,62 These findings have implications for DNA vaccination, indicating that longterm antigen expression should not be required and that, instead, a single strong pulse of antigen would be sufficient.

Antigenspecific CD8 T Cells are Exquisitely Sensitive to Antigen Contact

Our lab has recently shown that, even at the peak of the antiviral immune response, when some 50% of all CD8 T cells may be virusspecific,63,64 the great majority of these cells are not actively producing cytokines; however these virusspecific cells are exquisitely sensitive to antigen contact, initiating cytokine synthesis immediately upon encountering an infected cell, and terminating production the instant the contact is broken.65 This tight regulation is important because cytokines can be toxic to the host, and indeed systemic cytokine release is responsible for many of the symptoms of microbial infection, and for the extensive weight loss seen in certain cancers (reviewed, 66).

CD8 Memory T Cells are Effector Cells

In the preceding pages, we have stressed that vaccineinduced CD8 memory T cells are important components of antiviral vaccines. What are these vaccineinduced CD8 memory cells, and how do they exert their biological activities? We have previously shown that the outcome of infection in immunized individuals is determined by the presence, at early times, of detectable memory CD8 T cells; and we argued that the outcome of infection is decided within minutes or hours of virus challenge, even before any expansion of the memory cell population can occur.67 One inescapable corollary of this conclusion is that the memory cells present at the time of infection must be able to display at least some of their effector functions very soon after encountering virusinfected cells. However, contrary to this contention, memory cells have, for many years, been considered a dormant population, lacking the effector functions displayed by cells during the acute phase of infection (which we shall refer to as “acute cells”). Although there are some differencesfor example, memory cells are smaller than most acute cells, and usually contain less perforinthere are many similarities between the two cell populations. For example, surface markers such as CD11a, CD11b, CD44 and CD62L are present on “antigenexperienced” (acute or memory) CD8 T cells,68,73 but absent from naïve cells; this provides an inkling that memory and acute cells may share common functions. Furthermore, attempts to discriminate memory cells from acute cells using surface markers has been difficult, although a recent study showed differences in cellsurface expression of Oglycans.74 Several years ago, Selin & Welsh showed that a small population of virusspecific memory cells was capable of immediate lytic activity, without extensive antigenic restimulation75 and, more recently, others have shown that CD8 memory T cells harvested from nonlymphoid tissues were lytic, in contrast to their counterparts harvested from the spleen.76

Our lab has evaluated CD8 T cell effector functions during the acute and memory phases of virus infection, and has found similarities and differences.77 As shown in Figure 2 (top row), during the acute response to primary infection, virusspecific cells comprise two populations, IFNγTNFα (singlepositive) and IFNγTNFα (doublepositive). After infection is cleared, and the immune response enters the memory phase, the virusspecific cells are almost uniformly doublepositive. A similar pattern is observed after secondary virus challenge in immune mice (Figure 2, bottom row). Thus, the population of virusspecific T cells during active infection differs from the population present in the memory phase; but the relationship between the singlepositive and doublepositive cells is unknown. Importantly, we also found that, after encountering cognate antigen, memory cells initiated IFNγ production every bit as rapidly as did acute cells. During the acute phase of infection, many more virusspecific CD8 T cells are present than are found during the memory phase (Figure 3A); however, the rapidity with which the virusspecific cells can respond is very similar in the two populations (Figure 3B). Thus, CD8 memory cells can exert their effector functions immediately upon antigen contact. These observations are consistent with our hypothesis that the fate of an infected vaccinee is sealed very soon after infection; if the preexisting memory cells are sufficiently numerous, they can contain the infection immediately; if not, the virus disseminates, and can cause disease.

Figure 2. Cytokine production by effector and memory CD8 T cells.

Figure 2

Cytokine production by effector and memory CD8 T cells. Naïve mice were infected with LCMV (top row) and at the indicated times postinfection, virusspecific CD8 T cell responses were measured by ICCS. Splenocytes were gated on CD8 T cells, and (more...)

Figure 3. Acute and memory CD8+ T cells initiate IFNγ production at very similar rates.

Figure 3

Acute and memory CD8+ T cells initiate IFNγ production at very similar rates. Splenocytes were harvested from mice at 8 days (l) or 295 days (·) after LCMV infection, and were incubated with antigenic peptide for the indicated number of (more...)

How are CD8 T Cells Induced Following DNA Vaccination?

Surprisingly, this issue remains somewhat controversial. Doubtless, this topic will be addressed in other chapters in this book, and here we give our own perspectives on this important question. Soon after the discovery of DNA immunization, two hypotheses were proposed to explain the observation that CD8 T cells could be induced by DNA vaccines. First, that “nonspecialized” cells took up the DNA and expressed the encoded antigens; and these cells induced a primary CD8 T cell response. A priori, this hypothesis appeared unlikely to be correct, because most somatic cellsalthough usually expressing class I MHC moleculesdo not express the costimulatory signals needed to stimulate naïve antigenspecific T cells. Indeed, healthy muscle cells express negligible amounts of MHC molecules,78 rendering them even less likely candidates for the role of stimulator cells. The second hypothesis was that the DNAencoded antigen was presented to T cells by specialized antigenpresenting cells (APCs); and elegant studies showed incontrovertibly that APCs were required for successful induction of CD8 T cell responses by DNA vaccines.79,80 That APCs are important is generally accepted; and dendritic cells (DCs) are thought likely to play a key role. DCs expressing a foreign antigen are remarkably efficient at inducing CD8 T cells; only 1001000 antigenpresenting DCs are needed to induce a CD8 T cell response capable of protecting against subsequent virus challenge.81 Here we encounter the unresolved, and controversial, issueexactly how does the DNAencoded antigen gain access to the MHC class I pathway in the vaccinee's APCs? Again, two hypotheses have been proposed; (a) crosspriming, in which protein is transferred from transfected cells, to APCs and (b) uptake of DNA by APCs.

Does DNA Immunization Depend on Protein Transfer from in Vivo Transfected Cells, to APCs?

The first hypothesis states that protein produced by transfected somatic cells (e.g., by muscle cells following intramuscular injection of plasmid) is released, and is taken up by APCs (perhaps dendritic cells) which have the unusual ability to introduce exogenous proteins into their MHC class I pathway. The phenomenon of apparent protein transfer, first observed in studies of minor histocompatibility responses,82 is termed “crosspriming”. Some authors have suggested that crosspriming is important for83 [and perhaps, even, central to]84 the induction of CD8 T cell responses during virus infection; we do not believe that this stance is supported by the available evidence, but here we shall focus on CD8 T cell induction by DNA immunization. Several arguments have been advanced to support the idea that crosspriming underlies the successful induction of CD8 T cells by DNA vaccines.79,80 Perhaps the strongest argument is based on the following experimental approach. Nonlymphoid cells (e.g., muscle cells) expressing a given mouse MHC haplotype (say, H2b) are transfected in tissue culture with a plasmid expressing a wellcharacterized antigen (“X”) for which CD8 T cell epitopes are know for the “self” haplotype (H2b) and for one other MHC haplotype (say, H2d). Stablytransfected cells are selected, cultured for several passages, and these cellswhich, presumably, no longer contain any free plasmid DNAare inoculated into H2b/H2d recipient mice. Induction of H2brestricted CD8 T cell responses to antigen X would suggest that some form of antigen transfer to APCs is occurring, because (as stated above) it is unlikely that the injected muscle cells could directly stimulate a primary CD8 T cell response. An even more persuasive case can be made if H2drestricted CD8 T cell responses are induced; since the inoculated muscle cells do not express H2d alleles, the presence of such T cells is powerful evidence that antigen X is being presented by H2d MHC class I molecules, presumably on the host's APCs. Several studies similar to the above have shown that such CD8 T cells can be induced by inoculation of stablyexpressed cells and, as a result, many workers in the field believe that transfer of proteins from in vivo transfected cells is central to DNA immunization. Furthermore, plasmidencoded fusion proteins in which the antigen of interest is attached to an immunoglobulin Ig fragment,85 or to the extracellular domain of Flt3 ligand,86 are more immunogenic; the authors concluded that the enhanced immunogenicity resulted from specific uptake by DCs, although other interpretations are possible. Thus, a number of findings support the contention that DNA vaccines work by crosspriming. However, other studies run counter to this conclusion. For example, Zinkernagel's group found that disruption of stablytransfected fibroblasts prior to injection abrogated CD8 T cell induction, indicating that free proteins could not induce CD8 T cells via crosspriming.87 Furthermore, excision of a DNAinjected muscle within minutes of plasmid inoculation failed to prevent the induction of immunity, suggesting that protein production by muscle cells is unlikely to underlie successful DNA immunization.88 Finally, we89,91 and others92,95 have shown that targeting a DNAencoded protein for rapid degradation within the transfected cell leads to enhanced CD8 T cell responses, and pulsechase studies showed that, at any given time point, very little intact protein could be found in the transfected cell. It is difficult to reconcile this observation with the hypothesis that transfer of intact protein is important for successful DNA immunization.

Does DNA Immunization Depend on Uptake and Expression of Injected Plasmid by APCs?

An attractive alternative to crosspriming is that the requisite antigen presentation by APCs results simply from their taking up the inoculated DNA, expressing the encoded antigen, and thereby allowing the endogenouslysynthesized protein to enter the MHC class I pathway. DNA uptake and gene expression have been observed in dendritic cells in vivo following DNA immunization,96 and adoptive transfer of the in vivo transfected cells leads to CTL induction.97 Cotransfection studies also support the DNA uptake hypothesis.98 Furthermore, this idea is more consistent with the result of the study in which the injected muscle was rapidly ablated; presumably, some of the injected plasmid exited the muscle (either in the bloodstream, or by “leakage”) prior to its excision. The abovecited findings with DNAs encoding rapidlydegraded proteins also are more easily reconciled with this hypothesis than with crosspriming. Thus, although crosspriming probably plays some part in the induction of CD8 T cells following DNA immunization, we favor this second hypothesis. Note that the question is not merely academic; it is important that the dominant mechanism be identified, to allow us to optimize DNA immunization. If CD8 T cell induction occurs through protein transfer and crosspriming, then we should design plasmids which drive high levels of protein synthesis in the in vivo transfected cells (e.g., in muscle cells). On the other hand, if DNA immunization works via DNA uptake by APCs, we should focus on targeting the DNA to these cells, and enhancing gene expression in this cell type.

Enumeration and Characterization of CD8 T Cells Induced by DNA Immunization

Hundreds of papers have now been published which show, unequivocally, that DNA vaccines can induce CD8 T cell responses (as well as CD4 T cells & antibodies); we shall not attempt to review all of these in this chapter. In some cases, DNA vaccines have proven more immunogenic than other recombinant delivery systems (for example, recombinant vaccinia viruses, see ref. 99), and sometimes they appear to overcome a host's previous nonresponsiveness to a particular antigen.100 As a rule, DNA vaccines appear to induce better CD8 T cell responses than antibody responses (see, for example, ref. 101). This is true regardless of the route of immunization. CD8 T cell responses have been detected in many DNA vaccine studies, but in the vast majority of cases, the responses were not measured directly ex vivo; instead, DNAinduced CD8 T cells were first subjected to some form of secondary stimulation, and these restimulated cells were quantitated. The types of restimulation employed varied from study to study, but usually fell into one of two categories. (i) In vitro restimulation. Splenocytes from DNAimmunized animals were incubated for days (or sometimes weeks) in tissue culture, with stimulator cells expressing the cognate antigen, and then were assayed for cytolytic activity; or (ii) In vivo restimulation. Animals (usually mice) immunized with DNA encoding a particular antigen (often viral) were infected with the appropriate virus and, several days later, splenocytes were taken, and their cytolytic activity was measured. The use of extensive restimulation severely limited the conclusions which could be drawn from these studies; neither the number of cells which had been induced by the DNA vaccine, nor the functional attributes of the DNAinduced cells, could be confidently inferred. Therefore, it was important to develop T cell assays which would allow detection of low numbers of T cells, without extensive restimulation. For many years, in vitro cytotoxicity assays have been the “readout” of CD8 T cell function. This assay has been enormously valuable, and remains useful. However, it is not sufficiently sensitive to detect low numbers of effector cells and not all antigenspecific cells show high lytic activity. Analyses of antigenspecific CD8 T cell responses has been transformed by the advent of three additional techniques: first, MHC peptide tetramer technology, which allows the detection of T cells bearing receptors of defined antigen specificity. Second, intracellular cytokine staining (ICCS), in which cytokine production by T cells (which occurs within minutes of antigen contact; see refs. 65, 102) can be detected by antibody staining and subsequent flow cytometry. Third, cytokine ELIspot, in which cytokine production by immobilized antigenspecific cells can be detected.

DNAinduced CD8 T Cells can be Detected Directly ex Vivo

Our lab has made extensive use of ICCS, and we have found that DNA vaccineinduced CD8 T cells can be readily identified directly ex vivo without extensive restimulation;67,103,104 with ICCS, the cells are exposed to antigen within minutes of their being harvested from the animal, and antigen stimulation continues for only ˜6 hours, which is too short a time to allow significant expansion of CD8 T cell numbers. In this way, we can be confident that we are detecting the actual CD8 T cells induced by the DNA vaccine. Examples of ICCSbased detection of DNA vaccineinduced CD8 memory T cells are shown in Figure 4. Neonatal or adult mice were inoculated once with DNA (either pCMVNP, which encodes the LCMV nucleoprotein, or the “empty” plasmid pCMV, as a negative control), and a year later (without any interim boosting) the mice were sacrificed, and their splenocytes were assayed by ICCS. Antigenspecific CD8 T cells (identified by IFNg production in response to the 6 hour exposure to epitope peptide) were easily identified in the appropriate mice, and constituted ˜1% of total CD8 T cells. [In comparison, previous studies, in which DNAinduced memory cells were quantitated after restimulation, found much lower frequencies.]90,105 Therefore, a single DNA immunization induces very longlived memory cells, even in mice inoculated within hours of birth. This is consistent with the recent finding, that a single pulse of antigen is enough to cause naïve CD8 T cells to differentiate into memory cells. Others have shown that DNA vaccination of primates (rhesus macaques) can induce CD8 T cell response which are detectable directly ex vivo, and comprise ˜0.5% of CD8 T cells in the animals' peripheral blood.106 Thus, DNA vaccines induce CD8 T cell responses which, although lower than those found following virus infection or vaccination using a recombinant virus,103 are nevertheless impressive.

Figure 4. Typical CD8 T cell responses following a single DNA immunization.

Figure 4

Typical CD8 T cell responses following a single DNA immunization. Neonatal or adult mice were immunized with a single injection of plasmid DNA; either pCMVNP (top row) or the negative control plasmid pCMV (bottom row). One year later, splenocytes were (more...)

DNA-Induced CD8 T Cells are Qualitatively Similar to Those Induced by Virus Infection

Direct ex vivo detection of CD8 T cells induced by DNA immunization enables us to ask whether or not these cells are functionally equivalent to those induced by conventional vaccines. We have evaluated such cells using five criteria; cytokine production, perforin content, lytic ability, functional avidity, and protective efficacy. By all criteria, DNAinduced cells were indistinguishable from cells induced by other means.103,104

CD8 Cell Responses Induced by DNA Immunization in Humans

T cell responses have been identified in many animal models (see above); however, of the hundreds of papers published about DNA immunization, relatively few have reported results in humans. A review of clinical trials is included in another chapter in this volume, and here we provide only a short summary. A DNA vaccine encoding a malaria antigen was given to malarianaïve healthy human recipients, and CD8 T cell responses were detected against all 10 known epitopes, presented by at least 6 HLA alleles; promising though this may appear, the responses were not strong, and their induction required three doses of vaccine.107 Similarly, when 15 HIVinfected patients were given escalating doses of an HIV env/rev DNA vaccine, only weak responses, of arguable significance, were noted.108 Another study of DNA vaccination in HIVinfected individuals (3 immunizations over a 6month period) reported MHC restricted CD8 T cell responses in 8 of 9 vaccinees, although the response was transient in three patients.109 In a phase 1 trial, an HIV-1 env/rev DNA vaccine was administered (using either 100 μg or 300 μg doses) on 4 occasions to HIV1 seronegative individuals; antigenspecific lymphocyte proliferation and cytokine production was detected at least once in all highdose recipients, but the response did not persist in any of the individuals.110 In summary, so far, DNA vaccines (used alone) have not induced very strong immune responses in our species. There is, therefore, room for improvement.

Enhancing CD8 T Cell Induction by DNA Vaccines

We have recently summarized some of the approaches which might be used to enhance antibody and T cell responses to DNA immunization;111 here, we focus on improving CD8 T cell induction. Several approaches have been taken, each with some success.

Route of Immunization

CD8 T cell responses have been detected after DNA delivery by the intramuscular and intradermal routes but, perhaps surprisingly, the intravenous route appears less successful.112,113 A recent study suggested that the immunogenicity of inoculated DNA could be increased 100 to 1000fold by direct injection into lymph nodes.114 Several laboratories have reported success using attenuated Salmonella as a delivery vehicle for plasmid DNA, and one study showed that oral Salmonella followed by antigenexpressing DC was especially effective.115 Finally, in vivo electroporation (also called electropermeabilization)in which an electric current is applied in vivo to potentiate cellular uptake of DNAmarkedly increases in vivo transfection efficiency.116 There is a current surge in interest in applying this technique to DNA vaccination; and the method appears to enhance both antibody and CD8 T cell responses.117,123

Linking the Antigen to Heatshock Proteins

It has been reported that heatshock proteins may play a role in crosspriming, perhaps delivering antigenic peptides to the MHC class I pathway.124,125 Consequently, some labs have attached antigens to heat shock proteins, in an attempt to exploit this apparent pathway. For example, a fusion between Mycobacterium bovis Hsp65 and fragments of influenza NP led to more effective CTL induction.126 Fusion of human papilloma virus (HPV) E7 protein to Hsp70 from Mycobacterium tuberculosis led to a ˜30fold increase in E7specific CD8 T cells,127 and the same group reported similar results using Hsp linkage in a repliconbased RNA immunization system;128 whether this enhancement was due to more efficient crosspriming, or to a nonspecific “adjuvant” effect resulting from the coexpression of a highly immunogenic bacterial protein, remains to be determined.

Improving Codon Usage

Successful translation of mRNA requires that the appropriate quantities of charged tRNAs be available to “read” the codons; since prokaryotic codon usage differs from that seen in eukaryotes, optimal translation of a bacterial gene may require codon optimization. In one study, an oligonucleotide encoding a 9amino acid epitope from the intracellular bacterium Listeria monocytogenes was synthesized using the native bacterial sequence, or using codons optimized for murine expression; the latter construct was much more effectively translated in murine cells, and was more immunogenic when administered as a DNA vaccine.129 Similar conclusions were reached using an optimized gene encoding tetanus toxin,130 and in studies of Plasmodium yoelii.131 One would not necessarily predict that codon usage would be a consideration when designing antiviral vaccines, since viruses have evolved to exploit the eukaryotic translational machinery. However, codon usage differs among eukaryote genes, presumably as a means to subtly regulate protein expression, and therefore one cannot assume that all viral genes are optimally designed for translation. Altering the codon usage of HIV1 gp120 enhanced the antibody and CTL responses induced by a DNA vaccine,132 and codon optimization of the HIV gag gene yielded a DNA vaccine that was effective at a 100fold lower dose than a vaccine encoding the standard sequences.133 In contrast, others have carried out similar experiments using HIV gp160, and have concluded that little advantage accrued from codon optimization.134

Proteasomal Targeting to Increase Peptide Delivery to the Endoplasmic Reticulum

A certain proportion of proteins synthesized within the cell are destined to be delivered to the proteasome, a cytosolic organelle which hydrolyses the proteins, and the resulting peptides are transported (via the TAP transporters) into the endoplasmic reticulum (ER). It is thought that misfolded proteins, termed defective ribosomal products (or drips) may be preferentially targeted for proteasomal degradation;135 and proteasomal targeting requires the covalent attachment of multiple copies of the cellular protein ubiquitin, each attached to the other in a headtotail polyubiquitin array.136,137 Several laboratories have exploited the ubiquitin pathway to destabilize DNAencoded proteins, and thereby to enhance their delivery to the proteasome. Two subtly different tacks have been taken, and both have produced similar results. One approach exploits Varshavsky's “Nend” rule,138 which states that a protein's stability is determined, in large part, by the Nterminal amino acid residue of the mature polypeptide. Two groups have shown that, by using ubiquitin to replace a “stable” Nterminus with an “unstable” residue, one can increase protein turnover and thereby alter its immunogenicity.92,94 Another technique places a gene encoding a modified ubiquitin inframe with the antigen of interest in a DNA vaccine.139 We used this approach to show that, when the fusion protein is expressed in cells, the modified ubiquitin acts as the target for the addition of a polyubiquitin chain, and thus the antigen of interest is very efficiently delivered to, and degraded by, the proteasome. In this way, intracellular degradation is greatly enhanced, and the protective capacity of the DNA vaccines is improved.90,91,111 Similar success has been achieved using these ubiquitin plasmids in tumor models.89,95 However, the rapid degradation comes at a cost; antibody responses to the destabilized proteins are much reduced, presumably because there is insufficient intact protein to induce a biologicallysignificant antibody response.

Direct Delivery of the Encoded Materials to the ER

Ubiquitin indirectly increases peptide delivery to the ER, but other labs have taken a more direct approach, by attaching proteins, or isolated epitope minigenes, to signal sequences; these should ensure that nascent proteins, synthesized on the rough ER, will be translocated into the ER. For example, a DNA vaccine contain an HIV epitope minigene was markedly enhanced by the addition of the E3 leader sequence from adenovirus,140 and the immunogenicity of an SV40 CD8 T cell epitope expressed in a recombinant vaccinia virus was greatly increased using a similar approach.141

Do Protein Transduction Domains Enhance DNA Vaccination?

Sequences termed protein transduction domains (PTD), which can translocate attached materials across biological membranes, have been found in proteins encoded by viruses, bacteria, insects, and other organisms (reviewed in ref. 142). For example, a short highly basic region (YGRKKRRQRRR) allows the HIV1 nuclear transactivation protein (tat) to cross the cell membrane in a receptordependent manner,143,146 and importantly, for our purposes, tat or PTDlinked proteins are degraded inside the transduced cells, and their epitopes are presented by MHC class I molecules in a TAPdependent manner.147,148 We149 and others150 have attempted to exploit this attribute of PTD to improve antigen delivery following DNA immunization. The results are conflicting. Hung et al, using a herpesvirus PTD attached to GFP, concluded that intercellular spread of biologicallyactive protein was occurring.150 However, this finding is difficult to reconcile with other data which suggested that successful in vivo transduction of fulllength proteins required that the protein be denatured prior to transfer;144,145,151 and in our study we specifically sought, but were unable to detect, evidence of extensive intercellular protein transfer following transfection.149 However, in both studies, addition of the PTD resulted in much more effective stimulation of epitopespecific CD8 T cells, and enhanced in vivo protective efficacy of the DNA vaccine. Attachment of Pseudomonas aeruginosa exotoxin A to the HPV E7 protein led to a ˜30fold increase in the number of CD8 T cells primed by a DNA vaccine, with a parallel increase in biological efficacy.152

Primeboost Regimens

A number of recent studies have indicated that DNA vaccines may be valuable when used as part of a primeboost strategy (reviewed, ref. 153). For example, studies using a recombinant poxvirus delivery system showed that CTL could be induced, but that the effect of boosting was diminished by responses to the vector itself. However, if instead the vaccinee received a DNA injection, followed some time later with a recombinant poxvirus expressing the same antigens, a muchimproved outcome was noted; this was true for both HIV and malaria antigens.154,155 Studies with hepatitis C virus antigens showed that the CD8 T cell responses which followed recombinant poxvirus boosting were not only higher, but also were more diverse, than those seen following DNA vaccine priming;156 and the most effective CD8 T cell response against the papillomavirus E7 protein was induced by DNA prime / vaccinia boost.157 The approach also works with “string of beads” constructs, which were first conceived in 1993,22 and encode isolated epitopes. Also termed “polyepitope” or “polytope” vaccines,158,159 these vaccines induce responses when administered as plasmid DNA, and the responses are enhanced by subsequent poxviral boosting.160 The results are not always quite so clearcut, however; in one instance, poxvirus boosting after DNA priming led to increased cellular immunity, but decreased levels of antibody.161 Effective boosting of DNAprimed responses is not limited to recombinant poxviruses. DNA prime / protein boost strategies have met with some success.162 Boosting with additional doses of DNA also may be beneficial;163 we have found that, after 2 boosts, we can drive epitopespecific CD8 T cells to remarkably high levels (˜20% of a mouse's total CD8 T cells; Hassett & Whitton, unpublished).

Coadministration of Immunostimulatory Molecules

Many studies have employed immunostimulatory molecules to skew the immune responses (for example, towards Th1 or Th2 phenotypes) or to otherwise modulate them. Since a chapter in this book focuses on this topic, here we limit ourselves to presenting a table in which the effects on CD8 T cell responses are summarized. As can be seen, in several cases (e.g., GMCSF) the effect of an individual immunostimulatory molecule varies from study to study. In regard to CpG motifs, several studies (not cited in Table 1) have indicated that these sequences, if present in the administered DNA, provide a “builtin” adjuvant effect. One study has evaluated the effect of coadministering CpG oligonucleotides with a DNA vaccine, and the authors found no effect; however, that study also concluded that DNA vaccines did not induce longterm antiviral immunity, a result which differs very much from the conclusions reached by most laboratories.

Table 1. Modulating CD8 T cell responses by coadministration of immunostimulatory molecules.

Table 1

Modulating CD8 T cell responses by coadministration of immunostimulatory molecules.

Summary

Antigens encoded by DNA vaccines can induce all arms of the adaptive immune response, but to date they have proven most effective at inducing antigenspecific CD8 T cells. The great majority of experiments have been carried out in small animal models, where these vaccines work quite well; in a limited number of studies in primates (including humans), their effectiveness, although demonstrable, is somewhat diminished. Therefore, to accelerate the introduction of DNA vaccines into clinical and veterinary practice, it is important that their immunogenicity be enhanced. The rational modification of DNA vaccines requires that we have a basic understanding of the mechanisms which underpin successful DNA immunization. In this chapter, we review how DNA vaccines may work, and how this information permits us to exploit biological pathways to improve the outcome of genetic immunization. In addition to reviewing “rational” vaccine modification (based on, e.g., targeting antigens to specific antigen presentation pathways; or coadministering cytokines to modulate the vaccineinduced response) we also consider “empirical” approaches, such as using different primeboost regimens whichby a mechanism as yet unclearappear to greatly enhance antigenspecific memory in the vaccinee. Empirical studies proved the efficacy of essentially all vaccines in current use, and this old approach may once again prove useful in launching a new technology into the clinical arena.

Acknowledgements

We are grateful to Annette Lord for excellent secretarial support. This work was supported by NIH R01 award AI37186 (to JLW), and by grant number D/99/22463 from the Deutsche Krebshilfe e.V (to JAL). This is manuscript number 14145NP from the Scripps Research Institute.

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