Granulocyte-macrophage Colony-stimulating Factor Plasmids Research Articles

Intradermal Delivery of Cell Vaccine via Ice Microneedles for Cancer Treatment.

The living tumor cell vaccine (TCV) holds a promise for cancer immunotherapy. Microneedle arrays provide a tool to improve the immune response of vaccines by the intradermal administration in a painless manner. However, it remains challenges for microneedle arrays to deliver the living TCV intradermally. Here, an ice microneedle array delivered living TCVsis shown with sustained granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion for cancer treatment. The ice microneedle array is composed of ice microneedles and a matching polymer holder, which are customized fabricated by a static optical projection lithography (SOPL) technique. The living TCV consisted ofirradiated melanoma cells transfected with nanoparticle-mediated GM-CSF plasmids. After the living TCV is readily loaded into the ice microneedle via a cryopreservation process, it couldbe efficiently delivered into the dermis by the microneedle device. Compared to the subcutaneous injection, intradermal administration led to the recruitment of more dendritic cells at the vaccination site and the increased infiltration of CD8+ T cells in the tumor. The ice microneedle array deliveres intradermal TCVssignificantly inhibited melanoma growth and effectively prevented melanoma recurrence without obvious side effects. This work demonstrates a promising TCVs for melanoma treatment, which will inspire the future of cancer immunotherapy.

Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells.

Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Variable effects of the co-administration of a GM-CSF-expressing plasmid on the immune response to flavivirus DNA vaccines in mice

As a cytokine adjuvant, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been demonstrated to play central roles in the enhancement of the immune response and protection elicited by experimental vaccines. However, in our previous work, the co-administration of GM-CSF produced untoward effects on the immune response induced by a Japanese encephalitis virus DNA vaccine candidate. This study aimed to elucidate the adjuvant roles of GM-CSF in several Flaviviridae virus DNA vaccine candidates. Our results showed that the effects of GM-CSF were diverse: co-inoculated GM-CSF caused significant suppression to the dengue virus type 1 and type 2 prM-E DNA vaccinations and influenced protective efficiency against virus challenge. In contrast, GM-CSF showed little effect or an enhancement on the immune response elicited by hepatitis C virus C or E1 DNA vaccine candidates. Notably, these effects of GM-CSF were highly durable. Our results suggested that the adjuvant roles of the GM-CSF plasmid were complex and diverse, ranging from enhancement to suppression, depending on the immunogen of Flaviviridae virus DNA vaccine candidates. Therefore, the application of GM-CSF as a vaccine adjuvant or a therapeutic agent should be evaluated carefully.

Irreversible electroporation facilitates gene transfer of a GM-CSF plasmid with a local and systemic response

Electroporation uses an electric field to induce pores in the cell membrane that can transfer macromolecules into target cells. Modulation of electrical parameters leads to irreversible electroporation (IRE), which is being developed for tissue ablation. We sought to evaluate whether the application of IRE may induce a lesser electric field in the periphery where reversible electroporation may occur, facilitating gene transfer of a granulocyte macrophage colony-stimulating factor (GM-CSF) plasmid to produce its biologic response. Yorkshire pigs underwent laparotomy, and IRE of the liver was performed during hepatic arterial infusion of 1 or 7 mg of a naked human GM-CSF plasmid. The serum, liver, lymph nodes, and bone marrow were harvested for analysis. Human GM-CSF level rose from undetectable to 131 pg/mL in the serum at 24 hours after IRE and plasmid infusion. The liver demonstrated an ablation zone surrounded by an immune infiltrate that had greater macrophage intensity than when treated with IRE or plasmid infusion alone. This dominance of macrophages was dose dependent. Distant effects of GM-CSF were found in the bone marrow, where proliferating myeloid cells increased from 14% to 25%. IRE facilitated gene transfer of the GM-CSF plasmid and brought about a local and systemic biologic response. This technique holds potential for tumor eradication and immunotherapy of residual cancer.

Suppressive Effects on the Immune Response and Protective Immunity to a JEV DNA Vaccine by Co-administration of a GM-CSF-Expressing Plasmid in Mice

As a potential cytokine adjuvant of DNA vaccines, granulocyte-macrophage colony–stimulating factor (GM-CSF) has received considerable attention due to its essential role in the recruitment of antigen-presenting cells, differentiation and maturation of dendritic cells. However, in our recent study of a Japanese encephalitis virus (JEV) DNA vaccine, co-inoculation of a GM-CSF plasmid dramatically suppressed the specific IgG response and resulted in decreased protection against JEV challenge. It is known that GM-CSF has been used in clinic to treat neutropenia for repopulating myeloid cells, and as an adjuvant in vaccine studies; it has shown various effects on the immune response. Therefore, in this study, we characterized the suppressive effects on the immune response to a JEV DNA vaccine by the co-administration of the GM-CSF-expressing plasmid and clarified the underlying mechanisms of the suppression in mice. Our results demonstrated that co-immunization with GM-CSF caused a substantial dampening of the vaccine-induced antibody responses. The suppressive effect was dose- and timing-dependent and likely related to the immunogenicity of the antigen. The suppression was associated with the induction of immature dendritic cells and the expansion of regulatory T cells but not myeloid-derived suppressor cells. Collectively, our findings not only provide valuable information for the application of GM-CSF in clinic and using as a vaccine adjuvant but also offer further insight into the understanding of the complex roles of GM-CSF.

Co-expression of Japanese encephalitis virus prM–E–NS1 antigen with granulocyte-macrophage colony-stimulating factor enhances humoral and anti-virus immunity after DNA vaccination

Japanese encephalitis virus (JEV) is an agent of Japanese encephalitis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) is an attractive DNA vaccine adjuvant for its antigen presentation. In the present study, we have constructed DNA vaccines that carried JEV prM–E–NS1 genes with or without the GM-CSF gene. Immunization with the bicistronic plasmid pCAG-JEGM that co-expresses GM-CSF and viral prM–E–NS1, resulted in the highest IgG response and sufficient protection against virus-challenged BALB/c mice. However, much to our surprise, co-inoculation of the GM-CSF plasmid with the pCAG-JE plasmid expressing viral prM–E–NS1 lead to a low antibody titer and a relatively low survival rate. Moreover, anamnestic antibody-mediated protection played a dominant role in the mice JEV challenge model, according to the enhancement of post-challenge neutralizing antibody titers and further adoptive transfer experiments. Taken together, this study should encourage further development of JEV DNA vaccine strategies and caution against the use of cytokines as an adjuvant.

Vaccination with vif-deleted feline immunodeficiency virus provirus, GM-CSF, and TNF-α plasmids preserves global CD4 T lymphocyte function after challenge with FIV

Feline immunodeficiency virus (FIV) DNA vaccine approaches that included a vif-deleted FIV provirus (FIV-pPPRΔvif) and feline cytokine expression plasmids were tested for immunogenicity and efficacy by immunization of specific pathogen free cats. Vaccine protocols included FIV-pPPRΔvif plasmid alone; a combination of FIV-pPPRΔvif DNA and feline granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α expression plasmids; or a combination of FIV-pPPRΔvif and feline interleukin (IL)-15 plasmids. Cats immunized with FIV-pPPRΔvif, GM-CSF and TNF-α plasmids demonstrated an increased frequency of FIV-specific T cell proliferation responses compared to other vaccine groups. Immunization with FIV-pPPRΔvif and IL-15 plasmids was distinguished from other vaccine protocols by the induction of antiviral antibodies. Suppression of virus loads was not observed for any of the FIV-pPPRΔvif DNA vaccine protocols after challenge with the FIV-PPR isolate. However, prior immunization with FIV-pPPRΔvif, GM-CSF, and TNF-α plasmids resulted in preservation of CD4 T cell functions, including mitogen-induced cytokine expression and antigen-specific proliferation upon infection with FIV. These findings justify further examination of cytokine combinations as adjuvants for lentiviral DNA vaccines.

Gene therapy of neural cell injuries in vitro using the hypoxia-inducible GM-CSF expression plasmids and water-soluble lipopolymer (WSLP)

Non-viral polymeric gene carriers have been widely investigated but no promising biocompatible polymer was developed for the gene therapy of neural system injuries yet. This study evaluated the potential usage of water-soluble lipopolymer (WSLP) as a gene delivery vehicle in neural lineage cells of SK-N-BE(2)C, a neuroblastoma cell line and primary culture of mouse neural progenitor cells (mNPCs). When tested with the luciferase reporter (pSV-Luc), WSLP showed higher gene transfection efficiency by more than 8–10 folds yet with lower cytotoxicity than polyethylenimine of 1800 Da (PEI1800), a parental polymer, and Lipofectamine 2000. The optimum N/P ratios were 40:1 for WSLP and 10:1 for PEI1800, respectively. The transfection efficiency for both of WSLP and PEI1800 was higher overall in SK-N-BE(2)C cells than in mNPCs. WSLP was also used successfully for the delivery and hypoxia-inducible expression of luciferase reporter plasmid containing the erythropoietin (Epo) enhancer (pEpo-SV-Luc) or RTP801 promoter (pRTP801-Luc). The hypoxia-inducible system and WSLP were then successfully applied to the delivery of granulocyte macrophage colony-stimulating factor (GM-CSF) gene that was previously shown to have neuroprotective effect on neural cell death in vitro and in rat SCI model. The hypoxia-inducible GM-CSF plasmids (pEpo-SV-GM-CSF and pRTP801-GM-CSF) showed induced expression of GM-CSF under hypoxia and decrease in the hypoxia-induced cell death in SK-N-BE(2)C cells. In conclusion, this study demonstrated that WSLP could be an efficient gene delivery carrier for neural cells and gene therapy of GM-CSF using the hypoxia-inducible system could be a potential therapeutic intervention for neural injuries. Further studies are necessary to confirm the current findings in animal models of CNS injuries.

Novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 gag

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. Here we generated a novel codon-optimized murine GM-CSF gene as an adjuvant. The codon-optimized GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag. This novel codon-optimized GM-CSF gene offers a practical molecular strategy for potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 Gag

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. Here we generated a novel codon-optimized murine GM-CSF gene as an adjuvant. The codon-optimized GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag. This novel codon-optimized GM-CSF gene offers a practical molecular strategy for potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Evaluation of protective effect of DNA vaccination with genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against acute, chronical and congenital toxoplasmosis in mice

To develop a multiantigenic vaccine against toxoplasmosis, two Toxoplasma gondii antigens, SAG1 and GRA4 selected on the basis of previous immunological and immunization studies, were chosen. We showed that DNA-based immunization with plasmids expressing GRA4 (pGRA4) or SAG1 (pSAG1mut) reduced mortality of susceptible C57BL/6 mice upon oral challenge with cysts of the 76K type II strain (62% survival). Immunization with pGRA4 and pSAG1mut, enhanced the protection (75% survival). This protection was further increased by co-inoculation with a plasmid encoding the granulocyte-macrophage colony-stimulating factor (GM-CSF) (87% survival). This latter DNA cocktail provided significant protection of less susceptible outbred Swiss OF1 mice against the development of cerebral cysts. A significantly higher survival of newborns from immunized outbred mice exposed to infection during gestation was observed (4.25 ± 3.77 live pups/litter) in comparison to non-immunized mice (1.08 ± 2.15 live pups/litter) without preventing parasite vertical transmission. Analysis of the immune response showed that protected animals developed a specific humoral and cellular Th1 response to native T. gondii SAG1 and GRA4 antigens. Our data demonstrated that protection was improved by associating antigens (SAG1 and GRA4) and cytokine (GM-CSF) for further development of a multiantigenic vaccine against toxoplasmosis.

579. Generation of Potent Cellular Immune Responses Induced by Intradermal Delivery of Plasmid DNA Coexpressing Flt3 Ligand and GM-CSF

The protective efficacy of DNA vaccine has been demonstrated in murine models against infectious diseases or tumor growth. Recently, Fms-like tyrosine kinase 3-ligand (Flt3L) has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). Administration of Flt3L protein or recombinant DNA encoding Flt3L into mice results in the expansion of the DC population in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also critical for DC survival and differentiation in vitro. Previous studies have shown that codelivery of Flt3L-encoding and GM-CSF-encoding plasmids with antigen results in increase in DC numbers in comparison with codelivery of either plasmid alone. In this study, we constructed a bicistronic plasmid vector, Flt3L-IRES-GM-CSF plasmid, coexpressing murine Flt3L lacking intracellular domain and GM-CSF by using an internal ribosome entry site (IRES). Expressions of GM-CSF and Flt3L could be detected in COS-7 cells transfected with the plasmid. The immune stimulatory activity of plasmid vector coexpressing Flt3L and GM-CSF (coexpressed DNA vaccine) in comparison with codelivery of Flt3L and GM-CSF plasmids (codelivered DNA vaccine) was studied in mice. We first immunized mice by intradermal injection of various plasmid vectors, including 40 μg Flt3L-IRES-GM-CSF plasmid, 40 μg Flt3L plasmid plus 40 μg GM-CSF plasmid, 40 μg Flt3L plasmid alone, 40 μg GM-CSF plasmid alone, or 40 μg control plasmid. Expressions of Flt3L and GM-CSF were not detected in the sera of mice receiving intradermal injection of the plasmids. Next, we showed the enhanced recruitment of DCs to the immunization site and proliferation of DCs and T cells in the spleen and lymph node. The populations of DC, CD4+, and CD8+ T cells were increased significantly by approximately 50% to 60% in mice receiving coexpressed DNA vaccine compared with those in mice receiving control plasmid. Compared to mice treated with codelivered DNA vaccine, there were 15% and 41% increases in the populations of DC and CD4+ T cells, respectively. The CD4+ but not CD4+CD25+ T cells were found to be the major group in the increased T-cell population in the spleen and lymph node. We will investigate the function of the DCs harvested from mice receiving coexpressed DNA plasmid by mixed lymphocyte reaction and antigen-specific T cell proliferation. In addition, we found that there was an increase, ranging from 20% to 40%, in the populations of MHC class II+, CD4+, and CD8+ T cells in the spleen of mice bearing syngeneic bladder tumor that received coexpressed DNA vaccine compared with those that received codelivered DNA vaccine or control plasmid. Immunization of mice with coexpressed DNA vaccine delayed bladder tumor growth. Taken together, regarding the application of Flt3L and GM-CSF, our results indicate that coexpressed DNA vaccine induces at least equal or higher immune stimulatory response compared with codelivered DNA vaccine, while only half the amount of DNA is needed. Currently, we are using the coexpressed DNA vaccine in combination with plasmid DNA encoding tumor epitopes or tumor-specific oncolytic virus for immune-gene therapy of cancer.

Genetic immunization elicits antigen-specific protective immune responses and decreases disease severity in Trypanosoma cruzi infection.

Immunity to Trypanosoma cruzi requires elicitation of humoral and cell-mediated immune responses to extracellular trypomastigotes and intracellular amastigotes. In this study, the effectiveness of the T. cruzi trans-sialidase family (ts) genes ASP-1, ASP-2, and TSA-1 as genetic vaccines was assessed. Immunization of mice with plasmids encoding ASP-1, ASP-2, or TSA-1 elicited poor antigen-specific cytotoxic-T-lymphocyte (CTL) activity and T. cruzi-specific antibody responses. Codelivery of interleukin-12 and granulocyte-macrophage colony-stimulating factor plasmids with antigen-encoding plasmids resulted in a substantial increase in CTL activity and antibody production and in increased resistance to T. cruzi infection. In pooled results from two to four experiments, 30 to 60% of mice immunized with antigen-encoding plasmids and 60 to 80% of mice immunized with antigen-encoding plasmids plus cytokine adjuvants survived a lethal challenge with T. cruzi. In comparison, 90% of control mice injected with empty plasmid DNA died during the acute phase of infection. However, the pool of three ts genes provided no greater protection than the most effective single gene (ASP-2) either with or without coadministration of cytokine plasmids. Importantly, the extent of tissue parasitism, inflammation, and associated tissue damage in skeletal muscles during the chronic phase of T. cruzi infection in mice immunized with antigen-encoding plasmids plus cytokine adjuvants was remarkably reduced compared to mice immunized with only cytokine adjuvants or empty plasmid DNA. These results identify new vaccine candidates and establish some of the methodologies that might be needed to develop effective vaccine-mediated control of T. cruzi infection. In addition, this work provides the first evidence that prophylactic genetic immunization can prevent the development of Chagas' disease.

In vivo generation of dendritic cells by intramuscular codelivery of FLT3 ligand and GM-CSF plasmids.

Dendritic cells (DCs) are the major cells responsible for the uptake and the transport of antigens to regional lymphoid tissues and for the presentation of antigenic peptides to T cells. They are highly effective in immunotherapy. However, in lymphoid and other tissues, DCs constitute only a small population and are difficult to isolate in large numbers. Our objective was to devise a method with which to rapidly expand splenic DCs in vivo. We accomplished this by intramuscular injection of plasmids encoding mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and fms-like tyrosine kinase 3-ligand (FLT3-L). Gene transfer was amplified by electroporation. Both cytokine vectors significantly increased DC numbers, but they were more effective in combination. When either control plasmid (Blank), or FLT3-L or GM-CSF expression plasmids were injected individually, the mean numbers of CD11c(+)/MHC II(+) DCs in spleen cell suspensions were, respectively, 6, 11, and 23 million. When FLT3-L and GM-CSF plasmids were codelivered, this increased to 36 million. Peak levels occurred 7 days postinjection of DNA. To further characterize these DCs, we stained them with myeloid (CD11b, F4/80)- and lymphoid (CD8alpha)-related markers. FLT3-L cDNA favored lymphoid DC expansion and GM-CSF cDNA favored myeloid DC expansion, whereas combined treatment expanded both types with a myeloid predominance. We confirm the ability of these DCs to present antigen to CD4(+) T cells and to stimulate in mixed lymphocyte cultures. We demonstrate that DCs can be rapidly expanded by this simple gene transfer method, which has numerous potential applications.

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-γ and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein1 (pMSP1 42) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1 42 after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1 42 produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1 19 (yMSP1 19). Immunization also induced antigen specific T cell responses as measured by interferon-γ production, and by classical CTL chromium release assays. In addition, immunization with pMSP1 42 primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1 19. We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1 42. We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1 42 induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1 19. The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

A Plasmid Encoding Murine Granulocyte-Macrophage Colony-Stimulating Factor Increases Protection Conferred by a Malaria DNA Vaccine

AbstractUsing the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-γ responses of CD8+ T cells to the PyCSP 280–288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.