A. DNA Vaccines Against HIV-1
The human immunodeficiency virus-1 (HIV-1) is a retrovirus, which preferentially infects and kills CD4+ T cells and macrophages, ultimately resulting in immune system failure andmultipathogen infections. Recent breakthroughs in combination therapy using 3 or more different antiretroviral agents have generated optimism regarding the ability to control viral replication in vivo (21). However, this therapeutic regimen is costly, and it is too early to tell whether this approach can eradicate established infection (21,22). The costs and the stringent administration regimen requirements of these pharmaceutical agents make it clear that these drugs will only be effectively used in a limited part of the world population. Therefore, to address the worldwide problem of HIV-1 infection, there remains a need for a prophylactic vaccination strategy designed to control the epidemic through mass immunization campaigns (23).
One of the major obstacles in the development of a vaccine against HIV-1 is uncertainty regarding the exact immune correlates of protection (24). In studies of long-term non-progressor groups of HIV-infected individuals, evidence supports the notion that correlates of protection against HIV-1 could be provided by humoral, cellular, or even both arms of the immune response (25,26). High levels of type-specific neutralizing antibody have been observed in protected primates in some homologous challenge models (27-31). Neutralizing antibodies are susceptible to viral deception through antigenic diversity of HIV-1 envelope, and the ability of neutralizing antibody to prevent viral pathogenesis is still under considerable investigation (32-35).
One of the hallmarks of HIV-1 disease progression is the loss of cellular immune function, and the presence of strong cellular responses in some instances can correlate with control of viral replication (9,36). In cases of acute HIV-1 infection studied by several investigators, viral clearance was associated with specific CTL activity in each case (37,38). In addition, a subset (7 of 20) of occupationally exposed health care workers who were not infected possessed transient HIV-1-specific CTL response (39). HIV-1-specific CTLs were also found in a number of chronically exposed sex workers in Gambia who continue to resist infection with HIV-1 (40). In spite of these studies supporting the role of neutralizing antibodies and CTLs in conferring immunity to infection, some vaccinated primates exhibiting both neutralizing antibody and CTL responses were not protected from subsequent viral challenge in the pathogenic SIV model (41). Recently, the important role of CD4 helper responses in the anti-HIV immune response has been highlighted (42). Such responses likely have importance for both humoral and CD8 + effector responses.
The advantages of nucleic acid immunization listed above make it well suited as a potentially useful vaccination strategy against HIV-1. Within the HIV genome, there are several potential immunological targets for DNA vaccination (Fig. 4). The HIV-1 genome is organized into 3 major structural and enzymatic genes, 2 regulatory genes, and 4 accessory genes (43). The first major gene target is env, which codes for the outer viral envelope proteins. HIV enters the CD4+ cells via envelope-CD4 receptor complex. Following entry of HIV viral core, synthesis of a double-stranded DNA version of the HIV genome (called DNA provirus) begins by the viral DNA polymerase, reverse transcriptase (RT). The DNA provirus is then translocated to the nucleus as part of the protein-DNA preintegration complex and is integrated into the host cell genome with the help of the viral integrase (Int) enzyme. The provirus then replicates with the host DNA each time the cell divides. The gene gag codes for the core protein, and pol codes for the enzymatic proteins RT, Int, and protease (Pro). In general, these enzymatic proteins remain somewhat conserved and preserve their catalytic functions. Accordingly, these proteins may be less divergent immune targets than envelope proteins for CTL-mediated responses (44).
The regulatory genes tat and rev affect HIV-1 gene expression. Viral transcription is increased several hundred-fold by tat transactivation (45), making it an obvious target for therapeutic intervention. The rev protein increases the release of unspliced structural RNAs from the nucleus by displacing host splicing factors that otherwise prevent RNA transport from the nucleus to the cytoplasm (46). In addition, rev is a critical component in the production of the structural proteins of HIV-1. A report of a DNA vaccine study in humans shows the immunogenicity of the rev gene product, again supporting its importance as a vaccine target (47). In addition, a Tat DNA vaccine has been reported to influence SHIV viral challenge in a macaque model system, while human immune response against HIV infection includes Vpr as a target (48,49). However, studies with Vpr as a plasmid vaccine component report that its presence can negatively influence the resulting host immune response (50,51). Therefore, this target requires more study before being included as part of a plasmid vaccine cocktail. Nonetheless, further work on these targets could be important as they and Nef are the earliest HIV antigens expressed. Targeting these antigens may give the immune system early warning of HIV challenge.
In addition to the regulatory genes, HIV-1 carries an additional set of accessory genes, vif (virion infectivity factor), vpr (viral protein r), vpu (viral protein u), and nef (negative factor), which are potential targets for DNA immunization.
These accessory genes can be deleted from the viral genome without eliminating replication in vitro, suggesting that these gene products play a secondary rather than primary role in viral infection. The vif protein is located in the plasma membrane and may be important for production of infectious virions (52). In contrast to vif, vpu seems to facilitate the degradation of intracellular CD4 molecules (52). The vpr protein is found in the viral particle in high amounts and appears to have several biological activities, including the ability to increase viral transcription and to reactivate virus from cellular latency and arrest host cell division (53-55). Nef has never been critical for viral infection of cell lines in vitro (56), although experiments performed with the related simian immunodeficiency virus (SIV) found that rhesus macaques infected with virus having a deletion in nef had dramatically lower levels of viral replication (57). In addition, recent evidence suggests that Nef plays a crucial role in mediating bystander apoptosis through the upregulation of FasL expression on infected T cells (58). Accordingly, Nef expression in T cells also suppresses cell death by blocking ASKl-mediated cell death and by inducing signals that phosphorylate BAD (59,60). Developing DNA vaccine constructs directed against these accessory genes could provide additional arsenal in our battle against HIV-1.
DNA expression cassettes encoding for HIV-1 envelopes (strains HXB2, -MN, and -Z6) were among the first to be analyzedfor immunogenicity (Fig. 4) (61). Initial studies demonstrated that mice immunized with envelope constructs produced antibodies specific to recombinant gp160, gp120, and gp41 proteins. The antisera neutralized HIV-1 isolates in vitro at a low level (62). Neutralization of homologous isolates has also been reported after immunization with constructs based on the HIV-1 NL4-3 isolate in the presence of relatively low antienvelope IgG titers (63). Moreover, the pM160-MN construct not only demonstrated neutralization of homologous isolates but also showed lower, yet measurable neutralization of the heterologous HIV-1 Z6 isolate (62). In addition to the humoral responses, cellular immune responses were observed from envelope inoculated mice. Induction of T helper cell proliferative response against recombinant gp120 protein was observed (61). In addition, cytotoxic T lymphocyte responses have been observed against both targets infected with recombinant vaccinia-expressing envelope protein and targets prepared with envelope peptides (61,63,64).
In contrast to the high level of sequence divergence observed in the envelope glycoproteins of HIV-1, their gag and pol gene sequences appear to be less variable immunological targets. Thus, combining env constructs with gag/pol constructs could result in a more potent vaccination program. Expression cassettes encoding for both gag and pol elicited antigen-specific antibody, Th, and CTL responses (65). In addition to the DNA immunogen cassettes encoding env and gag/pol proteins, the DNA expression cassettes targeting nef and vif accessory proteins have been developed, and they have been shown to induce both antigen-specific humoral and cellular responses in mice (66,67).
For HIV plasmid vaccines, the issue of rev independence has appeared as a recent important issue. The structural genes of HIV are made as long unspliced transcripts that contain overlapping reading frames with the small regulatory genes of HIV tat and rev. These transcripts are retained in the nucleus and rapidly spliced to encode the small regulatory in the absence of rev, thus preventing the transport of full-length message to the ER where structural gene translation can occur. The messages are inhibited from transport to the ER by both known and cryptic rev-dependent sequences. In view of this, recent studies suggest that optimization of codons to the usage of highly expressed human genes, as well as deletion of residual inhibitory sequences, significantly enhanced Gag expression leading to potent augmentation of immune responses (68,69). In addition, a recent study suggested that alteration of AT-rich regions without changing the amino acid sequences can significantly enhance Gag expression through rev-independent mechanisms. These modifications led to enhanced Gag-specific immune responses, indicating that codon optimization may provide new avenues for enhancing vaccine potencies (70).
In addition to HIV-1, there is a growing list of DNA vaccines targeted against other viral pathogens. One of its central attractions is its flexibility in modulating immune responses. As a consequence, several vaccine cocktails have been created that specifically target highly immunogenic antigens, which result in potent antiviral immune responses.
The West Nile virus (WNV) is a vectorborne pathogen that induces brain inflammation and death in endemic regions. Recently, confirmed cases of infection and death have been reported in the Mid-Atlantic region of the United States. Because there is no specific therapy for the WNV infection at this moment, there is an increasing demand for the development of vaccine strategies to prevent disease from this virus. Currently, no human or veterinary vaccine is available to prevent WNV infection, and mosquito control is the only practical strategy to combat the spread of disease. Several vaccine com panies, including Acambis, Inc., and Baxter/immuno, have research and development programs on human vaccines (71). One major veterinary vaccine manufacturer (Ft. Dodge) is also developing formalin-inactivated and naked DNA vaccines.
A group from the Centers for Disease Control and Prevention has reported on the induction of protective immunity using a DNA vaccine that expressed the WNV prM and E proteins (72). In addition, as an extension of the multicompo-nent DNA vaccines strategy, a DNA vaccine expressing the WNV capsid (Cp) protein has been shown to induce immune responses in DNA vaccine-immunized mice (73). Collectively, these results support the potential utility of DNA vaccines as a tool for developing immunization strategies for WNV and other emerging pathogens.
Some of the earliest DNA vaccines were against influenza. In fact, the initial report by Ulmer et al. targeted the nucleopro-tein as an antigen and effectively generated CTLs to achieve protection against subsequent challenge with a heterologous strain of influenza A virus (74). Protection was also achieved through immunization against the hemagglutinin antigen (75). Interestingly, cross-protection of variant viral strains was also achieved in both mice and preclinical studies (76,77).
Other exciting prospects for DNA vaccines against viruses include complete protection against measles in a macaque model. Surprisingly, protection correlated with the generation of neutralizing antibodies rather than cytotoxic T cell induction (78).
Finally with the advent of recent bioterrorism scares, much interest has focused on the development of rapid mass stocks of vaccines to immunize the general public (79). In view of this, DNA vaccines' simplicity and ease of mass production effectively overcomes such obstacles. In addition, plasmid immunization against the protective antigen and lethal factor of anthrax effectively induced complete protection in preliminary mice experiments (80,81). Similar results were also achieved against the Ebola virus in both mice and primate experiments (82). These preliminary results highlight the potential efficacy of DNA vaccines for generating mass immunizations against potential bioterroristic weapons.
Although advances in science have led to countless theories and methods designed to combat human carcinoma, the battle is far from over. Surgical excision of tumors, drug therapies, and chemotherapy have been effective in certain cases but in other situations, particularly when the tumor has begun to metastasize, effective treatment is far more difficult and far less potent. Thus, researchers are continually investigating novel and more effective treatment strategies for various forms of cancer. Research, in recent years, has turned toward the use of vaccines to treat cancer. To this end, several proteins produced by tumor cells became a target for vaccine development. These tumor-associated antigens are predominantly expressed in a tissue-specific manner and are expressed at greatly increased levels in affected cells. Besides being impor tant diagnostic aids, these antigens represent appropriate targets for the development of cancer vaccines (83).
Tumor-associated antigens (TAAs) are proteins produced by tumor cells that can be presented on the cell surface in the context of major histocompatibility complexes (84). Recently, these antigens have been the focus of study as a viable option for immunotherapy of various types of cancer. In this chapter, we examine the progress in the investigation of the immuno-logical effects of 3 such TAAs, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), and human papillo-mavirus (HPV) type 16 E7 antigen.
Human CEA is a 180-kDa glycoprotein expressed in elevated levels in 90% of gastrointestinal malignancies, including colon, rectal, stomach, and pancreatic tumors, 70% of lung cancers, and 50% of breast cancers (84,85). CEA is also found in human fetal digestive organ tissue, hence the name carci-noembryonic antigen (86). It has been discovered that CEA is expressed in normal adult colon epithelium as well, albeit at far lower levels (87,88). Sequencing of CEA shows that it is associated with the human immunoglobulin gene superfam-ily and that it may be involved in the metastasizing of tumor cells (86).
The immune response to nucleic acid vaccination using a CEA DNA construct was characterized in a murine model. The CEA insert was cloned into a vector containing the cyto-megalovirus (CMV) early promoter/enhancer and injected intramuscularly. CEA-specific humoral and cellular responses were detected in the immunized mice. These responses were comparable to the immune response generated by rV-CEA (87). The CEA DNA vaccine was also characterized in a canine model, where sera obtained from dogs injected intramuscularly with the construct demonstrated an increase in antibody levels (89). Cellular immune responses quantified using the lymphoblast transformation assay also revealed proliferation of CEA-specific lymphocytes. Therefore, a CEA nucleic acid vaccine was able to induce both arms of the immune responses (89). CEA DNA vaccines are currently being investigated in humans.
Prostate cancer is the most common form of cancer and the second most common cause of cancer-related death in American men (90). The appearance of prostate cancer is much more common in men over the age of 50 (91). Three of the most widely used treatments are surgical excision of the prostate and seminal vesicles, external bean irradiation, and androgen deprivation. However, conventional therapies lose their efficacy once the tumor has metastasized, which is the case in more than half of initial diagnoses (92,93).
PSA is a serine protease and a human glandular kallikrein gene product of 240 amino acids, which is secreted by both normal and transformed epithelial cells of the prostate gland (94,95). Because cancer cells secrete much higher levels of the antigen, PSA level is a particularly reliable and effective diagnostic indicator of the presence of prostate cancer (96). PSA is also found in normal prostate epithelial tissue and its expression is highly specific (97).
The immune responses induced by a DNA vaccine encoding for human PSA has been investigated in a murine model
(97). The vaccine construct was constructed by cloning a gene for PSA into expression vectors under control of a CMV promoter. Following the injection of the PSA DNA construct (pCPSA), various assays were performed to measure both the humoral and cellular immune responses of the mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation CTL, and flow cytometry assays. Strong and persistent antibody responses were observed against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Immunization with pCPSA also induced MHC class I CD8 + T cell-restricted CTL response against tumor cell targets expressing PSA. The induction of PSA-specific humoral and cellular immune responses following injection with pCPSA was also observed in rhesus macaques
F. DNA Vaccines Against Cervical Cancer
HPV 16-associated proteins, including E6 and E7, are some of the most common proteins in cervical cancers and are ubi-quitiously expressed within these cells (99,100). However, DNA-based vaccine targeting these proteins seem to elicit minimal immune responses, and may necessitate potent adjuvants to provide efficacious tumor protection. Accordingly, recent studies suggest numerous targeting mechanisms (i.e., adjuvants) to enhance immunogenicity of these antigens.
Heat shock proteins (HSPs) have been found to induce tumor immunogenicity and their levels of expression are enhanced with highly immunogenic necrotic bodies (101-103). HSP-peptide complexes are also highly immunogenic and function effectively as adjuvants and cross-priming proteins (104-107). It has also been reported that the enhanced CTL responses are CD4 + independent and are limited only to antigenic peptides associated with HSPs (108-110). Importantly, evidence suggests that the HSPs may specifically target immature dendritic cells for the induction of proinflammatory reponses, further validating their potential role as vaccine adjuvants (111). In view of this, the HSP70 of mycobacterium tuberculosis was fused to the HPV-16 E7 antigen to construct a chimeric DNA vaccine. The E7-HSP70 DNA vaccine induced significantly enhanced levels of cellular responses, including a ratio of 435:14 (E7-HSP70 to E7) of E7-specific IFN-7 spot-forming CD8 + T cells via ELISPOT assays. In addition, data indicate the eradication of preexisting tumors and the resulting response was via CD4 + -independent mechanisms (111). The exact mechanism of HSP-mediated peptide processing is still yet unclear, although enhanced proteasomal processing may be involved in this cross-priming maneuver (111). In a similar fashion, another member of the HSP family that has aug mented the potency of DNA vaccines is calreticulin (112). The idea of calreticulin as an immune modulator was based on previous findings that calreticulin in conjugation with tumor peptides stimulates potent peptide-specific CD8+ T cell responses (113). In addition, calreticulin and its fragment vasostatin operate as inhibitors of angiogensis (114,115). Accordingly, when calreticulin was fused to HPV-16 E7 antigen as a DNA vaccine, a potent antitumor effect was provoked. The resulting response was attributed to both the enhanced immunogenicity against E7 and the generation of antian-giogenesis (112).
A more recent report within the clinics also suggests that immunization through DNA can also therapeutically attenuate the growth of neoplastic cells in humans. These studies specifically encapsulated DNA plasmids encoding HLA-A2-restricted epitopes of the HPV E7 antigen within biodegradable polymer microparticles. Early work suggests no adverse side effects while enhancing immune responses when implementing this specific therapy (116). All together, these promising results emphasize the potential of DNA vaccines as therapies.
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