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.

Preclinical prophylactic efficacy testing of Sm-p80-based vaccine in a nonhuman primate model of Schistosoma mansoni infection and immunoglobulin G and E responses to Sm-p80 in human serum samples from an area where schistosomiasis is endemic.

The prophylactic efficacy of a schistosome antigen (Sm-p80) was tested in a nonhuman primate model, the baboon. Using a total of 28 baboons, different vaccination strategies were used including recombinant Sm-p80 protein formulated in Toll-like receptor 7 and Toll-like receptor 9 agonists, and DNA priming followed by boosting with protein plus adjuvants. Recombinant protein approaches provided levels of prophylactic efficacy of 52%-58%, whereas prime-boost approaches conferred 38%-47% protection in baboons. An appropriately balanced pro-inflammatory (T-helper 17 [Th17] and Th1) and anti-inflammatory (Th2) type of response was generated; the Th1 and Th17 types of immune responses appear to be indicative of increased prophylactic efficacy. Production and expression of several cytokines (interleukin 2 [IL-2], interferon γ, IL-12α, IL-1ß, IL-6, and IL-22) were up-regulated in vaccinated animals. Human correlate studies revealed Sm-p80 reactivity with immunoglobulin G in human serum samples from schistosome-infected individuals. In addition, a complete lack of prevailing Sm-p80-specific immunoglobulin E in a high-risk or infected population was observed, thus minimizing the risk of hypersensitivity reaction following vaccination with Sm-p80 in humans. This study provided the proof of concept to move Sm-p80 forward into further preclinical development leading to human clinical trials.

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.

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.

Sm-p80-based DNA vaccine provides baboons with levels of protection against Schistosoma mansoni infection comparable to those achieved by the irradiated cercarial vaccine.

To date, no vaccine is available to prevent human schistosomiasis. We have targeted a protein of Schistosoma mansoni that plays an important role in the surface membrane renewal process, a mechanism widely believed to be utilized by the parasite as an immune evasion strategy. Sm-p80 antigen is a promising vaccine target because of its documented immunogenicity, protective efficacy, and antifecundity effects observed in both experimental murine and nonhuman primate models of this infectious disease. In the present study, we report that, in a vector approved for human use (VR1020), an Sm-p80-based DNA vaccine formulation confers a 46% reduction in the worm burden in a baboon (Papio anubis) model. Baboons vaccinated with Sm-p80-VR1020 had a 28% decrease in egg production after challenge with the infectious parasite. Sm-p80-VR1020 vaccine elicited robust immune responses to specific antigen Sm-p80, including immunoglobulin (Ig) G, its subtypes IgG1 and IgG2, and IgA and IgM in vaccinated animals. When stimulated in vitro with recombinant Sm-p80, peripheral blood mononuclear cells and splenocytes from baboons vaccinated with Sm-p80-VR1020 produced considerably higher levels of T helper 1 response-enhancing cytokines (interleukin [IL]-2 and interferon-gamma) than T helper 2 (Th2) response-enhancing cytokines (IL-4 and IL-10). Peripheral blood mononuclear cells produced a significantly higher number of spot-forming units for interferon-gamma than for IL-4 in enzyme-linked immunosorbent spot assays. A mixed T helper 1/T helper 2 type of humoral and T cell responses was generated after immunization with Sm-p80-VR1020. These findings again highlight the potential of Sm-p80 as a promising vaccine candidate for schistosomiasis.

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.

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.

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.

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.

Measles virus infection and vaccination: potential role in chronic illness and associated adverse events.

Over the last decade, a number of concerns have arisen related to safety issues that have had an adverse effect on the public's trust, particularly among parents whose children are the primary recipient of the vaccine. Historically, the live attenuated measles virus (MV) vaccine and the combination multivalent measles, mumps, and rubella (MMR) vaccine have had a major impact on the health of children worldwide and have been extremely successful at preventing infectious diseases associated with three childhood viral pathogens. In this report, we describe MV infection, replication, pathogenesis, and immunization. MV is a viral pathogen that exhibits a number of complex processes that can effect its replication, pathogenesis, and the induction of an effective antiviral immune response. We describe the published literature as it relates to MV infection and immunization and report adverse events in an attempt to provide a balanced discussion and an historical perspective of the MMR vaccine and autism.

Is HIV involved in the pathogenesis of non-infectious pulmonary complications in infected patients?

Pulmonary complications of AIDS are one of the major causes of morbidity and mortality in HIV-infected individuals, and are often direct indicators of enhanced progression to an AIDS-defining illness. HIV can be detected in the lung during asymptomatic disease, as well as during the advanced stages of AIDS. With prolonged survival of HIV-infected patients, non-infectious pulmonary complications are becoming more common and can lead to respiratory compromise and failure. The pathogenesis of non-infectious pulmonary complications, following HIV infection is not fully understood, and the role that HIV-induced immunosuppression plays in the development of these complications is not known. In this review, we will discuss non-infectious pulmonary complications of HIV infection, focusing on HIV-associated malignancies of the lung, inflammatory disorders, pulmonary hypertension and the effect of antiretroviral therapy on the incidence of these diseases. The possible role that HIV plays in the pathogenesis of these pulmonary disorders is also addressed.

Activation of focal adhesion kinase in adenovirus-infected human corneal fibroblasts.

PURPOSE: Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase with protean downstream influences on cell cycle regulation, cytoskeletal dynamics, and cell attachment, is activated by integrin binding and aggregation. Adenoviruses, including those associated with human keratitis, enter permissive cells by an integrin-mediated mechanism. Hence, a possible relationship between adenovirus infection and tyrosine phosphorylation of FAK in human corneal cells was explored. METHODS: Human corneal fibroblasts (HCFs) derived from donor corneas were infected for various periods with adenovirus type 19 (Ad19) or were mock infected with virus-free medium. Parallel experiments included echistatin, which is a snake venom disintegrin and a partial inhibitor of FAK. For immunoblot analysis, Triton-X-soluble and Triton-X-insoluble fractions were analyzed by SDS-PAGE and immunoblotted with phosphospecific antibodies. Expression of the interleukin (IL)-8 gene was analyzed by RT-PCR and ELISA. RESULTS: Ad19 infection of HCFs induced tyrosine phosphorylation of a protein at 125 kDa, which was evident within 15 minutes after infection and was established by immunoprecipitation to be FAK. Immunoblot with antibody to FAK tyrosine-397 confirmed phosphorylation of this key binding site for downstream signaling proteins. Immunoblot analysis further suggested a shift in the intracellular location of phosphorylated FAK from the cytosol (Triton-X-soluble cell lysate fraction) to the cytoskeleton (Triton-X-insoluble cell lysate pellet) on infection. Finally, treatment of HCFs with echistatin reduced virus-induced expression of the neutrophil chemotaxin IL-8, previously implicated in adenoviral pathogenesis. CONCLUSIONS: Ad19 infection of HCFs induces rapid phosphorylation of FAK, and a dramatic change in its intracellular distribution. Activation of FAK may contribute to the inflammatory response to adenovirus infection of the human cornea.

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.

Infection of baboon microglia with SIV-HIV recombinant viruses: role of CD4 and chemokine receptors.

Microglia constitute the primary cell type infected with HIV in the brain and play a major role in viral persistence in the CNS and in the development of AIDS dementia. Lack of a suitable animal model and limitations in the availability of human tissues hinder most HIV/AIDS studies investigating the neuropathogenesis of AIDS dementia. The aims of this study were to determine whether baboon microglia can be productively infected with SIV-HIV (SHIV) recombinant viruses in vitro and whether they express HIV-1 receptors and coreceptors. Our results show the presence of mRNA for CD4, CCR5, and CXCR4 chemokine receptors on baboon microglial cells. Microglia lacked mRNA for the CCR3 chemokine receptor. We also show productive infection of baboon microglial cells by two SHIV isolates, SHIV-KU and SHIV-89.6P, and blockade of the infection with soluble CD4 protein, CCR5, and CXCR4 monoclonal antibodies. This study demonstrating the feasibility of infecting baboon microglia with SHIV isolates is an important first step in using the baboon as an alternative nonhuman primate model to study HIV neuropathogenesis.

The conjunctival lymphoid follicle in mucosal immunology.

The conjunctiva forms a continuous mucosal surface from the eyelid margin to the cornea, and makes contact with airborne antigens and those on the adjacent eyelid skin and preocular tear film. Conjunctival lymphoid follicles (CLF) undergo hyperplasia upon conjunctival infection by a specific array of pathogens; infection-associated enlargement of draining preauricular lymph nodes suggests that CLF participate in the afferent limb of acquired immune responses for the ocular surface. In this review, we examine the evidence for classification of CLF as part of the common mucosal immune system, and explore the possible therapeutic implications.

Protective and antifecundity effects of Sm-p80-based DNA vaccine formulation against Schistosoma mansoni in a nonhuman primate model.

Schistosomiasis is an important parasitic disease for which there is no available vaccine. We have focused on a functionally important antigen of Schistosoma mansoni, Sm-p80, as a vaccine candidate because of its consistent immunogenicity, protective potential and antifecundity effect observed in murine models; and for its pivotal role in the immune evasion process. In the present study we report that an Sm-p80-based DNA vaccine formulation confers 38% reduction in worm burden in a nonhuman primate model, the baboon (Papio anubis). Animals immunized with Sm-p80-pcDNA3 exhibited a decrease in egg production by 32%. Sm-p80 DNA elicited specific immune responses that include IgG; its subtypes IgG1 and IgG2; and IgM in vaccinated animals. Peripheral blood mononuclear cells (PBMCs) from immunized animals when stimulated in vitro with Sm-p80 produced appreciably more Th1 response enhancing cytokines (IL-2, IFN-gamma) than Th2 response enhancing cytokines (IL-4, IL-10). PBMCs produced appreciably more spot-forming units for INF-gamma than for IL-4 in enzyme-linked immunosorbent spot (ELISPOT) assays. Overall it appears that even though a mixed (Th1/Th2) type of humoral antibody response was generated following immunization with Sm-p80; the dominant protective immune response is Th1 type. These data reinforce the potential of Sm-p80 as an excellent vaccine candidate for schistosomiasis.

Experimental vaccines in animal models for schistosomiasis.

Considerable morbidity and mortality results from the affliction of an estimated 200 million people worldwide by several species of schistosomes; 779 million are exposed to the disease in 74 different countries. Even though anti-parasitic drugs and other control measures, including public hygiene and snail control are available, the advent of an effective vaccine still remains the most potentially powerful means for the control of this disease. The putative vaccine could be administered to small children prior to the time when their contact with infected water is maximal, so as to prevent severe infection in the subsequent years. This review attempts to summarize the status of schistosome vaccine development with special emphasis on functionally important vaccine candidates. The importance of utilizing both murine and nonhuman primate models as a prerequisite for clinical trials is discussed.

Sperm protein 17 is a suitable target for adoptive T-cell-based immunotherapy in human ovarian cancer.

For ovarian cancer (OC) patients with advanced or metastatic disease, standard treatments (chemotherapy and radiotherapy) are not very effective and have undesirable side effects. Newer and more promising approaches in cancer treatment use components of the immune system. In this study, we applied an adoptive immunotherapy-based approach using a cancer testis antigen, sperm protein 17, as a target for the treatment of human metastatic OC in a NOD.CB17-PrkDCcid/J (nonobese, diabetic severe combined immunodeficient) mouse model. We used the human SK-OV-3A2.A3 OC cell line, endogenously expressing sperm protein 17, to induce tumor growth in mice. We provide direct evidence, for the first time, that in vitro cultured, monoclonal, cytotoxic T lymphocytes (derived either from advanced OC patients or from healthy donors), specific for sperm protein 17, can eradicate human metastatic OC cells. In addition, we observed no evidence of autoimmunity after histologic examination of the tissue sections adding to the safety profile of our approach.

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.