Cytokine-expressing Plasmids Research Articles

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

AbstractIL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12 transformed pathogen was safe upon immunization with low doses (103 CFU), induced IgG responses, and stimulated protective immune responses against Salmonella Typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring IL-12 secreting plasmids may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.

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.

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Systemic and mucosal immune responses in mice after rectal and vaginal immunization with HIV-DNA vaccine.

We examined the feasibility of inducing local and systemic human immunodeficiency virus (HIV)-specific immune responses by rectal and vaginal application of an HIV-DNA vaccine. Mice were immunized with an HIV-DNA vaccine preparation via a rectal or vaginal route. After several applications, HIV-specific antibodies were detected in sera, fecal extract solutions, and vaginal washes, and these antibodies were potent in inhibiting the syncytium formation of a CD4-positive human T cell line by a cell line capable of inducing HIV-1 infection. Spleen cells from rectally and vaginally immunized mice showed antigen-mediated IFN-gamma-inducing activity. In addition, with rectal immunization, mononuclear cells from both the spleen and the regional lymph nodes of the rectal region were found to be potent at inducing a cytotoxic T lymphocyte response. These humoral and cell-mediated immune responses were enhanced by augmenting the vaccine with granulocyte-macrophage colony-stimulating factor-expressing plasmids or IL-12-expressing plasmid. Our results demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunity and that these responses were enhanced by the addition of the above cytokine-expressing plasmids.

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.

DNA immunization of HLA transgenic mice with a plasmid expressing mycobacterial heat shock protein 65 results in HLA class I- and II-restricted T cell responses that can be augmented by cytokines.

Infection with Mycobacterium tuberculosis (MTB) remains a major cause of morbidity and mortality world-wide. An effective vaccination strategy is the immunization with plasmid DNA (pDNA), expressing an antigen (Ag) from a pathogen in vivo, which results in specific immune response against the encoded protein as well as the pathogen itself or cells infected with it. To test the ability to induce HLA-restricted T cell immune response against a mycobacterial antigen in humans by pDNA vaccination, we have used transgenic mice that express HLA class I (A*0201/Kb) or HLA class II (DRB1*0301) molecules. pDNA immunization with mycobacterial heat shock protein 65 (Mhsp65)-expressing plasmid (P3M.65) resulted in HLA-II-restricted, Ag-specific T cell-mediated immune responses characterized by proliferation and cytokine production. These T cell responses could be further augmented by the coinjection of P3M.65 and plasmid expressing murine GM-CSF. Furthermore, coimmunizing HLA-I transgenic mice with P3M.65 and a plasmid expressing murine IFN-gamma induced a specific cytotoxic T lymphocyte response restricted by HLA-A2. These results represent the first evidence of a concomitant in vivo induction of HLA class I- as well as class II-restricted T cell responses by pDNA immunization, which is induced or augmented by the codelivery of cytokine-expressing plasmids, supporting its potential use in clinical trials.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

Genetic immunization of BALB/c mice with a plasmid bearing the gene coding for a hybrid merozoite surface protein 1-hepatitis B virus surface protein fusion protects mice against lethal Plasmodium chabaudi chabaudi PC1 infection.

The genetic immunization of rodents with a plasmid coding for a Plasmodium chabaudi merozoite surface protein 1 (C terminus)-hepatitis B virus surface fusion protein (pPcMSP1(19)-HBs) provided protection of mice against subsequent lethal challenge with P. chabaudi chabaudi PC1-infected red blood cells. The percentage of survivor mice was higher in DNA-immunized mice than in animals immunized with a recombinant rPcMSP1(19)- glutathione S-transferase fusion protein administered in Freund adjuvant. In all mice immunized with the pPcMSP1(19)-HBs, a Th1-specific response, including the production of anti-MSP1(19)-specific immunoglobulins predominantly of the immunoglobulin G2a subtype and reacting almost exclusively against discontinuous epitopes, was elicited. The coinjection of Th1-type cytokine-expressing plasmids (gamma interferon, interleukin-2, and granulocyte-macrophage colony-stimulating factor) mostly abolished protection and boosting of MSP1(19)-specific antibodies. The inclusion of a lymph node-targeting signal did not significantly increase protection. These data provide further evidence that MSP1(19)-HBs DNA constructs might be useful as components of a genetic vaccine against the asexual blood stages of Plasmodium.

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.

To assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. Four chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/rev, gag/pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. There was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. Immunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.

Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer.

Intramuscular (i.m.) injections of a plasmid encoding human carcinoembryonic antigen (CEA) elicited both humoral and cellular immune responses in mice, but only partial inhibition of the growth of transplanted syngeneic CEA-positive P815 tumor cells (CEA/P815). Coinjection of the CEA vector with a vector encoding either interferon-gamma (IFN gamma) or IL-12 promoted IgG2a isotype anti-CEA antibody production, anti-CEA/P815 CTL activity and greater resistance to CEA/P815 tumor challenge. As well, CEA/P815-stimulated IFN gamma secretion in vitro was increased, but IL-4 diminished, consistent with a T-helper type 1 (Th1) response. In contrast, coinjection of the CEA vector with an IL-4 vector increased IgG1 production, but reduced CTL activity and resistance to tumor challenge. The latter treatment inhibited CEA/P815-dependent IFN gamma production but enhanced IL-4 secretion, consistent with a Th type 2 (Th2) response. Antitumor immunity was enhanced when the CEA and IL-12 plasmids were coinjected at the same muscle site, but not at separate sites despite increased serum IL-12 levels. Though the tumor cells expressed neomycin phosphotransferase, mice immunized with vectors encoding that protein (without CEA) were not protected against tumor growth, and produced no CTLs except for low levels when coinjected with an IL-12 vector. Thus, we show that immunity elicited by DNA vaccination against CEA can be biased to a protective type (high Th1 and CTL activity) or nonprotective type (high Th2 and low CTL activity) by i.m. coinjection of cytokine-expressing plasmids. IL-12 appears to act locally, but not systemically, through an adjuvant effect.

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations.

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.

Inhibitory effects of chronic ethanol consumption on cellular immune responses to hepatitis C virus core protein are reversed by genetic immunizations augmented with cytokine-expressing plasmids.

Chronic hepatitis C viral (HCV) infection is a major clinical problem in alcoholics with liver disease and may result from ethanol effects on the host immune response. To experimentally assess such effects, the DNA-based immunization approach was used to produce humoral and cellular immune responses against the HCV core protein in mice. Mice were fed an ethanol or isocaloric pair-fed control liquid diet followed by immunizations with HCV core DNA-expressing constructs. Chronic ethanol feeding was found to inhibit Th cell and CTL activities and substantially reduced cytokine secretion as well. In addition, a switch from Th1 to Th0 subtype was observed in proliferating CD4+ T cells derived from chronic ethanol-fed mice. These immunosuppressive effects were directly due to ethanol since crossover experiments to an isocaloric control diet restored the defects in cellular immunity. Furthermore, we determined if coadministration of an IL-2 or GM-CSF DNA expression plasmid with a plasmid expressing the HCV core protein (pHCV2-2) would reverse the inhibitory effects of chronic ethanol feeding on cellular immune responses. Coimmunization of chronic ethanol-fed mice with either IL-2 or GM-CSF expression plasmids restored cellular immunity and induced CD4+ inflammatory T cell and CD8+ CTL responses comparable with control mice immunized with pHCV2-2 alone. These studies provide evidence of how chronic ethanol feeding may effect cellular immune responses to a viral structural protein in the context of genetic immunization.

Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA-based vaccines augmented with cytokine-expressing plasmids.

Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for irradication of infection. DNA-based immunization is a promising approach to generate HCV-specific immune responses. Previous studies of DNA-based immunizations in mice using an HCV core DNA expression plasmid (pHCV2-2) demonstrated an efficient CTL response against HCV core epitopes; however, the humoral and Th cell proliferative responses were found to be weak. To enhance the immunogenicity of this nonsecreted viral structural protein at the B and T cell level, we coimmunized mice with pHCV2-2 and DNA expression constructs encoding for mouse IL-2, IL-4, and granulocyte-macrophage CSF proteins. Under these experimental conditions, a seroconversion frequency to anti-HCV core increased from 40 to 80% in immunized mice. The CD4+ inflammatory T cell proliferative responses as well as CD8+ CTL activity to HCV core protein were enhanced substantially after coimmunization with the IL-2 and granulocyte-macrophage CSF DNA expression constructs. In contrast, coimmunization with an IL-4-producing construct induced differentiation of Th cells toward a Th0 subtype and suppressed HCV core-specific CTL activity. Taken together, these studies emphasize that generation of antiviral immune responses using DNA-based immunization may be modified by local cytokine production at the site of Ag presentation.