Gene therapy using plasmid DNA-encoded anti-HER2 antibody for cancers that overexpress HER2.

Plasmid DNA-encoded antibodies, or DNA-based monoclonal antibodies (dMAbs), are delivered by intramuscular injection and in vivo electroporation (EP) and are effective in virus neutralization, although they have not been evaluated for tumor gene therapy. Here we investigated whether a dMAb was appropriate for tumor gene therapy. We constructed the expression plasmids coding for the heavy or light chain of a parental murine antibody of Herceptin with the antibody genes codon- and RNA-optimized and fused to the Kozak-IgE leader sequence in pVax1. Transfection of the plasmids into human muscle RD cells resulted in functional expression of the antibody, and this exhibited the same in vitro antiproliferative activity as Herceptin. A single intramuscular injection and in vivo EP of the plasmids (100 µg per head) resulted in high and sustained antibody expression in the sera of normal mice and in effective inhibition of tumor growth in nude mice bearing HER2-positive human breast carcinoma BT474 xenografts. The antitumor efficacy of the anti-HER2 dMAb was similar to that of four doses of intravenously injected 10 mg kg-1 Herceptin. The results demonstrate that the dMAb is effective in the treatment of HER2-positive breast cancer, suggesting that this dMAb may be applicable for tumor gene therapy.

Cutaneous Mycobacterium massiliense infection associated with cupping therapy.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms that are now seen as emerging human pathogens. NTM infections are very difficult to diagnose and treat, therefore a high index of clinical suspicion is needed for diagnosis. Cutaneous NTM infections have been primarily reported associated with previous invasive procedures. We report the case of a healthy 59-year-old woman who developed recurring abdominal skin lesions caused by Mycobacterium massiliense after she underwent noninvasive cupping therapy. We identified the pathogen using a PCR assay targeting the erm(41) gene of the bacterium. The patient was treated successfully by en bloc excision and long-term antibiotic treatment. This case shows that cutaneous infection with M. massiliense may occur in an immunocompetent person without an antecedent invasive procedure.

Impact of hemoglobin on survival of cervical carcinoma patients treated with concurrent chemoradiotherapy is dependent on lymph node metastasis findings by magnetic resonance imaging.

The objective of this study was to confirm whether hemoglobin (Hb) levels during chemoradiotherapy are associated with survival in patients with locally advanced cervical carcinoma and to assess impact of the Hb level on survival according to lymph node (LN) metastasis. A retrospective review of 85 cervical carcinoma patients treated with concurrent chemoradiotherapy was conducted. The stage of disease ranged between FIGO stage IB and stage IVA. Disease-free and overall survivals were evaluated by univariate and multivariate analyses. After median follow-up of 35.7 months, 24 patients developed recurrence of disease and 14 patients died from their disease. Stage, LN metastasis, and squamous cell carcinoma antigen and Hb levels during chemoradiation were correlated significantly with survival (P < 0.05). Maintenance of Hb above 10.0 g/dL was associated with better survival (P < 0.05). However, no such benefits were observed in patients with LN metastasis by magnetic resonance imaging (MRI). Multivariate Cox regression hazard model showed that Hb levels during chemoradiation were an independent prognostic factor in patients without LN metastasis by MRI. Maintenance of Hb during chemoradiation is of benefit in cervical carcinoma patients without LN metastasis but not with LN metastasis by MRI.

Natural killer cell activity and quality of life were improved by consumption of a mushroom extract, Agaricus blazei Murill Kyowa, in gynecological cancer patients undergoing chemotherapy.

A mushroom extract, Agaricus blazei Murill Kyowa (ABMK), has been reported to possess antimutagenic and antitumor effects. Here, we investigate the beneficial effects of ABMK consumption on immunological status and qualities of life in cancer patients undergoing chemotherapy. One hundred cervical, ovarian, and endometrial cancer patients were treated either with carboplatin (300 mg / m(2)) plus VP16 (etoposide, 100 mg / m(2)) or with carboplatin (300 mg / m(2)) plus taxol (175 mg / m(2)) every 3 weeks for at least three cycles with or without oral consumption of ABMK. We observed that natural killer cell activity was significantly higher in ABMK-treated group (ANOVA, n = 39, P < 0.002) as compared with nontreated placebo group (n = 61). However, no significant difference in lymphokine-activated killer and monocyte activities was observed in a manner similar to the count of specific immune cell populations between ABMK-treated and nontreated groups. However, chemotherapy-associated side effects such as appetite, alopecia, emotional stability, and general weakness were all improved by ABMK treatment. Taken together, this suggests that ABMK treatment might be beneficial for gynecological cancer patients undergoing chemotherapy.

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo.

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.

Enhancement of VP1-specific immune responses and protection against EMCV-K challenge by co-delivery of IL-12 DNA with VP1 DNA vaccine.

It has been reported that co-delivery of IL-12 DNA with a DNA vaccine further enhances antigen (Ag)-specific protective immunity in pathogenic challenge models. However, the enhancing effects of antibody by IL-12 have been controversial. To clarify this issue, we constructed an IL-12 expression vector, co-immunized IL-12 DNA with an encephalomyocarditis virus (EMCV)-D VP1 plasmid vaccine, and then evaluated immune modulatory effects and protection against lethal EMCV-K challenge. We observed that VP1-specific IgG production, as well as seroconversion rates, were significantly enhanced by IL-12 co-injection, indicating that IL-12 can enhance antibody responses in this model system. In particular, co-injection with VP1 plus IL-12 DNA into the same leg enhanced systemic Ag-specific IgG production to a significantly greater extent than either the separate leg injection of VP1 and IL-12 DNA or VP1 DNA vaccine alone. This suggests that local co-expression of IL-12 along with antigens is more important for enhanced antibody production. Furthermore, IgG2a isotype was significantly enhanced by IL-12 DNA co-injection, indicating a Th1 bias. In addition, co-delivery of IL-12 DNA was demonstrated to enhance VP1-specific Th cell proliferative responses. When animals were challenged with a lethal dose of EMCV-K, IL-12 DNA-co-immunized animals exhibited enhanced survival, as compared to VP1 DNA vaccine alone. These studies suggest that IL-12 plays an important role in increasing Ag-specific Th1 type antibody and cellular responses, resulting in enhanced protection against lethal EMCV-K challenge.

Interleukin 7 can enhance antigen-specific cytotoxic-T-lymphocyte and/or Th2-type immune responses in vivo.

Interleukin 7 (IL-7) protein has been reported to be important in the development of cytotoxic-T-lymphocyte (CTL) responses. However, other studies also support a partial Th2 phenotype for this cytokine. In an effort to clarify this unusual conflict, we compared IL-7 along with IL-12 (Th1 control) and IL-10 (Th2 control) for its ability to induce antigen (Ag)-specific CTL and Th1- versus Th2-type immune responses using a well established DNA vaccine model. In particular, IL-7 codelivery showed a significant increase in immunoglobulin G1 (IgG1) levels compared to IgG2a levels. IL-7 coinjection also decreased production of Th1-type cytokine IL-2, gamma interferon, and the chemokine RANTES but increased production of the Th2-type cytokine IL-10 and the similarly biased chemokine MCP-1. In herpes simplex virus (HSV) challenge studies, IL-7 coinjection decreased the survival rate after lethal HSV type 2 (HSV-2) challenge compared with gD plasmid vaccine alone in a manner similar to IL-10 coinjection, whereas IL-12 coinjection enhanced the protection, further supporting that IL-7 drives immune responses to the Th2 type, resulting in reduced protection against HSV-2 challenge. Moreover, coinjection with human immunodeficiency virus type 1 env and gag/pol genes plus IL-12 or IL-7 cDNA enhanced Ag-specific CTLs, while coinjection with IL-10 cDNA failed to influence CTL induction. Thus, IL-7 could drive Ag-specific Th2-type cellular responses and/or CTL responses. These results support that CTLs could be induced by IL-7 in a Th2-type cytokine and chemokine environment in vivo. This property of IL-7 allows for an alternative pathway for CTL development which has important implications for host-pathogen responses.

LFA-3 plasmid DNA enhances Ag-specific humoral- and cellular-mediated protective immunity against herpes simplex virus-2 in vivo: involvement of CD4+ T cells in protection.

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses. To investigate specific roles of adhesion molecules in immune induction we coimmunized LFA-3 and ICAM-1 cDNAs with a gD plasmid vaccine and then analyzed immune modulatory effects and protection against lethal herpes simplex virus (HSV)-2 challenge. We observed that gD-specific IgG production was enhanced by LFA-3 coinjection. However, little change in IgG production was observed by ICAM-1 coinjection. Furthermore, both Th1 and Th2 IgG isotype production was driven by LFA-3. LFA-3 also enhanced Th cell proliferative responses and production of interleukin (IL)-2, interferon-gamma, IL-4, and IL-10 from splenocytes. In contrast, ICAM-1 showed slightly increasing effects on T-cell proliferation responses and cytokine production. beta-Chemokine production (RANTES, MIP-1alpha, and MCP-1) was also influenced by LFA-3 or ICAM-1. When animals were challenged with a lethal dose of HSV-2, LFA-3-coimmunized animals exhibited an enhanced survival rate, as compared to animals given ICAM-1 or gD DNA vaccine alone. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. These studies demonstrate that adhesion molecule LFA-3 can play an important role in generating protective antigen-specific immunity in the HSV model system through increased induction of CD4+ Th1 T-cell subset.

Macrophage colony-stimulating factor can modulate immune responses and attract dendritic cells in vivo.

Studies have indicated that professional APCs in the periphery, such as dendritic cells and macrophages, play an important role in initiating DNA vaccine-specific immune responses. To engineer the immune response induced by DNA vaccines in vivo we investigated the modulatory effects of codelivering growth factor genes for the hematopoietic APCs along with DNA vaccines. Specifically, we examined the effects on the antigen-specific immune responses following the codelivery of the gene expression cassettes for M-CSF, G-CSF, and GM-CSF along with HIV-1 DNA immunogen constructs. We observed that coimmunization with GM-CSF increased the antibody response and resulted in a significant enhancement of lymphoproliferative response. Furthermore, among all coinjection combinations, we found that M-CSF coinjections resulted in a high level of CTL enhancement. This enhancement of CTL responses observed from the coinjection with M-CSF was CD8+ T cell dependent and was associated with the presence of CD11c+ cells at the site of injection and with the antigen-specific induction of the beta-chemokine MIP-1beta, suggesting a role for this chemokine in CTL induction. These results suggest that hematopoietic growth factors should be further studied as potential adjuvants for in vivo modulators of immune responses.

Engineering of DNA vaccines using molecular adjuvant plasmids.

These studies support the view that additional goals of enhancing DNA vaccine technology will probably be at several levels. The ability to deliver antigens more efficiently to professional APCs is likely to have important implications for our studies of basic principles of immunology. Furthermore, there are simple practical approaches to vaccine enhancement that can be tested with the present group of DNA vaccines. These studies should include the use of cytokine molecular adjuvants as well as possible co-stimulatory molecules. It is expected that the delivery of these "adjuvanted" DNA vaccines will require additional safety evaluation; however, it is clear that studies can be easily designed to address the important safety issues associated with these novel vaccine adjuvants. Overall, the results indicate that further more precise quantitative studies and combination studies examining these additional promising adjuvant candidates are warranted.

Improving DNA vaccines targeting viral infection.

DNA vaccination techniques have been recently under intensive investigation both preclinically and in human studies aimed at impacting viral infection. Collectively, DNA vaccines expressing viral antigens induce both antigen-specific humoral and cellular immune responses which in model systems are capable of impacting viral infection. However, in clinical settings the potency of this approach is still under investigation. Efficacy is improved in specific circumstances through the addition of immunomodulatory molecules including cytokines as plasmid cassettes or through modification of the numbers of specific CpG sequences present in the backbone. Furthermore, combined vaccination schemes have been an important research focus for generating enhanced immunogenicity against viral infections. The ultimate utility of these approaches to prevent viral infection will require more work. However, improvements in the potency and focus of DNA vaccines present us with new opportunities for both basic research into protective immunity as well as novel strategies for immune therapy and prophylaxis.

Protective immune correlates can segregate by vaccine type in a murine herpes model system.

A central tenet of vaccine development is to identify immune correlates of protection. Both plasmid-encoded gD as well as recombinant protein gD can protect mice from lethal herpes simplex virus (HSV) challenge. It is known that different vaccine modalities should induce different immune phenotypes. Yet, paradoxically, it is also thought that the basis for protection should rely on exploitation of vulnerabilities of the pathogen and therefore that the overlapping properties of these different vaccines would reveal insight into common immune mechanisms responsible for protection. We sought to investigate this question by comparing two different vaccine modalities in the HSV-2 mouse model. We observed that gD protein was a strong inducer of T(h)2-type immune responses, and overall antibody titers of IgG, IgE and IgA were significantly higher than those induced by plasmid gD vaccines. In contrast, the plasmid gD vaccine induced a strong T(h)1 bias. Following high-dose challenge the gD protein was most effective at providing protection. However, at lower lethal dose challenge, while both vaccines were protective with regards to survival, only the plasmid-vaccinated animals were protected from HSV-2 infection-induced morbidity. These studies suggest that these different vaccine modalities induce protection through unique non-overlapping mechanisms, supporting that vaccine correlates are associated with the types of immunogen rather than solely the pathogen.

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.

It has previously been reported that herpes simplex virus (HSV)-2 gD DNA vaccine preferentially induces T-helper (Th) 1-type cellular immune responses, whereas the literature supports the view that subunit vaccines tend to induce potent antibody responses, supporting a Th2 bias. Here, using an HSV gD vaccine model, we investigated whether priming and boosting with a DNA or protein vaccine could induce both potent antibody and Th1-type cellular immune responses. When animals were primed with DNA and boosted with protein, both antibody and Th-cell proliferative responses were significantly enhanced. Furthermore, production of Th1-type cytokines (interleukin-2, interferon-gamma) was enhanced by DNA priming-protein boosting. In contrast, protein priming-DNA boosting produced antibody levels similar to those following protein-protein vaccination but failed to further enhance Th-cell proliferative responses or cytokine production. DNA priming-protein boosting resulted in an increased IgG2a isotype (a Th1 indicator) profile, similar to that induced by DNA-DNA vaccination, whereas protein priming-DNA boosting caused an increased IgG1 isotype (a Th2 indicator) profile similar to that seen after protein-protein vaccination. This result indicates that preferential induction of IgG1 or IgG2a isotype is determined by the type of priming vaccine used. Thus, this study suggests that HSV DNA priming-protein boosting could elicit both potent Th1-type cellular immune responses and antibody responses, both of which likely are important for protection against HSV infection.

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.

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.

Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses from the codelivery of Thl cytokines (interleukin-2 [IL-2] and IL-12), Th2 cytokines (IL-4 and IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes along with a DNA vaccine construct encoding for simian immunodeficiency virus (SIV) gag/pol proteins. We observed that coinjection with IL-2, IL-4, IL-10, and GM-CSF resulted in increased levels of antigen-specific antibodies. In addition, we found that coinjection with cytokine genes drove the immune responses toward a more Thl or Th2 phenotype. We also observed that coadministration of IL-2, IL-12, and GM-CSF genes resulted in a dramatic enhancement of Th proliferation responses. Moreover, coimmunization with IL-12 genes resulted in a dramatic enhancement of antigen-specific cytotoxic T lymphocyte (CTL) responses. These results support the potential utility of molecular adjuvants in DNA vaccine regimens.

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.

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4(+)-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.

Enhancement of protective humoral (Th2) and cell-mediated (Th1) immune responses against herpes simplex virus-2 through co-delivery of granulocyte-macrophage colony-stimulating factor expression cassettes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) could in theory attract antigen-presenting cells in muscle following intramuscular DNA immunization, resulting in enhanced antigen-specific immune responses. Thus, such adjuvants could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of GM-CSF cDNA as a vaccine adjuvant for herpes simplex virus (HSV)-2 in a mouse challenge model. GM-CSF cDNA co-injection enhanced levels of specific IgG, IgE and IgA against HSV-2 gD protein significantly higher than gD plasmid vaccination alone. Moreover, GM-CSF co-injection induced a dramatic increase in IgG1 levels, as compared to IgG2a levels, suggesting a Th2 bias in the response. T helper cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by GM-CSF cDNA co-injection. When challenged with a lethal dose of HSV-2, GM-CSF co-injection increased survival rates to 90%, an improvement as compared to gD vaccination alone (60-63%). Furthermore, GM-CSF cDNA co-injection reduced herpetic lesions and resulted in a faster recovery from lesions. These data indicate that GM-CSF cDNA enhances both humoral and cellular immune responses and enhances vaccine efficacy, resulting in reduced HSV-2-derived morbidity as well as mortality.

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.

Coadministration of IL-12 or IL-10 expression cassettes drives immune responses toward a Th1 phenotype.

Cytokines are important regulators of the immune response. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation as well as allergic responses. In a model system in mice, we have studied the effect of plasmids expressing interleukin (IL)-10 or IL-12 on the modulation of antigen-specific responses. Coadministration of IL-12 or IL-10 genes with DNA immunogens directed the antigen-specific immune response toward a T helper (Th1)-type immunity. In addition to the modulation of antigen-specific immune responses, we studied the induction of delayed-type hypersensitivity (DTH) to contact allergens as an in vivo model of the Th1 response. We found that IL-12 and IL-10 gene-containing plasmids, and not the bacterial plasmid alone, upregulate this response. Our cytokine gene delivery technique demonstrates an important level of control of the magnitude and direction of induced immune responses and could be advantageous in a wide variety of immunotherapeutic strategies.

Protective immunity against heterologous challenge with encephalomyocarditis virus by VP1 DNA vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene.

For DNA vaccination studies, recombinant VP1 protein of encephalomyocarditis virus (EMCV) was produced from Escherichia coli, and eukaryotic VP1 expression vector, pCT-Gs-VP1, was generated and used as a DNA vaccine. Mice were immunized intramuscularly (i.m.) with pCT-Gs-VP1 in the presence or absence of plasmid DNA expressing granulocyte-macrophage colony stimulating factor (GM-CSF), and were subsequently analyzed for their anti-VP1 immune responses with recombinant VP1 in ELISA. Immunization of mice with pCT-Gs-VP1 resulted in VP1-specific immune response and 43% protection from subsequent lethal heterologous challenge of EMCV. Coinjection of mice with pCT-Gs-VP1 and plasmid DNA encoding GM-CSF was shown to increase the seroconversion rate of the immunized mice with a single DNA injection, and enhanced to a higher degree VP1-specific immunity, which appeared to result in better protection (about 80%) from lethal virus challenge. Thus, our results provide evidence for the potential use of GM-CSF to induce better immune response and resistance against viral infection in DNA vaccination.