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(Journal of Leukocyte Biology. 2000;68:793-806.)
© 2000 by Society for Leukocyte Biology

DNA vaccination: antigen presentation and the induction of immunity

Devon J. Shedlock and David B. Weiner

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Correspondence: David B. Weiner, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 505 Stellar-Chance Lab., 422 Curie Dr., Philadelphia, PA 19104. E-mail: dbweiner{at}mail.med.upenn.edu


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ABSTRACT
 
DNA vaccination, or genetic immunization, is a novel vaccine technology that has great potential for reducing infectious disease and cancer-induced morbidity and mortality worldwide. Since their inception, DNA vaccines have been used to stimulate protective immunity against many infectious pathogens, malignancies, and autoimmune disorders in animal models. Plasmid DNA encoding a polypeptide protein antigen is introduced into a host where it enters host cells and serves as an epigenetic template for the high-efficiency translation of its antigen. An immune response, which is mediated by the cellular and/or humoral arms of the immune system and is specific for the plasmid-encoded antigen, ensues. It is thought that "professional" antigen-presenting cells play a dominant role in the induction of immunity by presenting vaccine peptides on MHC class I molecules, following direct transfection or "cross"-presentation, and MHC class II molecules after antigen capture and processing within the endocytic pathway. The correlates of immunity can be manipulated according to many immunization parameters, including the method of vaccine delivery, presence of genetic adjuvants, and vaccine regimen. DNA vaccines first advanced to the clinic five years ago, and the initial picture of their utility in humans is emerging. However, further analysis is required to determine their ultimate efficacy and safety in human beings. This technology has acquired a strong foothold in the field of experimental immunotherapy, and it is hoped that it will eventually represent the next generation of prophylactic and therapeutic vaccines.

Key Words: DNA vaccines • genetic immunization • genetic vaccination • immunotherapy • DNA plasmid • cross-presentation • cross-priming • genetic adjuvant • prime-boost


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INTRODUCTION
 
Genetic immunization is a novel vaccine strategy that conceptually combines some of the most desirable attributes of standard vaccine approaches. Although traditional live-attenuated or killed vaccines have proven their effectiveness in the eradication of many microbial infections, today’s safety requirements and resilient pathogens require vaccine modalities of significant complexity that will overcome current technological inadequacies. Strong humoral immune responses alone, induced by some conventional vaccines, do not provide prophylaxis against many current intruders including Herpes simplex virus (HSV), malaria, and the human immunodeficiency virus (HIV), for which the induction of a strong cellular immune response is likely required. The fulfillment of this demand is under intensive investigation currently in our laboratory and many others throughout the world using DNA vaccine technology.

Since their inception in the early 1950s [1 ], a period of about 40 years elapsed before Will et al. [2 ], Dubensky et al. [3 ], and Wolff et al. [4 ] demonstrated that the administration of recombinant DNA into an animal resulted in the expression of the protein encoded by that plasmid. Not soon after, it was shown that the expression of foreign protein from delivered DNA elicited a humoral immune reaction that was specific for the encoded antigen, by Tang et al. [5 ] and then simultaneously by Ulmer et al. [6 ] and Fynan et al. [7 ] that immunization with a DNA plasmid could protect mice against a lethal influenza challenge. Moreover, Wang et al. [8 ] showed that a plasmid vaccine could induce protective immune responses against HIV-1 antigen-expressing targets. Altogether, the implications of these findings served to establish genetic immunization as an approach to induce an immune reaction against infectious agents. Since that time, over 1000 manuscripts have been published on the ability of DNA vaccines to induce strong immune responses against proteins from infectious agents such as malaria [9 10 11 ], tuberculosis (TB) [12 13 14 ], rabies virus [15 ], hepatitis B virus (HBV) [16 , 17 ], HSV [18 ], Ebola virus [19 ], and HIV [20 21 22 23 ].

The strategy of most of these investigations is relatively simple: A DNA plasmid encoding a desired protein is injected into the muscle or skin of an animal, where it thereupon enters host cells and directs the synthesis of its polypeptide antigen. Once the plasmid-antigen is processed and presented by transfected host cells, a cellular and humoral immune response against the antigen is provoked. The plasmid’s immunogenicity may be enhanced in part by the presence of repeated immunostimulatory motifs that are recognized by the immune system as foreign. The DNA vector is bacterial-derived and equipped with eukaryotic or viral promoter/enhancer transcription elements that direct the high-efficiency transcription of the plasmid-antigen within the nucleus of the host cell.

Genetic immunization exhibits many advantages over traditional vaccines that use live-attenuated or killed pathogen, proteins, or synthetic peptides, which are listed in Table 1 . Humoral and cellular-immune responses can be achieved in animal models at extremely low dosages of DNA vaccine. Unlike immunization with proteins, the intracellular synthesis of plasmid protein results in antigen likely to be folded in its native conformation, correctly glycosylated, and normal post-translational modifications to occur similar to natural infection, favoring the production of relevant neutralizing antibodies. In addition, they are safer conceptually than live vaccines because of the inability to revert into virulence, and they do not require the use of toxic chemical inactivation methods. Current techniques in molecular biology enable the easy manipulation of plasmid vectors, which are able to accommodate virtually any gene or its derivatives. At relatively low costs, these recombinant plasmids can be produced at large scale in bacteria and isolated simply using commercially available reagents. DNA vaccines are also considered more temperature-stable than conventional vaccines, boasting a longer shelf-life. This is to be of great significance likely, because it would impact the requirement of a cold chain, a costly and difficult issue, and thereby enhance vaccine storage and mobility.


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  		Table 1. Commendable Qualities of DNA Vaccines

DNA vaccine safety
The risks associated with DNA plasmid inoculation are being assessed currently in many animal models and Phase I clinical trials. The suspicions that plasmid DNA may cause tumorgenesis, integrate into the host chromosome [24 ], or induce anti-DNA autoimmune responses in the host [25 , 26 ] raise warranted concern, yet little evidence has substantiated the occurrence of these phenomenon, particularly in humans or primate experimental models. Mutation rates occurring from the integration of plasmid DNA into the host chromosome have been calculated in animal studies and found to be much lower than the spontaneous mutation rate for mammalian genomes [27 , 28 ]. A study conducted in fish has confirmed also that the administration of DNA plasmids can elicit immunity effectively without the initiation of nucleic-acid autoimmunity or host-chromosome integration [29 ].

Our laboratory has described the safe and well-tolerated administration of HIV-1 DNA plasmid constructs in adult, pregnant, and infant chimpanzees, with the induction of humoral and cellular immunity [30 ]. We have also carried out the first human trial of a therapeutic DNA vaccine for HIV-1 infection, which generated encouraging results [20 , 31 , 32 ]. Vaccine administration induced no local or systemic reactions, no anti-DNA antibody, nor muscle-enzyme elevations, and increases were noted in cytotoxic T lymphocyte activity against HIV surface antigen-bearing targets [20 , 31 , 32 ]. These findings suggest that the inoculation of plasmid DNA into animals and humans is considerably safe and an effective means of generating immune responses against plasmid-encoded antigen. Recently, another clinical study has demonstrated that the intramuscular (i.m.) administration of a malaria DNA vaccine of up to three doses of 2500 µg plasmid DNA was well-tolerated also, thereby expanding the safety limits of genetic vaccine dosages in humans [33 ].

DNA vaccination in alternative immunotherapies
Another facet of DNA vaccine technology focuses on immune-related diseases, such as autoimmunity and cancer [34 ]. By manipulating the balance of T helper (Th) 1 and 2 lymphocytes using DNA plasmid immunization, many of the pathogenic qualities of autoimmune disease may be potentially addressed. Protective immunity against an experimental autoimmune encephalomyelitis (EAE) model has been induced by using a DNA vaccination method that favors the induction of a Th2-type response [35 ]. Conversely, suppression of a Th2 response by the induction of a Th1-type response against allergens associated in an immunoglobulin (Ig)E antibody-mediated allergic response has been shown to neutralize the dysregulated production of Th2 cytokines and diminish allergic reactions [36 37 38 39 ]. These findings demonstrate the functional utility of DNA vaccines in the realm of autoimmune therapy.

Additionally, DNA immunization has proven an effective candidate in the fight against certain cancers. The difficulty involved with antitumor vaccination is that immunity must be activated to already present tumor cells in the setting of minimal residual disease [40 ]. This has been accomplished by breaking the tolerance to tumor-specific self-antigens and activating a self-directed immune response [41 ]. The growth of human tumor cells that produce and secrete a target protein has been retarded or inhibited by a DNA vaccine construct encoding a subunit of that target protein [42 ]. Although breaking the tolerance to self-antigens may provide some form of protection against tumor growth, one study raises concerns about the possibility of adverse-coupled autoimmune reactions [43 ]. For instance, immunization with homologous DNA in mice broke tolerance to a well-characterized melanoma differentiation antigen and additionally accompanying an unwanted autoimmune manifestation of coat depigmentation [43 ]. Clearly, for human studies, a thorough discussion of safety as well as the potential risk-benefit of any immunization target must be considered. Nevertheless, the results of many tumor, allergy, and autoimmunity investigations are encouraging and offer hope in the alleviation of these morbid conditions.


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DNA VACCINE DELIVERY
 
The most popular method of administering DNA vaccines has been parenterally, which includes needle injection into muscle or skin and gas-powered, DNA-covered particle bombardment using a "gene-gun" (g.g.). Although these forms of delivery require either a needle or ballistic device to mechanically force plasmid through or into the skin, noninvasive routes of delivery have been demonstrated that entail the topical application of pure DNA plasmid to skin or mucosae. Each one of these methods of delivery introduces vaccine to distinct areas of immune surveillance and therefore primes the immune system in distinct ways.

The use of a needle to inject an aqueous solution of DNA plasmid into tissue is a relatively simple and effective way of vaccine administration, resulting in the direct transfection of some cells and the uptake by others in the vicinity of the inserted needle. Injection intradermally (i.d.) or into the skin results in the transfection of mainly skin fibroblasts and keratinocytes, whereas i.m. injection transfects largely myocytes. In g.g.-mediated delivery, gold particles covered with plasmid DNA are propelled by helium or CO2 pressure into tissue [44 ]. This method of delivery is very effective at driving plasmid into the cells of the epidermis and requires far less DNA than needle injection.

Noninvasive methods of plasmid delivery involve the topical application of plasmid to the skin or mucosae. The induction of antigen-specific immune responses has been shown following the application of a plasmid solution to various mucosal surfaces including intranasal [45 46 47 ], oral [48 ], and intravaginal [30 , 49 ]. It has been shown also that the topical application of DNA plasmid directly to the skin transfects the superficial layers of the epidermis surrounding hair follicles, generates reporter-gene activity at levels comparable to that of i.d. injection [50 ], is dependent on the presence of normal hair follicles, and induces antigen-specific immune responses that display Th2 features [51 ]. Unpublished findings from our laboratory suggest that the uptake of plasmid DNA through hair follicles is asymmetrical. This technique of delivery may be ideal for targeting genes to the skin for the treatment of cutaneous disorders or in the context of wound healing.

The correlates of immunity resulting from each of these methods of delivery are determined usually by the mode and site of plasmid administration. Forms of delivery targeting the skin, including i.d. injection, g.g. bombardment, and topical application, have been shown to elicit a humoral response primarily, characterized by a rapid progression to a Th2-type response, associated with the production of an IgA and IgG1 antibody isotype [51 52 53 ]. Conversely, injection into muscle results in the induction of a strong cellular-mediated response, or Th1 type, that primes antigen-specific cytolytic T lymphocytes (CTLs) and is associated with the production of IgG2a antibody [54 ]. Although each of these methods elicits systemic weak or strong cellular and humoral immune responses, they may not induce mucosal immunity, such as inoculation at the mucosal surface [55 ]. This may be important in preventing the entry of a pathogen into the host, as opposed to after it has infiltrated the body [56 ]. The extent of protection elicited by these various modes of vaccine administration is determined most likely by the network of antigen-presenting cells (APCs) residing in the target tissue and the quantity of DNA plasmids administered (Fig. 1 ) [57 ]. For example, APCs are more prevalent in the skin than in muscle, so less plasmid DNA may be required to induce a response of similar magnitude. However, the quality of the immune responses suggests that the APCs transfected in these different locations are functionally distinct and therefore prime the immune response uniquely. These particular features suggest further evaluation of each compartment could be important for future vaccine design.



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  		Figure 1. DNA vaccination results in DC maturation and migration. Immunostimulatory motifs present in plasmid DNA and microinjury caused by parenteral delivery stimulate the maturation and migration of antigen-loaded DCs. Langerhans cells, networked extensively in the dermis, and interstitial DCs, residing at low numbers within muscle, encounter vaccine and then travel to lymph nodes via afferent lymph, where they interact with T cells.


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MECHANISM OF IMMUNE INDUCTION
 
DNA vaccines elicit strong and long-lasting humoral and cell-mediated immune responses in many animal models. Although there has been much speculation regarding the mechanisms underlying DNA vaccine function, the mechanisms are complex and have yet to be fully elucidated. Progressively dissecting the cellular and immunological processes of genetic immunization that are responsible for the induction of immune responses will lead ultimately to further advances in this technology. At the cellular level, the efficacy of DNA vaccination depends on the interaction between their polypeptide products and the two major groups of cells that mediate immunity: lymphocytes and APCs.

The intracellular transcription and translation of plasmid DNA are thought to mimic the replication of a virus during infection. Both systems must traverse the plasma membrane initially and require the cellular machinery to translate their encoded proteins. In transfected nonhaematopoietic cells, intracellularly synthesized plasmid product is processed effectively via the transporters associated with antigen processing (TAP)-dependent, endogenous-processing pathway. In addition, soluble or secreted vaccine antigen may be phagocytosed by APCs and gain entry into the major histocompatibility complex (MHC) class II exogenous pathway. So, like the viral proteins produced by a replicating virus, plasmid product may gain access to both pathways simultaneously, affecting its presentability to the immune system (Fig. 2 ).



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  		Figure 2. DNA vaccination mimics viral infection. Both systems must traverse the plasma membrane and require the cellular machinery to translate their encoded proteins. Viral and plasmid-encoded proteins are processed via the TAP-dependent intracellular pathway and presented in association with MHC class I molecules. These polypeptides can gain access to the exogenous processing pathway in phagocytic cells if they are soluble. Although killed protein vaccination results in only a humoral response, DNA vaccination induces a strong humoral and cellular immune response against the plasmid-encoded antigen.

Because T cell-mediated responses are normally dependent on the recognition of antigen-associated molecules on APCs, how then is it possible for transfected somatic cells to stimulate protective CTL responses? This appears counterintuitive, because somatic cells lack the proper costimulatory signals in conjunction with MHC class I molecules that are required for the expansion of naïve antigen-specific CD8+ T cells. The presence of lymphokines and proinflammatory factors produced by activated CD4+ T cells is another requirement for the activation of CTLs also. Insofar, what remains to be the functional role of the plasmid-harboring somatic cell? It is well established that these cells become low-level, antigen-producing "factories" for long periods post-transfection. But, one study demonstrates that surgical ablation of a muscle bundle at the site of plasmid injection 10 min after administration did not affect the magnitude or longevity of an Ab response [58 ]. These findings imply the expression of plasmid-antigen by somatic cells does not lead solely to the induction of immunity; other cells interspersed in recipient tissue may be of greater significance in the generation of a CTL response.

Roles of dendritic cells (DCs) in immune induction
The popularity of APCs as the key inducers of immunity in genetic immunization is expanding, distinguishing them as the immunological bridge between somatic cells and T cells by the trafficking of antigen between the site of delivery to secondary lymphoid organs. Macrophages and DCs are "professional" APCs derived from bone marrow and have the ability of present antigen to naïve T cells within secondary lymphoid tissues. DC have been identified in virtually all the lymphoid and connective tissues and are characterized uniquely by the expression of the molecules DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN) [59 ] and a subfamily of DC-associated C-type lectins (dectin-1 and –2) [60 ], which have all been discovered recently.

Delivery of DNA plasmid i.d. and g.g. targets the skin, which harbors two populations of DC: epidermal Langerhans cells (LC) and interstitial DC [61 ]. Conversely, immunization by i.m. injection targets muscle, which has fewer DCs and lacks LCs altogether. Within the periphery, immature DCs exist in a highly phagocytic state and are characterized by the low-level expression of MHC class I, MHC class II, and costimulatory molecules, rendering them poor initiators of immune responses. These DCs are stimulated vigorously by proinflammatory cytokines, bacterial proteins, and certain viruses and are known to migrate via afferent lymph to draining lymph nodes where they can efficiently activate antigen-specific naïve T cells, which are otherwise strictly dependent on CD4+ T cell help [62 63 64 ]. During this migratory process, DCs undergo maturation, whereby all of the previously mentioned molecules are up-regulated and DC numbers in the regional lymph nodes consequently increase because of influx or expansion from precursors [65 ]. During this period of activation and presentation, communication with CD4+ cells induces the ability of the DCs to activate naïve CD8+ cells via CD40 signaling [62 63 64 ] and also induces the establishment of memory CD4+ T cells that are capable of long-life existence without repeated antigenic stimulation. The significance of resident DCs in genetic immunization has been demonstrated in transplantation studies that show the induction of a CTL response is restricted to the MHC haplotype of bone marrow-derived APCs and not to the haplotype of transfected somatic cells, following i.m. [66 ] and g.g. [67 ] administration of plasmid DNA.

DC maturation can be induced also by the method of plasmid delivery and by the immunostimulatory qualities of plasmid DNA. The act of g.g. bombardment and needle injection during the administration of DNA plasmids are forms of physical microinjury that result in local irritation, which has been shown to stimulate the recruitment of transfected and nontransfected LCs from skin to draining lymph nodes [65 ]. In this way, the physical stress associated with invasive DNA delivery acts as a type of immunological adjuvant.

In some animal model systems, the DNA plasmids themselves have been shown to possess adjuvant qualities that are determined by the presence of immunostimulatory sequences within the DNA vector backbone. Repeated immunostimulatory unmethylated CpG motifs act to initiate the innate immune response, generating the familiar cytokine mediators interferon (IFN)-{alpha}, IFN-ß, interleukin (IL)-12, and IL-18 from macrophages and monocytes, and IL-18 and IFN-{gamma} from natural killer (NK) cells, thereby promoting the differentiation of naïve T cells to Th1 cells [68 69 70 ]. They have been associated also with the maturation of DCs, followed by the production of IL-12 and the elevated expression of CD86, CD40, and MHC class II molecules [71 ].

DCs are extremely potent activators of naïve T cells and are thought to play at least three distinct roles in priming the immune system to vaccine antigen: (1) MHC class II-restricted presentation of antigen captured from transfected cells, (2) MHC class I-restricted presentation by directly transfected DCs, and (3) MHC class I-restricted "cross"-presentation of vaccine antigen, illustrated in Figure 3 [57 ]. Although each mechanism is significant in stimulating at least one facet of immunity, it remains debatable as to which one plays the dominant role in the induction of protection. It is most likely that each is important in genetic vaccination and that one is not exclusively responsible for evoking potent humoral and cell-mediated responses.



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  		Figure 3. Roles of APCs/DCs in the induction of immunity against plasmid-encoded antigen. DCs can display vaccine antigen in at least three ways: (1) MHC class I- and II-restricted presentation following direct transfection, (2) MHC class II-restricted presentation of secreted antigen processed in the exogenous pathway, and (3) MHC class-I restricted cross-presentation of antigen acquired from transfected apoptotic cells. Immunostimulatory qualities of plasmid vaccine stimulate DC maturation and migration to lymph nodes, where they can interact with T cells.

The foremost-mentioned function of DCs in inducing immunity relies on their phagocytic ability to capture secreted forms of the vaccine antigen expressed by plasmid-transfected cells. These are processed in the exogenous pathway and loaded onto MHC class II molecules. When these DCs receive the proper maturation signal, they up-regulate costimulatory molecules and then migrate to and communicate with antigen-specific helper T cells, which are then induced to secrete Th2 cytokines. Usually, antibody responses occur only when antigen is secreted from cells. This chain of events implies that the transfected cells have a functional role of secreting antigen, serving as antigen "factories". Although antigen secretion may help to augment the development of a strong humoral response, it has not been shown to induce CTLs and, therefore, cannot act independently of the other two roles of DCs in the induction of immunity proposed in this review. In fact, the use of a nonsecreting antigen model has demonstrated the failure of transfected somatic cells to activate fresh naïve transgenic T cells injected three weeks post-administration, supporting the conclusion that long-term expression of antigenic material is not the dominant mechanism of immune induction [72 ].

Direct transfection of DC
How then is antigen associated with DC MHC I molecules? The second proposed mechanism of induction requires the direct transfection of resident DCs within the tissue at the site of administration. Many lines of supporting evidence for this mechanism have been provided by several investigators. Plasmid DNA has been isolated directly from DCs originating within the local lymph nodes and skin following i.m. and i.d. administration [73 ]. Green fluorescent protein (GFP)-expressing LCs were isolated in draining lymph nodes after g.g. administration of plasmid-encoding GFP protein [74 ], supporting the idea that skin LCs are transfected directly. For example, M. Chattergoon et al. [75 ] have shown that macrophages and DCs can be directly transfected following i.m. injection, are highly activated, migrate to the regional lymph nodes, and can be found in the peripheral blood. DCs isolated from skin at the site of plasmid administration following g.g. bombardment were shown to be sufficient in the presentation of antigen to T cells [65 , 73 ]. Collectively, these observations imply that a small number of directly transfected, tissue-resident professional APCs first express, process, and present the vaccine antigen and then migrate to draining lymph nodes where they interact with naïve T cells. When CD4+ T cells are activated in this manner, they have been shown to migrate to the spleen, where memory T cells have been shown to persist for up to a year in the absence of a source of persistent antigen [72 ]. It is interesting that extremely small numbers of transfected mature DCs—as little as 500 DCs transfected in vitro and then injected—are capable of producing humoral and cell-mediated immune responses [76 ]. It has been estimated that according to the densities of LCs within the skin and the area to which g.g. bombardment delivers DNA plasmid, <1% of total area LCs would have to be transfected directly to be sufficient in eliciting an immune response in the proper stimulatory environment [57 ].

Cross-presentation of vaccine antigen
The final proposed mechanism of DC immune induction involves the "cross"-presentation of exogenous vaccine antigen to T cells in a MHC class I-restricted fashion and is illustrated in Figure 4 . It was known previously that DCs acquire antigens from dying monocytes, which are processed in the MHC class I cascade and presented to naïve virus-specific CD8+ T cells, during viral infection [77 , 78 ]. In fact, the first observation, made more than 20 years ago, was that exogenous antigen could gain entry into the MHC class I-restricted pathway under certain conditions [79 ]. The effective cross-priming of naïve T cells to exogenous antigen requires also the active involvement of CD4+ helper T cells [80 ]. Such a mechanism is theorized to be important in the induction of protection against a tissue-tropic virus that does not infect APCs, thereby ensuring presentation of viral antigen to antigen-specific T cells.



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  		Figure 4. Cross-presentation of plasmid-encoded antigen by DCs. During high levels of cell death, such as viral infection or tissue damage, apoptotic-transfected somatic cells are cleared by activated DCs. Plasmid-encoded antigen can then enter into the TAP-dependent, intracellular, antigen-processing pathway and be presented in an MHC class I-restricted manner. Following migration to lymph nodes, cross-presenting DCs can expand (cross-prime) or delete (cross-tolerate) antigen-specific naïve CD8+ T cells in the periphery, depending on the presence of "danger" signals. Such stimuli may affect the outcome of cross-presentation by evoking CD4+ T cell help or by rendering CD8+ T cells sensitive to surface receptor-mediated death.

Evidence for this hypothesis has been provided by many studies focusing on this phenomenon. The transplantation of stably transfected myoblasts into chimeric mice was able to induce a protective antibody and CTL response that was restricted to the MHC haplotype of the bone morrow-derived APCs, not unlike the immune responses generated by plasmid immunization alone [81 , 82 ]. Also, plasmid-transfected tumor cells were used to demonstrate the same result [83 ]. Altogether, it appears that exogenous antigens are captured and processed by APCs first and then delivered to sites of naïve T cell circulation, bound as peptides to MHC class I molecules.

In what forms are vaccine antigens acquired by DCs, and what signals are then responsible for DC maturation? It has been shown that when massive apoptosis is triggered in the presence of "danger" signals, such as viral infection, DCs and not macrophages can present antigens derived from apoptotic cells to CD8+ T cells. However, apoptotic cells themselves, in the absence of "danger" signals, have been shown to stimulate DC maturation sufficiently and generate similar results [84 ]. During the normal apoptosis of single cells in living tissues, DCs are not required usually, because scavenging macrophages clear apoptotic mass efficiently while secreting DC maturational-inhibitory factors, such as IL-10 [61 ]. But, when the level of apoptosis exceeds the clearing ability of macrophages, DCs release their own maturative factors, such as IL-1ß and tumor necrosis factor {alpha} (TNF-{alpha}), and stimulate their participation in clearance and immune induction [84 ]. Another study revealed that the engulfment of even low numbers of apoptotic cells that were opsinized by specific antibodies triggered the secretion of DC-maturative factors and promoted migration to lymph nodes [85 ]. Successful cross-priming of antigens acquired in this manner requires a "danger" signal most likely or a proinflammatory environment, such as those produced in events of excessive cellular apoptosis and opsinization. However, the cross-presentation of antigens was not observed after cell necrosis, potentially implying the importance of the ordered processes associated with apoptosis, possible reorganization, and changes in the plasma membrane [84 ]. In this regard, it is important to consider that there is little evidence of substantial necrosis from the live oral polio vaccine, yet it induces a strong cellular response, suggesting that human viral infections driving apoptosis can be immunologically important.

Immunity or tolerance?
The mechanism of cross-presentation is implicated in both the occurrences of cross-priming and cross-tolerance [78 ]. If the function of cross-presentation were directed only toward the induction of immunity, then the cross-presentation of self-antigens would result in autoimmunity. Fortunately, it has been shown that the cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8+ T cells [86 ]. In this study, adoptively transferred ovalbumin (OVA)-specific CD8+ T cells were activated by cross-presented tissue antigens in transgenic mice expressing a membrane-bound form of OVA, leading to proliferation and then deletion of these cells [86 ]. Normally, peripheral self-antigens may be ignored because of their denied entry into the cross-presentation pathway. Whether or not cross-presentation of antigens results in immunity or cross-tolerance may depend on their association with cells actively producing large amounts of viral antigens, which may stimulate the appropriate CD4+ T cell help [87 ]. Cross-tolerance may be a mechanism functioning to delete extra-thymic autoreactive CD8+ T cells. Although it is possible that ignorant autoreactive CD8+ T cells could accumulate and increase the potential of eliciting autoimmunity, cross-tolerance may be a mechanism that normally helps to keep these cells at lower levels [86 ].

In the cross-presentation of exogenous antigen by DCs to naïve T cells, what determines the course of immunity or tolerance? It appears as if the environment in which the DC captured the antigen may determine how that antigen is cross-presented to specific T cells, leading them to expansion or deletion. One study demonstrated that cross-presentation by DCs of exogenous antigen led to the up-regulation of the fas molecule, activation-induced T cell-death mediator, or CD95 on the CD8+ T cells that were specific for that antigen. As a result, these T cells were rendered more receptive to CD95-induced death, which may depend on the presence of inflammatory signals associated with infection [88 ]. TNF-related, apoptosis-inducing ligand (TRAIL)-mediated apoptosis is another signaling pathway that may be responsible for the stimulation or deletion of reactive CD8+ T cells [89 ]. It was found that the production of IFN stimulated DCs to express TRAIL, granting them the ability to kill TRAIL-sensitive cells [89 ]. So, CD8+ T cells may be sensitive to TRAIL-mediated killing, depending on the presence of "danger" or signals, which would render them TRAIL-resistant.


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ENGINEERING IMMUNE RESPONSES
 
Vaccines that elicit prophylactic immune responses are specifically constructed and administered to provide optimal protection at the sites most frequently encountering pathogens. For example, effective mucosal immunity is desired when protecting against infectious agents transmitted by aerosols, such as TB. Ideally, vaccine regimens must be tailored to neutralize pathogens before the onset of infection and disease. Because experiments in primates suggest that DNA vaccines alone may not be as immunogenic in these species as they are in rodents [11 , 90 ], their coadministration with genetic and chemical adjuvants may bolster their immunogenicity and efficacy. In addition, the use of particular adjuvants can help direct the magnitude and direction of prophylactic- and therapeutic-immune correlates that target bugs at pivotal stations within the pathogen/host interaction.

Many strategies involving the combination of DNA immunization and adjuvants are currently under investigation. Specifically, vaccine immunogenicity can be modulated by factors that attract professional APCs, provide additional costimulation, or heighten the uptake of plasmid DNA. In these ways, the direction of an immune response can be guided toward a cell-mediated, Th1-type response or an antibody-mediated, Th2-type response, driven by the differential expression of cytokine patterns by their distinctive T cell subsets [91 ].

Cytokine-encoding plasmids
Cytokines are molecules secreted by bone marrow-derived cells that regulate the intensity and duration of the immune response in lymphocytes and other immune cells expressing a particular cytokine receptor. In 1993, Raz et al. [92 ] inoculated a group of mice with several DNA plasmids encoding cytokines in an effort to improve the approaches of somatic gene therapy involving the direct administration of cytokines. Expression of these plasmids was observed to induce systemic immunological effects characteristic of the specific functions of the respective cytokine proteins and also could enhance the immune response to an exogenous antigen that was delivered at a different site.

The coadministration of DNA vaccines with cytokine-encoding adjuvants can manipulate the differentiation and expansion of Th1 and Th2 cytokine producers effectively. For example, protection from certain viruses or tumors would require the production of Th1-inducing cytokines, such as IL-2, IL-12, IL-15, IL-18, and IFN-{gamma}, which promote cell-mediated immune responses. We have shown that the plasmid codelivery of IL-12 with DNA immunogens can drive the immune responses toward a Th1 phenotype [93 ] and increase the survival rate of mice, following a lethal dose challenge in an HSV-2 model [94 , 95 ]. We have also shown that the codelivery of IL-12, IL-18, and IFN-{gamma} genes, along with HIV-1 vaccine constructs, enhanced the level of antigen-specific Th-proliferative responses in mice and rhesus macaques [96 ]. Furthermore, the chemokine RANTES (regulated on activation, normal T expressed and secreted) enhanced the levels of antigen-specific Th1 and CTL responses [97 ].

Conversely, protection from antibody-mediated pathologies may benefit from the use of Th2-inducing cytokines such as IL-4, IL-5, and IL-10 to drive humoral immunity. Sin and co-workers [98 ] demonstrated that increased levels of antigen-specific antibodies were associated with codelivery of IL-4, IL-10, and the chemokine macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}) [97 ] with an HIV-1 construct. In addition, we have tested the effects of the proinflammatory cytokines IL-1{alpha}, TNF-{alpha}, and TNF-ß and found that TNF-{alpha} enhances dramatically antigen-specific Th-cell proliferation and CTL responses [99 ]. In a tumor challenge model, the coadministration of cytokine-gene adjuvants IL-2, IL-6, IL-7, or IL-12 along with a plasmid encoding the model tumor-associated antigen were shown to reduce significantly the number of established metastases [100 ]. The administration of cytokine-encoding DNA has been shown also to augment antitumor immunity in tumor-bearing mice, identified by tumor eradication and increased mouse survival [101 ]. Recently, codelivery of a plasmid expressing an IL-2/Ig fusion protein was shown to augment HIV-1 and simian immunodeficiency virus (SIV) DNA vaccine-elicited antibody- and cellular-mediated immune responses in rhesus monkeys [102 , 103 ]. Collectively, these findings suggest that the plasmid expression of cytokines involved in the activation and proliferation of lymphocyte populations may improve the efficacy of DNA vaccines.

Another method of enhancing the immune response using genetic cytokine adjuvants is the expansion of the professional APC pool, particularly DCs and macrophages, at the site of inoculation. The expression of the haematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) and a DNA vaccine have been shown to boost the activity of B- and T-helper cells toward rabies glycoprotein and improves the protective response against a lethal challenge [104 ]. This boosting effect of plasmid-expressed GM-CSF on immune responses against vaccine antigen has been confirmed by the coinoculation of this gene adjuvant with vaccine-encoding HIV-1 env protein [105 ], encephalomyocarditis virus [106 ], and hepatitis C virus [107 ]. It is most likely that the expression of this growth factor attracts APCs and stimulates their maturation.

Although the coadministration of genetic cytokine adjuvants have shown much promise in enhancing immune responses, the issue of safety should still be taken into account toward their implementation in clinical trials. The administration of protein cytokines by themselves can spur side effects associated with increased systemic levels for long periods of time. Although most of the codelivery studies have been performed in rodents, recent studies have moved into nonhuman primates to evaluate the safety and efficacy of codelivering cytokine gene adjuvants with DNA vaccines for prophylaxis and immune therapy [96 , 102 ]. The initial results are encouraging and support the importance of this approach for human studies. However, each application should be reviewed for specific safety issues relevant to the cytokine adjuvant.

Costimulatory molecule-encoding plasmids
The codelivery of plasmids encoding costimulatory molecules is a method theorized to improve the antigen-presenting capabilities of transfected host cells. Molecules such as B7.1 (CD80), B7.2 (CD86), and CD40 are expressed by APCs and serve as a "second signal" next to antigen-loaded MHC class I molecules necessary for the assembly of the T cell receptor and the subsequent activation and expansion of antigen-specific T cells. We have shown that the codelivery of a B7.2-encoding plasmid with an HIV-1 DNA construct enhanced antigen-specific Th cell proliferation and CTLs [108 ] and moreover that it could possibly enable nonbone marrow-derived cells to directly prime and expand CTLs (Fig. 5 ) [109 ]. Although we did not achieve similar results using a B7.1 genetic adjuvant [109 ], others have found it to enhance CTL. The disagreement between these findings may reflect a functional role of the plasmid-encoded immunogen, based on antigen type, in determining the efficiency of the coexpressed B7.1 to elicit immune responses. It is speculated that the presence of B7.2 on somatic cells facilitates the direct stimulation of T cells or induces a proinflammatory environment that enhances antigen presentation via cross-priming by increasing the level of apoptosis and T cell reactivity. Although these studies suggest that B7.2 and not B7.1 plays a central role in the generation of antigen-specific CTL responses, the mechanisms underlying this functional transformation remain unclear and warrant further investigation.



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  		Figure 5. The expression of B7.2, in conjunction with vaccine antigen-loaded MHC class I and CD40 surface molecule can stimulate the expansion of T cells. It is possible that the expression of B7.2 along with the vaccine immunogen may facilitate the ability to expand immunogen-specific CD8+ T cells directly. Another possibility is that expression of B7.2 may stimulate a proinflammatory environment that enhances apoptosis, and thereby cross-priming, and CD8+ T cell reactivity.

Another strategy currently under development is the coadministration of cell-surface molecules that induce cellular apoptosis. Theoretically, this technique targets vaccine antigen to the cross-priming pathway by delivering antigen associated with apoptotic cells to DCs and thereby guiding immune responses toward a Th1 phenotype. Recently, we codelivered the fas receptor with HIV-1 gp160 and observed that fas-mediated death of antigen-bearing cells resulted in (1) targeted delivery of antigen to DCs, (2) enhancement of antigen-specific CTL responses, and (3) expression of Th1-type cytokines and chemokines [110 ]. The interpretation of these results is that the amplification of cell death by the coexpression of an apoptosis-inducing genetic adjuvant strengthens cellular immunity by heightening the cross-presentation of antigen to T cells by phagocytic APCs.

Fusion protein-encoding plasmids
The expression of fusion proteins from a DNA vaccine is an attractive means of modulating an antigen-specific immune response without the use of potentially toxic chemical adjuvants. The targeting of plasmid-encoded proteins to the class I antigen-processing pathway has been demonstrated to modulate the immunogenicity of DNA vaccines. The up-regulation of MHC class I presentation of vaccine peptides on transfected cells increases their ability to communicate with antigen-specific T cells by virtue of heightened epitope exhibition. The intracellular proteolysis of plasmid-encoded proteins in the cytosolic pathway can be modulated by structural alterations that target them to a multifunctional protease complex or proteasome. By increasing the entry of vaccine proteins into the intracellular pathway and modulating the concentration of peptides within the endoplasmic reticulum, it is hypothesized that MHC class I maturation will increase also.

Plasmid-encoded proteins can be targeted for rapid degradation within the proteasome by fusing them to ubiquitin (Ub) protein. This protein plays a pivotal role in intracellular polypeptide degradation by covalently linking to a lysine residue on a target protein and priming the formation of a poly-Ub chain, which then targets proteins to the cytosolic proteolytic system. Because only a small percentage of cellular protein becomes ubiquitinated normally, the increased delivery of vaccine antigens to the proteasome should enhance their presentation by MHC class I molecules. Two strategies increasing the degradation of plasmid antigen have been shown that render plasmid antigen-Ub fusion proteins unstable.

The first strategy involves the synthesis of an antigen that is transiently linked to Ub. The coding sequence of a vaccine antigen with a nonmethionine N-terminus is fused in frame to the C-terminus of the coding sequence of a Ub monomer. This fusion protein mimics a poly-Ub preprotein and is recognized by Ub hydrolases that normally cleave at the Ub C-terminus to create functional Ub monomers. Following cleavage of this conjugate, a nonconjugated vaccine antigen with an altered N-terminus is the product. The N-end rule dictates that a protein in this state is unstable and may not require ubiquitination to be degraded [111 , 112 ]. It has been confirmed that in some systems this destabilized product is degraded rapidly and causes the induction of a protective antigen-specific CTL response in a lethal tumor challenge model [113 , 114 ].

A second fusion protein-encoding plasmid strategy involves the rapid degradation of noncleavable Ub conjugates. Although the previous system relied on protein destabilization following cleavage by Ub hydrolayses, this one takes advantage of the destabilizing properties of a fixed, uncleavable, Ub-conjugated protein. A particular base-pair mutation in Ub renders it resistant to cleavage, and it retains its normal function of tagging proteins for proteasome cleavage [115 ]. Rapid degradation of this uncleavable conjugate has been demonstrated to enhance antigen-specific CTL responses, conferring viral protection at the expense of antibody production [116 ]. Because of its rapid intracellular turnover, little protein is found in the cytoplasm at any one time, and none is secreted, which effectively abolishes its entry into the APC exogenous pathway [116 ]. However, another study using a Ub-conjugated influenza nitrophenylhydroxyacetate (NP) protein has observed reduced antibody responses without the enhancement of CTL responses [117 ]. This suggests that there are antigen-specific issues that limit this approach and prevent its generalization. This loss of antibody induction may not be suitable for some vaccination approaches, where antibodies are necessary for the clearance and protection against certain pathogens.

Other fusion protein strategies involve the expression of fusion antigen that is directed to sites of immune induction by the addition of a targeting signal. It has been shown that fibroblasts transfected with a DNA plasmid encoding a fusion of antigen and a melanosomal transport signal can target epitopes to the MHC class II presentation pathway and induce CD4+ T cell responses [118 ]. This strategy would undoubtedly enhance the humoral arm of immunity. The fusion of IgG to one of two ligands, L-selectin or cytotoxic T-lymphocyte antigen 4 (CTLA-4), which binds receptors on APCs, was shown to enhance humoral and cell-mediated immune responses [119 ]. Also, the addition of an endoplasmic reticulum-targeting leader sequence was shown to improve drastically antibody production and cellular immune responses to a Borrelia burgdorferi protein [120 ].


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COMBINATION VACCINATION APPROACHES
 
The combination of DNA and conventional vaccines in various ways aims to couple the benefits of each vaccine component thereby rendering stronger correlates of protective immunity. Although DNA vaccines alone induce strong immune responses, live-attenuated or killed vaccines may be required to boost these responses to a protective level, such as in the case of malaria. Although repeated inoculations of protein or live-vector immunogens have failed to increase the potency or durability of immune responses, the development of DNA vaccine combination approaches is hoped to surpass this barrier. Many heterologous prime-boost strategies use DNA vaccines, recombinant virus, and protein in different assortments, which "prime" the immune system to the vaccine antigen first, followed by a subsequent "boost" immunization, which enhances the preliminary response. Strategies gaining the most attention are DNA vaccine-priming followed by protein or recombinant virus-boosting.

Although protein subunit vaccines by themselves induce a Th2-type immune response primarily [121 , 122 ], they remain unfit to elicit protective immunity to some pathogens. But, studies by Sin et al. [54 ] have shown that when they are combined with DNA vaccines in a DNA-priming, protein-boosting approach, antibody and cellular responses are enhanced in a murine HSV-2 surface glycoprotein D-vaccine model. Moreover, immunization with an HIV-1 DNA vaccine-boosted by an HIV-1 DNA vaccine plus recombinant HIV-1 protein has been shown to protect monkeys from infection after challenge with a chimeric simian and HIV (SHIV) [123 ]. A similar study that uses g.g. bombardment instead of i.m. injection has failed to achieve protection against SHIV, which supports a unique and important difference between the types of immunity induced by these distinct modes of delivery [124 ].

Another method of prime-boosting that is proving effective uses DNA-priming followed by live-vectored-boosting. This strategy differs from protein-boosting mainly by the utilization of a recombinant agent that is able to replicate within host cells and thereby gain access to the MHC I pathway, among other immunostimulatory benefits of infection. DNA-priming and -boosting with recombinant vaccinia have been shown to be more immunogenic and protective in a mouse malaria model than the DNA vaccine alone [125 ]. Furthermore, boosting with recombinant modified vaccinia virus Ankara has been shown to induce complete protection in this same model [126 , 127 ]. It is interesting that the efficacy of this powerful combination can be improved strongly by the coadministration of a plasmid-expressing murine GM-CSF along with the DNA vaccine primer [128 ].

An important finding was made using this approach when an i.d.-delivered DNA vaccine followed by a recombinant fowl pox virus (rFPV) vaccine-booster expressing common HIV proteins was shown to be able to contain attenuated SIV infections in rhesus macaques with low-to-undetectable levels of neutralizing antibodies [129 , 130 ]. In a study performed by Robinson and colleagues [130 ], containment of an extremely virulent SHIV was achieved following a two-stage challenge with a weakly pathogenic SHIV [130 ]. Therefore, it is difficult to determine whether the DNA-rFPV vaccine alone or the combination of the vaccine and the attenuated SHIV was responsible for protection. In either account, it has been suggested that this protection may be provided by a noncytolytic, secreted, CD8+ T cell antiviral activity [131 ]. However, this will require further study. Such an HIV inhibitory antiviral response may not require human leukocyte antigen compatibility nor involve cell killing and may even target multiple stages of the viral life-cycle [132 ]. Importantly, one conclusion that can be drawn from this study is that protection can occur in the presence of an exceedingly low neutralizing antibody response. Although the implications of these findings will not be discussed in this review, they serve to demonstrate the importance and utility of DNA vaccines in the advancement of basic and applied science.


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CONCLUSION AND FUTURE DIRECTIONS
 
Genetic immunization is a novel vaccine technology that may greatly reduce human morbidity and mortality in the future. Conceptually, it combines many of the desirable attributes of conventional vaccines and preserves the ability to elicit strong, and sometimes protective, immune responses in rodents and nonhuman primates. Although the basic mechanisms of immune induction are still under intense investigation, it is likely that APCs and especially DCs play a paramount role in the presentation of vaccine antigen to the immune system. Further understanding of this interplay will enable the rational design of DNA vaccines and methods of delivery that are capable of stimulating prophylactic- or therapeutic-immune correlates. Currently, many promising techniques of immune enhancement are being developed that modulate the intensity and direction of responses, such as the use of genetic adjuvants and DNA vectors of greater immunostimulation. It is likely that the flexibility of this approach will spawn future vaccines that are highly customized and specifically tailored for a particular disease. It is clear that DNA vaccination technology has established itself in the field of experimental immunotherapy and through prudent design and experimentation, may represent an important component of the next generation of prophylactic and therapeutic vaccines that are efficacious and economically accessible to peoples worldwide.


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ACKNOWLEDGEMENTS
 
D. J. S. is a graduate student in the Cell and Molecular Biology group at the University of Pennsylvania. This work was supported in part by grants from the National Institutes of Health as well as research funding from Wyeth-Lederle Vaccines program to D. B. W.

Received September 15, 2000; accepted September 15, 2000.


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REFERENCES
 
    1
  1. Stasney, J., Cantarow, A., Paschkis, K. E. (1950) Production of neoplasms by fractions of mammalian neoplasms Cancer Res 10,775-782
    2. 2
  2. Will, H., Cattaneo, R., Koch, H. G., Darai, G., Schaller, H., Schellekens, H., van Eerd, P. M., Deinhardt, F. (1982) Cloned HBV DNA causes hepatisis in chimpanzees Nature 299,740-742[Medline]
    3. 3
  3. Dubensky, T. W., Campbell, B. A., Villarreal, L. P. (1984) Direct transfection of viral and plasmic DNA into the liver or spleen of mice Proc. Natl. Acad. Sci. USA 81,7529-7533[Abstract/Free Full Text]
    4. 4
  4. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., Felgner, P. L. (1990) Direct gene transfer into mouse muscle in vivo Science 247,1465-1468[Abstract/Free Full Text]
    5. 5
  5. Tang, D. C., DeVit, M., Johnston, S. A. (1992) Genetic immunization is a simple model for eliciting an immune response Nature 356,152-154[Medline]
    6. 6
  6. Ulmer, J. B., Donnelly, J. J., Parker, S. E., Rhodes, G. H., Felgner, P. L., Dwarki, V. J., Gromkowski, S. H., Deck, R. R., DeWitt, C. M., Friedman, A., Hawe, L. A., Leander, K. R., Martinez, D., Perry, H. C., Shiver, J. W., Montgomery, D. L., Liu, M. A. (1993) Heterologous protection against influenza by injection of DNA encoding a viral protein Science 259,1745-1749[Abstract/Free Full Text]
    7. 7
  7. Fynan, E. F., Webster, R. G., Fuller, D. H., Haynes, J. R., Santoro, J. C., Robinson, H. L. (1993) DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations Proc. Natl. Acad. Sci. USA 90,11478-11482[Abstract/Free Full Text]
    8. 8
  8. Wang, B., Merva, M., Dang, K., Ugen, K. E., Boyer, J., Williams, W. V., Weiner, D. B. (1994) DNA inoculation induces protective in vivo immune responses against cellular challenge with HIV-1 antigen-expressing cells AIDS Res. Hum. Retrovir. 10(Suppl. 2),S35-S41
    9. 9
  9. Hoffman, S. L., Doolan, D. L., Sedegah, M., Wang, R., Scheller, L. F., Kumar, A., Weiss, W. R., Le, T. P., Klinman, D. M., Hobart, P., Norman, J. A., Hedstrom, R. C. (1997) Toward clinical trials of DNA vaccines against malaria Immunol. Cell Biol. 75,376-381[Medline]
    10. 10
  10. Kalinna, B. H. (1997) DNA vaccines for parasitic infections Immunol. Cell Biol. 75,370-375[Medline]
    11. 11
  11. Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charonvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A., Hoffman, S. L. (1998) Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine Science 282,476-480[Abstract/Free Full Text]
    12. 12
  12. Strugnell, R. A., Drew, D., Merciece, J., DiNatale, S., Firez, N., Dunstan, S. J., Simmons, C. P., Vadolas, J. (1997) DNA vaccines for bacterial infections Immunol. Cell Biol. 75,364-369[Medline]
    13. 13
  13. Kaufmann, S. H. (1995) Immunity to intracellular microbial pathogens Immunol. Today 16,338-342[Medline]
    14. 14
  14. Lowrie, D. B., Silva, C. L., Tascon, R. E. (1997) DNA vaccines against tuberculosis Immunol. Cell Biol. 75,591-594[Medline]
    15. 15
  15. Xiang, Z. Q., Spitalnik, S., Tran, M., Wunner, W. H., Cheng, J., Ertl, H. C. J. (1994) Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus Virology 199,132-140[Medline]
    16. 16
  16. Davis, H. L., Michel, M. L., Mancini, M., Schleef, M., Whalen, R. G. (1994) Direct gene transfer in skeletal muscle: plasmid DNA based immunization against the hepatitis B virus surface antigen Vaccine 12,1503-1509[Medline]
    17. 17
  17. Tacket, C. O., Roy, M. J., Widera, G., Swain, W. F., Broome, S., Edelman, R. (1999) Phase I safety and immune response studies of a DNA vaccine encoding hepatitis B surface antigen delivered by a gene delivery device Vaccine 19,2826-2829
    18. 18
  18. Kriesel, J. D., Spruance, S. L., Daynes, R. A., Araneo, B. A. (1996) Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease J. Infect. Dis. 173,536-541[Medline]
    19. 19
  19. Xu, L., Sanchez, A., Yang, Z., Zaki, S. R., Nabel, E. G., Nichol, S. T., Nabel, G. J. (1998) Immunization for ebola virus infection Nat. Med. 4,37-42[Medline]
    20. 20
  20. Boyer, J. D., Chattergoon, M. A., Ugen, K. E., Shah, A., Bennett, M., Cohen, A., Nyland, S., Lacy, K. E., Bagatazzi, M. L., Higgins, T. J., Baine, Y., Ciccarelli, R. B., Ginsberg, R. S., MacGregor, R. R., Weiner, D. B. (1999) Enhancement of cellular immune response in HIV-1 seropositive individuals: a DNA-based trial Clin. Immunol. 90,100-107[Medline]
    21. 21
  21. Wang, B., Boyer, J. D., Srikantan, V., Coney, L., Carrano, R., Phan, C., Merva, M., Dang, K., Agadjanyan, M. G., Ugen, K. E., Williams, M. V., Weiner, D. B. (1993) DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and non-human primates DNA Cell Biol 12,799-805[Medline]
    22. 22
  22. Wang, B., Ugen, K. E., Srikantan, V., Agadjanyan, M. G., Dang, K., Refaeli, Y., Sato, A., Boyer, J., Williams, W. V., Weiner, D. B. (1993) Gene inoculation generates immune responses against human immunodeficiency virus type 1 Proc. Natl. Acad. Sci. USA 90,4156-4160[Abstract/Free Full Text]
    23. 23
  23. Boyer, J. D., Ugen, K. E., Wang, B., Agadjanyan, M., Gilbert, L., Bagarazzi, M. L., Chattergoon, M., Frost, P., Javadian, A., Williams, W. V., Refaeli, Y., Ciccarelli, R. B., McCallus, D., Coney, L., Weiner, D. B. (1997) Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination Nat. Med. 3,526-532[Medline]
    24. 24
  24. Nichols, W. W., Ledwith, B. J., Manam, S. V., Troilo, P. J. (1995) Potential DNA vaccine integration into host cell genome Ann. N. Y. Acad. Sci. 772,30-39[Medline]
    25. 25
  25. Katsumi, T., Nobuhiko, E., Abe, A., Haseegawa, Y., Ito, M., Sato, H. (1994) Humoral and cellular immunity to an encoded protein induced by direct DNA injection Hum. Gene Ther. 5,1335-1339[Medline]
    26. 26
  26. Donnelly, J. J., Ulmer, J. B., Shiver, J. W., Liu, M. A. (1997) DNA vaccines Annu. Rev. Immunol. 15,617-648[Medline]
    27. 27
  27. Nichols, W. W., Ledwith, B. J., Manam, S. V., Troilo, P. J. (1995) Potential DNA vaccine integration into host cell genome Ann. N. Y. Acad. Sci. 772,30-39
    28. 28
  28. Martin, T., Parker, S. E., Hedstrom, R., Le, T., Hoffman, S. L., Norman, J., Hobart, P., Lew, D. (1999) Plasmid DNA malaria vaccine: the potential for genomic integration following intramuscular injection Hum. Gene Ther. 10,759-768[Medline]
    29. 29
  29. Kanellos, T., Sylvester, I. D., Ambali, A. G., Howard, C. R., Russell, P. H. (1999) The safety and longevity of DNA vaccines for fish Immunology 96,307-313[Medline]
    30. 30
  30. Bagarazzi, M. L., Boyer, J. D., Ugen, K. E., Javadian, M. A., Chattergoon, M., Shah, A., Bennett, M., Ciccarelli, R., Carrano, R., Coney, L., Weiner, D. B. (1998) Safety and immunogenicity of HIV-1 DNA constructs in chimpanzees Vaccine 16,1836-1841[Medline]
    31. 31
  31. MacGregor, R. R., Boyer, J. D., Ugen, K. E., Lacy, K. E., Gluckman, S. J., Bagarazzi, M. L., Chattergoon, M. A., Baine, Y., Higgins, T. J., Ciccarelli, R. B., Coney, L. R., Ginsberg, R. S., Weiner, D. B. (1998) First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response J. Infect. Dis. 178,92-100[Medline]
    32. 32
  32. Ugen, K. E., Nyland, S. B., Boyer, J. D., Vidal, C., Lera, L., Rasheid, S., Chattergoon, M., Bagarazzi, M. L., Ciccarelli, R., Higgins, T., Baine, Y., Ginsberg, R., Macgregor, R. R., Weiner, D. B. (1998) DNA vaccination with HIV-1 expressing constructs elicits immune responses in humans Vaccine 16,1818-1821[Medline]
    33. 33
  33. Le, T. P., Coonan, K. M., Hedstrom, R. C., Charoenvit, Y., Sedegah, M., Epstein, J. E., Kumar, S., Wang, R., Doolan, D. L., Maguire, J. D., Parker, S. E., Hobart, P., Norman, J., Hoffman, S. L. (2000) Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers Vaccine 18,1893-1901[Medline]
    34. 34
  34. Chen, C., Ji, H., Suh, K. W., Choti, M. A., Pardoll, D. M., Wu, T. (1999) Gene gun-mediated DNA vaccination induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the liver and lungs Gene Ther 6,1972-1981[Medline]
    35. 35
  35. Ramshaw, I. A., Fordham, S. A., Bernard, C. C., Maguire, D., Cowden, W. B., Willenborg, D. O. (1997) DNA vaccines for the treatment of autoimmune disease Immunol. Cell Biol. 75,409-413[Medline]
    36. 36
  36. Raz, E., Tighe, H., Sato, Y., Corr, M., Dudler, J. A., Roman, M., Swain, S. L., Spiegelberg, H. L., Carson, D. A. (1996) Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization Proc. Natl. Acad. Sci. USA 93,5141-5145[Abstract/Free Full Text]
    37. 37
  37. Hsu, C. H., Chua, K. Y., Tao, M. H., Lai, Y. L., Wu, H. D., Huang, S. K., Hsieh, K. H. (1996) Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization Nat. Med. 2,540-544[Medline]
    38. 38
  38. Jahn-Schmid, B., Siemann, U., Zanker, A., Bohle, B., Messner, P., Unger, F. M., Sleytr, U. B., Scheiner, O., Kraft, D., Ebner, C. (1997) Bet v 1, the major birch pollen allergen, conjugated to crystalline bacterial cell surface proteins, expands allergen-specific T cells of the Th1/Th0 phenotype in vitro by induction of IL-12 Int. Immunol. 9,1867-1874[Abstract/Free Full Text]
    39. 39
  39. Hartl, A., Kiesslich, J., Weiss, R., Bernhaupt, A., Mostböck, S., Scheiblhofer, S., Ebner, C., Ferreira, F., Thalhamer, J. (1999) Immune responses after immunization with plasmid DNA encoding Bet v 1, the major allergen of birch pollen J. Allergy Clin. Immunol. 103,107-113[Medline]
    40. 40
  40. Stevenson, F. K. (1999) DNA vaccines against cancer: from genes to therapy Ann. Oncol. 10,1413-1418[Abstract/Free Full Text]
    41. 41
  41. Davis, H. L., Brazolot Millan, C. L., Mancini, M., McCluskie, M. J., Hadchouel, M., Comanita, L., Tiollais, P., Whalen, R. G., Michel, M. L. (1997) DNA-based immunization against hepatitis B surface antigen (HbsAg) in normal and HbsAg-transgenic mice Vaccine 15,849-852[Medline]
    42. 42
  42. Geissler, M., Wands, G., Gesien, A., Monte, S., Bellet, D., Wands, J. R. (1997) Genetic immunization with the free human chorionic gonadotropin ß subunit elicits cytotoxic T lymphocyte responses and protects against tumor formation in mice Lab. Investig. 76,859-871[Medline]
    43. 43
  43. Weber, L. W., Bowne, W. B., Wolchok, J. D., Srinivasan, R., Qin, J., Moroi, Y., Clynes, R., Song, P., Lewis, J. J., Houghton, A. N. (1998) Tumor immunity and autoimmunity induced by immunization with homologous DNA J. Clin. Invest. 102,1258-1264[Medline]
    44. 44
  44. Williams, R. S., Johnston, S. A., Riedy, M., DeVit, M. J., McElligott, S. G., Sanford, J. C. (1991) Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles Proc. Natl. Acad. Sci. USA 88,2726-2730[Abstract/Free Full Text]
    45. 45
  45. Klavinskis, L. S., Barnfield, C., Gao, L., Parker, S. (1999) Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts J. Immunol. 162,243-262
    46. 46
  46. Hamajima, K., Sasaki, S., Fukushima, J., Kaneko, T., Xin, K. Q., Kudoh, I., Okuda, K. (1998) Intranasal administration of HIV-DNA vaccine formulated with a polymer, carboxymethylcellulose, augments mucosal antibody production and cell-mediated immune response Clin. Immunol. Immunopathol. 88,205-210[Medline]
    47. 47
  47. Kuklin, N., Daheshia, M., Karem, K., Manickan, E., Rouse, B. T. (1997) Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization J. Virol. 71,3138-3145[Abstract/Free Full Text]
    48. 48
  48. Etchart, N. R., Buckland, R., Liu, M. A., Wild, T. F., Kaiserlian, D. (1997) Class I-restricted induction by mucosal immunization with naked DNA encoding measles virus haemagglutinin J. Gen. Virol. 78,1577-1580[Abstract]
    49. 49
  49. Wang, B., Dang, K., Agadjanyan, M. G., Srikantan, V., Li, F., Ugen, K. E., Boyer, J., Merva, M., Williams, W. V., Weiner, D. B. (1997) Mucosal immunization with a DNA vaccine induces immune responses against HIV-1 at a mucosal site Vaccine 15,821-825[Medline]
    50. 50
  50. Yu, W. H., Kashani-Sabet, M., Liggitt, D., Moore, D., Heath, T. D., Debs, R. J. (1999) Topical gene delivery to murine skin J. Investig. Dermatol. 112,370-375[Medline]
    51. 51
  51. Fan, H., Lin, Q., Morrissey, G. R., Khavari, P. A. (1999) Immunization via hair follicles by topical application of naked DNA to normal skin Nat. Biotechnol. 19,870-872
    52. 52
  52. Boyle, J. S., Silva, A., Brady, J. L., Lew, A. M. (1997) DNA immunization: induction of higher avidity antibody and effect of route on T cell cytotoxicity Proc. Natl. Acad. Sci. USA 94,14626-14631[Abstract/Free Full Text]
    53. 53
  53. Fuller, D. H., Haynes, J. R. (1994) A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 120 vaccine AIDS Res. Hum. Retrovir. 10,1433-1441[Medline]
    54. 54
  54. Sin, J. I., Bagarazzi, M., Pachuk, C., Weiner, D. B. (1999) DNA priming-protein boosting enhances both antigen-specific antibody and Th1-type cellular immune responses in a murine herpes simplex virus-2 gD vaccine model DNA Cell Biol 18,771-779[Medline]
    55. 55
  55. McCluskie, M. J., Davis, H. L. (1999) Novel strategies using DNA for the induction of mucosal immunity Crit. Rev. Immunol. 19,303-329[Medline]
    56. 56
  56. McCluskie, M. J., Davis, H. L. (1999) Mucosal immunization with DNA vaccines Microbes Infect 1,685-698[Medline]
    57. 57
  57. Takashima, A., Morita, A. (1999) Dendritic cells in genetic immunization J. Leukoc. Biol. 66,350-356[Abstract]
    58. 58
  58. Torres, C. A. T., Iwanasaki, A., Barber, B. H., Robinson, H. L. (1997) Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations J. Immunol. 158,4529-4532[Abstract]
    59. 59
  59. Geijtenbeek, T. B. H., Torensma, R., Vliet, S. J. V., Duijnhoven, G. C. F. V., Adema, G. J., Kooyk, Y. V., Figdor, C. G. (2000) Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses Cell 100,575-585[Medline]
    60. 60
  60. Ariizumi, K., Shen, G. L., Shikano, S., Ritter, R., 3rd, Zukas, P., Edelbaum, D., Morita, A., Takashima, A. (2000) Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms J. Biol. Chem. 275,11957-11963[Abstract/Free Full Text]
    61. 61
  61. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity Nature 392,245-252[Medline]
    62. 62
  62. Ridge, J. P., Di Rosa, F., Matzinger, P. (1998) A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell Nature 393,474-478[Medline]
    63. 63
  63. Bennett, S. R. M., Carbone, F. R., Karamalis, F., Flavell, R. A., Miller, J. F. A. P., Heath, W. R. (1998) Help for cytotoxic-T-cell responses is mediated by CD40 signaling Nature 393,478-480[Medline]
    64. 64
  64. Schoenberger, S. P., Toes, R. E. M., van der Voort, E. I. H., Offringa, R., Melief, C. J. M. (1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions Nature 393,480-483[Medline]
    65. 65
  65. Porgador, A., Irvine, K. R., Iwasaki, A., Barber, B. H., Restifo, N. P., Germain, R. N. (1998) Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization J. Exp. Med. 188,1075-1082[Abstract/Free Full Text]
    66. 66
  66. Corr, M., Lee, D., Carson, D. A., Tighe, H. (1996) Gene vaccination with naked plasmid DNA: mechanism of CTL priming J. Exp. Med. 184,1555-1560[Abstract/Free Full Text]
    67. 67
  67. Iwasaki, A., Torres, C. A. T., Ohashi, P. S., Robinson, H. L. (1997) The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites J. Immunol. 159,11-14[Abstract]
    68. 68
  68. Sato, Y., Roman, M., Tighe, H., Lee, D., Corr, M., Nguyen, M. D., Silverman, G. J., Lotz, M., Carson, D. A., Raz, E. (1996) Immunostimulatory DNA sequences necessary for effective intradermal gene immunization Science 273,352-354[Abstract]
    69. 69
  69. Klinman, D. M., Yi, A., Beaucage, S. L., Conover, J., Krieg, A. M. (1996) CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma Proc. Natl. Acad. Sci. USA 93,2879-2883[Abstract/Free Full Text]
    70. 70
  70. Barry, M. A., Johnston, S. A. (1997) Biological features of genetic immunization Vaccine 15,788-791[Medline]
    71. 71
  71. Jakob, T., Walker, P. S., Kreig, A. M., Udey, M. C., Vogel, J. C. (1998) Activation of cutaneous dendritic cells by CpG-containing oligodeoxy-nucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA J. Immunol. 161,3042-3049[Abstract/Free Full Text]
    72. 72
  72. Akbari, O., Panjwani, N., Garcia, S., Tascon, R., Lowrie, D., Stockinger, B. (1999) DNA vaccination: transfection and activation of dendritic cells as key events for immunity J. Exp. Med. 189,169-177[Abstract/Free Full Text]
    73. 73
  73. Casares, S., Inaba, K., Brumeanu, T. D., Steinman, R. M., Bona, C. A. (1997) Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope J. Exp. Med. 186,1481-1486[Abstract/Free Full Text]
    74. 74
  74. Condon, C., Watkins, S. C., Celluzzi, C. M., Thompson, K., Falo, L. D., Jr (1996) DNA-based immunization by in vivo transfection of dendritic cells Nat. Med. 10,1122-1128
    75. 75
  75. Chattergoon, M. A., Robinson, T. M., Boyer, J. D., Weiner, D. B. (1998) Specific immune induction following through in vivo transfection and activation of macrophages/antigen-presenting cells J. Immunol. 160,5707-5718[Abstract/Free Full Text]
    76. 76
  76. Timares, L., Takashima, A., Johnston, S. A. (1998) Quantitative analysis of the immunopotency of genetically transfected dendritic cells Proc. Natl. Acad. Sci. USA 95,13147-13152[Abstract/Free Full Text]
    77. 77
  77. Albert, M. L., Pearce, S. F. A., Francisco, L. M., Sauter, B., Roy, P., Silverstein, R. L., Bhardwaj, N. (1998) Immature dendritic cells phagocytose apoptotic cells via {alpha}vß5 and CD36, and cross-present antigens to cytotoxic T lymphocytes J. Exp. Med. 188,1359-1368[Abstract/Free Full Text]
    78. 78
  78. Albert, M. L., Sauter, B., Bhardwaj, N. (1998) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs Nature 392,86-89[Medline]
    79. 79
  79. Bevan, M. J. (1976) Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay J. Exp. Med. 143,1283-1288[Abstract/Free Full Text]
    80. 80
  80. Bennett, S. R. M., Carbone, F. R., Karamalis, F., Miller, J. F. A. P., Heath, W. R. (1997) Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help J. Exp. Med. 186,65-70[Abstract/Free Full Text]
    81. 81
  81. Ulmer, J. B., Deck, R. R., DeWitt, C. M., Fu, T. M., Donnelly, J. J., Caulfield, M. J., Liu, M. A. (1997) Expression of a viral protein by muscle cells in vivo induces protective cell-mediated immunity Vaccine 15,839-841[Medline]
    82. 82
  82. Ulmer, J. B., Deck, R. R., Dewitt, C. M., Donnelly, J. J., Liu, M. A. (1996) Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells Immunology 89,59-67[Medline]
    83. 83
  83. Huang, A. Y., Golumbek, P., Ahmadzadeh, M., Jaffee, E., Pardoll, D., Levitsky, H. (1994) Role of bone marrow-derived cells in presenting MHC class I-mediated tumor antigens Science 264,961-965[Abstract/Free Full Text]
    84. 84
  84. Rovere, P., Vallinoto, C., Bondanza, A., Crosti, M. C., Rescigno, M., Ricciardi-Castagnoli, P., Ruglari, C., Manfredi, A. A. (1998) Bystander apoptosis triggers dendritic cell maturation and antigen presenting function J. Immunol. 161,4467-4471[Abstract/Free Full Text]
    85. 85
  85. Rovere, P., Sabbadini, M. G., Vallinoto, C., Fascio, U., Rescigno, M., Crosti, M., Ricciardi-Castagnoli, P., Balestrieri, G., Tincani, A., Manfredi, A. A. (1999) Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context Arthritis Rheum 42,1412-1420[Medline]
    86. 86
  86. Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F. A. P., Heath, W. R. (1997) Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8+ T cells J. Exp. Med. 186,239-245[Abstract/Free Full Text]
    87. 87
  87. Kurts, C., Miller, J. F. A. P., Subramaniam, R. M., Carbone, F. R., Heath, W. R. (1998) Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction J. Exp. Med. 188,409-414[Abstract/Free Full Text]
    88. 88
  88. Kurts, C., Heath, W. R., Kosaka, H., Miller, J. F. A. P., Carbone, F. R. (1998) The peripheral deletion of autoreactive CD8+ T cells induced by cross-presentation of self-antigens involves signaling through CD95 (fas, apo-1) J. Exp. Med. 188,415-420[Abstract/Free Full Text]
    89. 89
  89. Fanger, N. A., Maliszewski, C. R., Schooley, K., Griffith, T. S. (1999) Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRIAL) J. Exp. Med. 190,1155-1164[Abstract/Free Full Text]
    90. 90
  90. Fuller, D. H., Corb, M. M., Barnett, S., Steimer, K., Haynes, J. R. (1997) Enhancement of immunodeficiency virus-specific immune responses in DNA-immunized rhesus macaques Vaccine 15,924-926[Medline]
    91. 91
  91. O’Garra, A., Murphy, K. (1994) Role of cytokines in determining T-lymphocyte function Curr. Opin. Immunol. 6,458-466[Medline]
    92. 92
  92. Raz, E., Watanabe, A., Baird, S. M., Eisenberg, R. A., Parr, T. B., Lotz, M., Kipps, T. J., Carson, D. A. (1993) Systemic immunological effects of cytokine genes injected into skeletal muscle Proc. Natl. Acad. Sci. USA 90,4523-4527[Abstract/Free Full Text]
    93. 93
  93. Kim, J. J., Maguire, H. C., Jr, Nottingham, L. K., Morrison, L. D., Tsai, A., Sin, J. I., Chalian, A. A., Weiner, D. B. (1998) Coadministration of IL-12 or IL-10 expression cassettes drives immune responses toward a Th1 phenotype J. Interferon Cytokine Res. 18,537-547[Medline]
    94. 94
  94. Sin, J. I., Kim, J. J., Arnold, R. L., Shroff, K. E., McCallus, D., Pachuk, C., McElhiney, S. P., Wolf, M. W., Pompa-de Bruin, S. J., Higgins, T. J., Ciccarelli, R. B., Weiner, D. B. (1999) IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge J. Immunol. 162,2912-2921[Abstract/Free Full Text]
    95. 95
  95. Sin, J. I., Kim, J. J., Boyer, J. D., Ciccarelli, R. B., Higgins, T. J., Weiner, D. B. (1999) In vivo modulation of vaccine-induced immune responses toward a Th1 phenotype increases potency and vaccine effectiveness in a herpes simplex virus type 2 mouse model J. Virol. 73,501-509[Abstract/Free Full Text]
    96. 96
  96. Kim, J. J., Nottingham, L. K., Tsai, A., Lee, D. J., Maguire, H. C., Oh, J., Dentchev, T., Manson, K. H., Wyand, M. S., Agadjanyan, M. G., Ugen, K. E., Weiner, D. B. (1999) Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-{gamma}, IL-12, or IL-18 gene adjuvants J. Med. Primatol. 28,214-223[Medline]
    97. 97
  97. Boyer, J. D., Kim, J., Ugen, K., Cohen, A. D., Ahn, L., Schumann, K., Lacy, K., Bagarazzi, M. L., Javadian, A., Ciccarelli, R. B., Ginsberg, R. S., MacGregor, R. R., Weiner, D. B. (1999) HIV-1 DNA vaccines and chemokines Vaccine 19,S53-S64
    98. 98
  98. Kim, J. J., Simbiri, K. A., Sin, J. I., Dang, K., Oh, J., Dentchev, T., Lee, D., Nottingham, L. K., Chalian, A. A., McCallus, D., Ciccarelli, R., Agadjanyan, M. G., Weiner, D. B. (1999) Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV J. Interferon Cytokine Res. 19,77-84[Medline]
    99. 99
  99. Kim, J. J., Trivedi, N. N., Nottingham, L. K., Morrison, L., Tsai, A., Hu, Y., Mahalingam, S., Dang, K., Ahn, L., Doyle, N. K., Wilson, D. M., Chattergoon, M. A., Chalian, A. A., Boyer, J. D., Agajanyan, M. G., Weiner, D. B. (1998) Modulation of amplitude and direction of in vivo immune responses by coadministration of cytokine gene expression cassettes with DNA immunogens Eur. J. Immunol. 28,1089-1103[Medline]
    100. 100
  100. Irvine, K. R., Rao, J. B., Rosenberg, S. A., Restifo, N. P. (1996) Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases J. Immunol. 156,238-245[Abstract]
    101. 101
  101. Sun, W. H., Burkholder, J. K., Sun, J., Culp, J., Turner, J., Lu, X. G., Pugh, T. D., Ershler, W. B., Yang, N. S. (1995) In vivo cytokine gene transfer by gene gun reduces tumor growth in mice Proc. Natl. Acad. Sci. USA 92,2889-2893[Abstract/Free Full Text]
    102. 102
  102. Barouch, D. H., Craiu, A., Kuroda, M. J., Schmitz, J. E., Zheng, X. X., Santra, S., Frost, J. D., Krivulka, G. R., Lifton, M. A., Crabbs, C. L., Heidecker, G., Perry, H. C., Davies, M., Xie, H., Nickerson, C. E., Steenbeke, T. D., Lord, C. I., Montefiori, D. C., Strom, T. B., Shiver, J. W., Lewis, M. G., Letvin, N. L. (2000) Augmentation of immune responses to HIV-1 and simian immunodeficiency virus DNA vaccines by IL-2/Ig plasmid administration in rhesus monkeys Proc. Natl. Acad. Sci. USA 97,4192-4197[Abstract/Free Full Text]
    103. 103
  103. Kim, J. J., Yang, J. S., VanCott, T. C., Lee, D. J., Manson, K. H., Wyand, M. S., Boyer, J. D., Ugen, K. E., Weiner, D. B. (2000) Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants J. Virol. 74,3427-3429[Abstract/Free Full Text]
    104. 104
  104. Xiang, Z., Ertl, H. C. (1995) Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines Immunity 2,129-135[Medline]
    105. 105
  105. Kim, J. J., Yang, J. S., Lee, D. J., Wilson, D. M., Nottingham, L. K., Morrison, L., Tsai, A., Oh, J., Dang, K., Dentchev, T., Agadjanyan, M. G., Sin, J. I., Chalian, A. A., Weiner, D. B. (2000) Macrophage colony-stimulating factor can modulate immune responses and attract dendritic cells in vivo Hum. Gene Ther. 11,305-321[Medline]
    106. 106
  106. Sin, J. I., Sung, J. H., Suh, Y. S., Lee, A. H., Chung, J. H., Sung, Y. C. (1997) Protective immunity against heterologous challenge with encephalomyocarditis virus by VP1 DNA vaccination: effect of co-injection with a GM-CSF gene Vaccine 15,1827-1833[Medline]
    107. 107
  107. Geissler, M., Gesien, A., Tokushige, K., Wands, J. R. (1997) Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccine augmented with cytokine-expressing plasmids J. Immunol. 158,1231-1237[Abstract]
    108. 108
  108. Kim, J. J., Ayyavoo, V., Bagarazzi, M. L., Chattergoon, M., Boyer, J. D., Wang, B., Weiner, D. B. (1997) Development of a multicomponent candidate vaccine for HIV-1 Vaccine 15,879-883[Medline]
    109. 109
  109. Agadjanyan, M. G., Kim, J. J., Trivedi, N., Wilson, D. M., Monzavi-Karbassi, B., Morrison, L. D., Nottingham, L. K., Dentchev, T., Tsai, A., Dang, K., Chalian, A. A., Maldonaod, M. A., Williams, W. V., Weiner, D. B. (1999) CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo J. Immunol. 162,3417-3427[Abstract/Free Full Text]
    110. 110
  110. Chattergoon, M. A., Kim, J. J., Yang, J. S., Robinson, T. M., Lee, D. J., Dentchev, T., Wilson, D. M., Ayyavoo, V., Weiner, D. B. (2000) Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis Nat. Biotechnol. 18,974-979[Medline]
    111. 111
  111. Bachmair, A., Varshavsky, A. (1989) The degradation signal in a short-lived protein Cell 56,1019-1032[Medline]
    112. 112
  112. Michalek, M. T., Grant, E. P., Rock, K. L. (1996) Chemical denaturation and modification of ovalbumin alters its dependence on ubiquitin conjugation for class I antigen presentation J. Immunol. 157,617-624[Abstract]
    113. 113
  113. Rodriguez, F., Zhang, J., Whitton, J. L. (1997) DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction J. Virol. 71,8497-8503[Abstract/Free Full Text]
    114. 114
  114. Townsend, A., Bastin, J., Gould, G., Brownlee, M., Andrew, M., Coupar, B., Boyle, D., Chan, S., Smith, G. (1988) Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen J. Exp. Med. 168,1211-1224[Abstract/Free Full Text]
    115. 115
  115. Ecker, D. J., Stadel, J. M., Butt, T. R., Marsh, J. A., Monia, B. P., Powers, D. A., Gorman, J. A., Clark, P. E., Warren, F., Shatzman, A. (1989) Increasing gene expression in yeast by fusion to ubiquitin J. Biol. Chem. 264,726-731[Abstract/Free Full Text]
    116. 116
  116. Tobery, T., Siliciano, R. F. (1999) Induction of enhanced CTL-dependent protective immunity in vivo by N-end rule targeting of a model tumor antigen J. Immunol. 162,639-642[Abstract/Free Full Text]
    117. 117
  117. Fu, T. M., Guan, L., Friedman, A., Ulmer, J. B., Liu, M. A., Donnelly, J. J. (1998) Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens Vaccine 16,1711-1717[Medline]
    118. 118
  118. Wang, S., Bartido, S., Yang, G., Qin, J., Moroi, Y., Panageas, K. S., Lewis, J. J., Houghton, A. N. (1999) A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses J. Immunol. 163,5820-5826[Abstract/Free Full Text]
    119. 119
  119. Boyle, J. S., Brady, J. L., Lew, A. M. (1998) Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction Nature 392,408-411[Medline]
    120. 120
  120. Weiss, R., Dürnberger, J., Mostböck, S., Scheiblhofer, S., Harlt, A., Breitenbach, M., Strasser, P., Dorner, F., Livey, I., Crowe, B., Thalhamer, J. (1999) Improvement of the immune response against plasmid DNA encoding OspC of borrelia by an ER-targeting leader sequence Vaccine 18,815-824[Medline]
    121. 121
  121. Sin, J. I., Ayyavoo, V., Boyer, J., Kim, J., Ciccarelli, R., Weiner, D. B. (1999) Protective immune correlates can segregate by vaccine type in a murine herpes model system Int. Immunol. 11,1763-1773[Abstract/Free Full Text]
    122. 122
  122. Montefiori, D. C., Evans, T. G. (1999) Toward an HIV-type 1 vaccine that generates potent, broadly cross-reactive neutralizing antibodies AIDS Res. Hum. Retrovir. 15,689-698[Medline]
    123. 123
  123. Letvin, N. L., Montefiori, D. C., Yasutomi, Y., Perry, H. C., Davies, M. E., LeKutis, C., Alroy, M., Freed, D. C., Lord, C. I., Handt, L. K., Liu, M. A., Shiver, J. W. (1997) Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination Proc. Natl. Acad. Sci. USA 94,9378-9383[Abstract/Free Full Text]
    124. 124
  124. Putkonen, P., Quesada-Rolander, M., Leandersson, A. C., Schwartz, S., Thorstensson, R., Okuda, K., Wahren, B., Hinkula, J. (1998) Immune responses but no protection against SHIV by gene-gun delivery of HIV-1 DNA followed by recombinant subunit protein boosts Virology 250,293-301[Medline]
    125. 125
  125. Sedegah, M., Jones, T. R., Kaur, M., Hedstrom, R., Hobart, P., Tine, J. A., Hoffman, S. L. (1998) Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine Proc. Natl. Acad. Sci. USA 95,7648-7653[Abstract/Free Full Text]
    126. 126
  126. Dégano, P., Schneider, J., Hannan, C. M., Gilbert, S. C., Hill, A. V. (1999) Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models Vaccine 18,623-632[Medline]
    127. 127
  127. Schneider, J., Gilbert, S. C., Hannan, C. M., Dégano, P., Prieur, E., Sheu, E. G., Plebanski, M., Hill, A. V. S. (1999) Induction of CD8+ T cells using heterologous prime-boost immunisation strategies Immunol. Rev. 170,29-38[Medline]
    128. 128
  128. Sedegah, M., Weiss, W., Sacci, J. B., Jr, Charoenvit, Y., Hedstrom, R., Gowda, K., Majam, V. F., Tine, J. A., Kumar, S., Hobart, P., Hoffman, S. L. (2000) Improving protective immunity induced by DNA-based immunization: priming with antigen and GM-CSF-encoding plasmid DNA and boosting with antigen-expressing recombinant poxvirus J. Immunol. 164,5905-5912[Abstract/Free Full Text]
    129. 129
  129. Kent, S. J., Zhao, A., Best, S. J., Chandler, J. D., Boyle, D. B., Ramshaw, I. A. (1998) Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus J. Virol. 72,10180-10188[Abstract/Free Full Text]
    130. 130
  130. Robinson, H. L., Montefiori, D. C., Johnson, R. P., Manson, K. H., Kalish, M. L., Lifson, J. D., Rizvi, T. A., Lu, S., Hu, S., Mazzara, G. P., Panicali, D. L., Herndon, J. G., Glickman, R., Candido, M. A., Lydy, S. L., Wyand, M. S., McClure, H. M. (1999) Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations Nat. Med. 5,526-534[Medline]
    131. 131
  131. Levy, J. A., Mackewicz, C. E., Barker, E. (1996) Controlling HIV pathogenesis: the role of the moncytotoxic anti-HIV response of CD8+ T cells Immunol. Today 19,217-224
    132. 132
  132. Tomaras, G. D., Lacey, S. F., McDanal, C. B., Ferrari, G., Weinhold, K. J., Greenberg, M. L. (2000) CD8+ T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indicating effects on virus gene expression Proc. Natl. Acad. Sci. USA 97,3503-3508[Abstract/Free Full Text]



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[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Peng, H. Ji, C. Trimble, L. He, Y.-C. Tsai, J. Yeatermeyer, D. A. K. Boyd, C.-F. Hung, and T.-C. Wu
Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6
J. Virol., August 15, 2004; 78(16): 8468 - 8476.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Shen and K. L. Rock
Cellular protein is the source of cross-priming antigen in vivo
PNAS, March 2, 2004; 101(9): 3035 - 3040.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. H. Day, Y. Zhang, P. Clair, K. H. Grabstein, M. Mazel, A. R. Rees, M. Kaczorek, and J. Temsamani
Induction of Antigen-Specific CTL Responses Using Antigens Conjugated to Short Peptide Vectors
J. Immunol., February 1, 2003; 170(3): 1498 - 1503.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
I. D. Davis, M. Jefford, P. Parente, and J. Cebon
Rational approaches to human cancer immunotherapy
J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. Coban, K. J. Ishii, D. J. Sullivan, and N. Kumar
Purified Malaria Pigment (Hemozoin) Enhances Dendritic Cell Maturation and Modulates the Isotype of Antibodies Induced by a DNA Vaccine
Infect. Immun., July 1, 2002; 70(7): 3939 - 3943.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
A. Campos-Neto, J. R. Webb, K. Greeson, R. N. Coler, Y. A. W. Skeiky, and S. G. Reed
Vaccination with Plasmid DNA Encoding TSA/LmSTI1 Leishmanial Fusion Proteins Confers Protection against Leishmania major Infection in Susceptible BALB/c Mice
Infect. Immun., June 1, 2002; 70(6): 2828 - 2836.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
C.-F. Hung, L. He, J. Juang, T.-J. Lin, M. Ling, and T.-C. Wu
Improving DNA Vaccine Potency by Linking Marek's Disease Virus Type 1 VP22 to an Antigen
J. Virol., March 15, 2002; 76(6): 2676 - 2682.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. F. Lipscomb and B. J. Masten
Dendritic Cells: Immune Regulators in Health and Disease
Physiol Rev, January 1, 2002; 82(1): 97 - 130.
[Abstract] [Full Text] [PDF]

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