The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen.

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.

CD4+ T lymphocytes are critical mediators of tumor immunity to simian virus 40 large tumor antigen induced by vaccination with plasmid DNA.

A mechanistic analysis of tumor immunity directed toward the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory for scenarios of recombinant Tag immunization in BALB/c mice. In the present study, we performed a preliminary characterization of the immune components necessary for systemic tumor immunity induced upon immunization with plasmid DNA encoding SV40 Tag as a transgene (pCMV-Tag). Antibody responses to SV40 Tag were observed via indirect enzyme-linked immunosorbent assay following three intramuscular (i.m.) injections of pCMV-Tag and were typified by a mixed Th1/Th2 response. Complete tumor immunity within a murine model of pulmonary metastasis was achieved upon two i.m. injections of pCMV-Tag, as assessed by examination of tumor foci in mouse lungs, without a detectable antibody response to SV40 Tag. Induction-phase and effector-phase depletions of T cell subsets were performed in vivo via administration of depleting rat monoclonal antibodies, and these experiments demonstrated that CD4(+) T lymphocytes are required in both phases of the adaptive immune response. Conversely, depletion of CD8(+) T lymphocytes did not impair tumor immunity in either immune phase and resulted in the premature production of antibodies to SV40 Tag. Our findings are unique in that a dominant role could be ascribed to CD4(+) T lymphocytes within a model of DNA vaccine-induced tumor immunity to Tag-expressing tumor cells. Additionally, our findings provide insight into the general mechanisms of vaccine-induced tumor immunity directed toward tumors bearing distinct tumor-associated antigens.

Vaccines and immunotherapeutics for the treatment of malignant disease.

The employment of the immune system to treat malignant disease represents an active area of biomedical research. The specificity of the immune response and potential for establishing long-term tumor immunity compels researchers to continue investigations into immunotherapeutic approaches for cancer. A number of immunotherapeutic strategies have arisen for the treatment of malignant disease, including various vaccination schemes, cytokine therapy, adoptive cellular therapy, and monoclonal antibody therapy. This paper describes each of these strategies and discusses some of the associated successes and limitations. Emphasis is placed on the integration of techniques to promote optimal scenarios for eliminating cancer.

Role of the innate immune response and tumor immunity associated with simian virus 40 large tumor antigen.

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.

Tumor immunity against a simian virus 40 oncoprotein requires CD8+ T lymphocytes in the effector immune phase.

The required activities of CD4(+) T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8(+) T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8(+) T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-gamma), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8(+) T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8(+) T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8(+) T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.

INTS6/DICE1 inhibits growth of human androgen-independent prostate cancer cells by altering the cell cycle profile and Wnt signaling.

BACKGROUND: The gene encoding integrator complex subunit 6 (INTS6), previously known as deleted in cancer cells 1 (DICE1, OMIM 604331) was found to be frequently affected by allelic deletion and promoter hypermethylation in prostate cancer specimens and cell lines. A missense mutation has been detected in prostate cancer cell line LNCaP. Together, these results suggest INTS6/DICE1 as a putative tumor suppressor gene in prostate cancer. In this study, we examined the growth inhibitory effects of INTS6/DICE1 on prostate cancer cells. RESULTS: Markedly decreased INTS6/DICE1 mRNA levels were detected in prostate cancer cell lines LNCaP, DU145 and PC3 as well as CPTX1532 as compared to a cell line derived from normal prostate tissue, NPTX1532. Exogenous re-expression of INTS6/DICE1 cDNA in androgen-independent PC3 and DU145 cell lines substantially suppressed their ability to form colonies in vitro. This growth inhibition was not due to immediate induction of apoptosis. Rather, prostate cancer cells arrested in G1 phase of the cell cycle. Expression profiling of members of the Wnt signaling pathway revealed up-regulation of several genes including disheveled inhibitor CXXC finger 4 (CXXC4), frizzled homologue 7 (FZD7), transcription factor 7-like 1 (TCF7L1), and down-regulation of cyclin D1. CONCLUSION: These results show for the first time a link between INTS6/DICE1 function, cell cycle regulation and cell-cell communication involving members of the Wnt signaling pathway.

Evidence of simian virus 40 exposure in a colony of captive baboons.

Simian virus 40 (SV40) is a polyomavirus for which non-human primates are the permissive host. The baboon (Papio spp.) is an old world monkey that is used in a variety of research investigations; however, natural infection of SV40 among baboons has not been thoroughly examined or reported. Initially, we were interested in determining the prevalence of SV40 infection among a captive colony of baboons based on the presence of antibodies to SV40 large T-antigen (Tag). An overall seroprevalence rate of >50% was found after screening sera from 142 baboons in the colony based on ELISA. Endpoint titer values for serum antibody binding to SV40 Tag reached as high as 1280 for 5 out of 142 baboons. Peptide binding assays revealed that a range of SV40 Tag epitopes are immunogenic in the baboon, and that individual animals differ in their humoral immune responses to SV40 Tag based on epitope recognition. Specificity to SV40 Tag and not some other primate polyomavirus encoded large Tag was further examined by serologic reactivity to peptide epitopes unique to SV40 Tag. Additional serology was performed to assess SV40 Tag reactivity by Western blot and whether antibodies were capable of neutralizing SV40 infectivity in vitro. Although antibodies with high levels of SV40 neutralization were observed in a number of the baboons, there was a lack of correlation between viral neutralization and antibodies to SV40 Tag. Further examination using molecular-based diagnosis and SV40 Tag specific real-time quantitative PCR determined that some of the baboons appeared to be exposed to SV40. DNA sequence analysis of the PCR products confirmed that SV40 Tag specific sequences were detected in baboons.

A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17.

No experimental animal model employing a primary human ovarian carcinoma (OC) cell line is presently available that tracks the progression of this cell line with an identifiable marker. This hinders investigations related to developing new approaches for treating OC. Here, we describe the development of a tumor model in NOD/SCID mice for human OC that makes use of the endogenously expressed tumor specific sperm protein 17 (Sp17) cancer testis antigen. In this model, human SKOV-3 OC cell lines were intra-peritoneally seeded. Subsequently viable SKOV-3 cells were recovered from primary organ cell cultures from the liver ovaries, abdomen, and ascitic fluid, and their presence was confirmed by the detection of Sp17 mRNA by RT-PCR and Sp17 protein by immunocytochemistry and FACS analysis. When SKOV-3 tumor cells were administered intravenously the mice developed primarily lung tumor foci. This model makes it possible to evaluate new immunotherapeutic strategies for the treatment of human OC based on the biomarker Sp17.

Fc gamma receptors play a dominant role in protective tumor immunity against a virus-encoded tumor-specific antigen in a murine model of experimental pulmonary metastases.

Simian virus 40 (SV40) large tumor antigen (Tag) represents a virus-encoded tumor-specific antigen expressed in many types of human cancers and a potential immunologic target for antitumor responses. Fc receptors are important mediators in the regulation and execution of host effector mechanisms against conditions including infectious diseases, autoimmunity, and cancer. By examining tumor protection in SV40 Tag-immunized wild-type BALB/c mice using an experimental pulmonary metastasis model, we attempted to address whether engagement of the immunoglobulin G Fc receptors (FcgammaRs) on effector cells is necessary to mediate antitumor responses. All immunized BALB/c FcgammaR-/- knockout mice developed anti-SV40 Tag antibody responses prior to experimental challenge with a tumorigenic cell line expressing SV40 Tag. However, all mice deficient in the activating FcgammaRI (CD64) and FcgammaRIII (CD16) were unable to mount protective immunologic responses against tumor challenge and developed tumor lung foci. In contrast, mice lacking the inhibitory receptor FcgammaRII (CD32) demonstrated resistance to tumorigenesis. These results underscore the importance of effector cell populations expressing FcgammaRI/III within this murine tumor model system, and along with the production of a specific humoral immune response, antibody-dependent cell-mediated cytotoxicity (ADCC) may be a functioning mechanism of tumor clearance. Additionally, these data demonstrate the potential utility of ADCC as a viable approach for targeting vaccination strategies that promote FcgammaRI/III scavenging pathways against cancer.

DNA vaccines: successes and limitations in cancer and infectious disease.

Vaccination with plasmid DNA is an active area of investigation that is being applied to diseases including cancer and microbial pathogens associated with infectious diseases. Since its discovery, great progress has been made with the administration of DNA vaccines to initiate specific and effective immune responses against targeted illnesses. However, many obstacles still face its use in prophylactic and therapeutic vaccination scenarios. The nature of these difficulties alongside the successes and future of plasmid DNA will be discussed.

Surrogate tumor antigen vaccination induces tumor-specific immunity and the rejection of spontaneous metastases.

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection.

In vitro simian virus 40 large tumor antigen expression correlates with differential immune responses following DNA immunization.

Simian virus 40 (SV40) contains an essential protein, large tumor antigen (Tag), which assists in viral replication and causes cell transformation and immortalization. Our laboratory has examined plasmid DNA, expressing SV40 Tag under two different promoters, for use in potential cancer vaccination strategies. One plasmid, pSV3-neo, failed to induce SV40 Tag antibody, produced a weak cell-mediated response, and only partial protection in murine experimental tumor challenge systems. The second plasmid, pCMV-Tag, induced antibodies to SV40 Tag, produced a robust cell-mediated response, and invoked complete tumor immunity in vivo. The induction of CD4+ and CD8+ T cell responses following plasmid DNA immunization and tumor cell challenge reflected a type 1 cytokine secretion profile. Our hypothesis for this differential immune response is that pCMV-Tag exhibits a higher level of transgene expression due to a more efficient promoter. We determined that pCMV-Tag levels of SV40 Tag mRNA and protein expression were higher when compared to pSV3-neo. A threshold amount of SV40 Tag may be required to stimulate antibody production and provide complete systemic tumor immunity.

Characterization of the immune response to DNA vaccination strategies for schistosomiasis candidate antigen, Sm-p80 in the baboon.

Even though schistosomicidal agents and other control measures, including public hygiene and snail control exist, development of an efficacious vaccine still remains the most potentially powerful method for control of schistosomiasis. In our continuing efforts to develop a vaccine against schistosomiasis, we have selected a vaccine candidate (Sm-p80), which plays an important role in the immune evasion process of the parasite. Sm-p80 has been shown to confer up to 60% protection in mice following experimental infection. In this initial study, we have used Sm-p80 plus the Th1 response promoting cytokine, interleukin-2 (IL-2), in a DNA immunogen formulation. The vaccine was tested for its safety and immunogenicity in a baboon model of schistosomiasis. The vaccine generated a Th1 type Sm-p80-specific response in baboons with IgG(1)/IgG(2) ratios of less than 1.0. No detectable IgG(3) or IgG(4) anti-Sm-p80 responses were present in the immunized baboons. The antibodies to Sm-p80 were able to kill up to 35% schistosomula in vitro in the presence of complement. These results although preliminary suggest the potential of Sm-p80 as a viable vaccine candidate for schistosomiasis.

Anti-idiotype responses abrogate anti-CD4-induced tolerance to a tumor-specific antigen and promote systemic tumor immunity.

PURPOSE: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. METHODS: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. RESULTS: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. CONCLUSIONS: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4+ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.

A role for antibodies in tumor immunity.

Recent advances have demonstrated the clinical utility of specific monoclonal antibodies that recognize tumor-associated antigens on the surface of the tumor cell in the treatment of breast cancer and B cell lymphoma in humans. In addition to these studies, an experimental tumor model, where antibodies that recognize a viral-encoded tumor-specific antigen play a major role in tumor immunity, will be discussed. Together, these studies implicate antibodies as a means of providing tumor immunity against some cancers. The necessity for designing cancer vaccines that induce antibodies with specificity for antigens on the tumor cell will be discussed.

Cutting edge: class I presentation of host peptides following HIV infection.

Class I MHC molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes. Identification of peptides presented by class I molecules during infection is therefore a priority for detecting and targeting intracellular pathogens. To understand which host-encoded peptides distinguish HIV-infected cells, we have developed a mass spectrometric approach to characterize HLA-B*0702 peptides unique to or up-regulated on infected T cells. In this study, we identify 15 host proteins that are differentially presented on infected human T cells. Peptides with increased expression on HIV-infected cells were derived from multiple categories of cellular proteins including RNA binding proteins and cell cycle regulatory proteins. Therefore, comprehensive analysis of the B*0702 peptide repertoire demonstrates that marked differences in host protein presentation occur after HIV infection.

CD4+ T lymphocytes play a critical role in antibody production and tumor immunity against simian virus 40 large tumor antigen.

The role of CD4+ T lymphocytes in antitumor immunity has been largely attributed to providing signals required for the priming of MHC class I-restricted CD8+ cytotoxic T lymphocytes, and CD8+ cytotoxic T lymphocytes are thought to serve as the predominant mediators of tumor killing in vivo. We decided to evaluate the role of T lymphocyte subsets in tumor immunity induced by recombinant SV40 large tumor antigen (Tag) within an experimental murine pulmonary metastasis model of SV40 Tag-expressing tumors. Studies in BALB/c mice used in vivo depletion of either CD4+ or CD8+ T cells in the induction phase of the immune response to SV40 Tag. These studies indicate that CD4+ T cells but not CD8+ T cells were critical in the production of antibodies to SV40 Tag and in tumor immunity as the result of recombinant SV40 Tag immunization. On the basis of the predominance of the IgG1 isotype in the antibody response to SV40 Tag immunization, Th2 type CD4+ T cells appeared to be involved. SV40 Tag immunization was not as effective in the induction of tumor immunity in therapeutic modalities when compared with the prophylactic setting. Our results suggest that CD4+ T cells, along with antibody responses, play a role in the induction of tumor immunity to a viral-encoded tumor antigen.

Synthetic AIDS vaccine by targeting HIV receptor.

A class of synthetic peptide immunogens for the cell surface HIV receptor complex has been developed to elicit antibodies that block viral entry by inhibiting gp120-CD4 interaction. These peptides extend our HIV receptor-directed approach from passive immunotherapy with mAb B4 (Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 10367) to active immunization by a synthetic peptide-based vaccine. A peptide site from CD4 was identified as a B cell epitope capable of mimicking a susceptible site on the HIV receptor complex, and then rendered immunogenic. An effective target antigenic site (B cell epitope) for the cell surface HIV receptor complex was selected by epitope mapping from among diverse CD4 and chemokine receptor peptides. It is a cyclized sequence modified from the CDR2-like domain of CD4 (AA 39-66), that was predicted to produce steric hindrance of the discontinuous recognition site of mAb B4. The immunogenicity of the targeted epitope was augmented by tandem combination with promiscuous T helper cell epitopes (Th). The antibody response to this class of immunogens attained sufficient concentrations and affinities of the correct specificity to block the interactions of HIV env glycoprotein with the cellular receptor, and prevent infection. The polyclonal antibodies generated against these fusion constructs in multiple animal species neutralized a broad array of HIV-1 primary isolates from clades A to E. Despite eliciting antibodies to the key CD4 immunomodulatory molecule, the site-specific and chemically defined immunogens displayed no overt immunotoxicity in baboons and have potential for the immunotherapy and immunoprophylaxis of HIV infection.

SV40 Tag-specific cytotoxic T lymphocytes generated from the peripheral blood of malignant pleural mesothelioma patients.

Malignant pleural mesothelioma (MPM) is an aggressive cancer, with survival of less than one year following diagnosis and treatment with current protocols. Recent studies have demonstrated the presence of the simian virus 40 (SV40)-like, large tumor antigen (Tag) in nearly 60% of MPMs. SV40 Tag is a viral-encoded tumor-specific antigen, and thus a potential target for the induction of anti-tumor immunity and the development of therapeutic vaccines. We describe here evidence for the existence of SV40 Tag-specific immune responses in patients with MPM whose tumors express Tag. Humoral immunity was demonstrated by the detection of IgG titers against Tag in serum samples from 1/3 of patients examined. CTLs were generated from the peripheral blood of an HLA-A2(+) MPM patient with a synthetic peptide representing an HLA-A2 binding epitope in SV40 Tag. The CTLs demonstrated epitope fine specificity, in that other peptides from SV40 Tag and a peptide from influenza virus were not recognized in the context of HLA-A2. Moreover, the CTLs were capable of recognizing mesothelioma tumor cells that expressed SV40 Tag, in an MHC class I restricted manner.