Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses.
(41/2315)
The injection of plasmid DNA encoding hepatitis B virus (HBV) envelope proteins in mouse muscle leads to the induction of specific humoral and cellular immune responses. Most studies on DNA-based immunization have used viral promoters to drive antigen expression. In this study, we compared the efficiency of a muscle-specific promoter, the human desmin gene promoter, with the commonly used cytomegalovirus (CMV) early gene promoter. We showed that increased in vitro expression of HBV envelope proteins from the human desmin gene promoter has no effect on the in vivo immune response even after the injection of as little as 10 micrograms of DNA. The injection of vectors encoding HBV envelope proteins under the control of either the human desmin gene promoter or the CMV promoter induced humoral and cytotoxic immune responses at comparable levels and of the same duration. The recruitment of antigen-presenting cells to the DNA injection site by pretreatment of muscle with a necrotizing agent increases the precocity and the intensity of the responses, particularly when the nonspecific CMV vector was used. (+info)
Equine arteritis virus derived from an infectious cDNA clone is attenuated and genetically stable in infected stallions.
(42/2315)
Virus derived from an infectious cDNA clone of equine arteritis virus (EAV030H) was intranasally inoculated into two stallions, neither of which subsequently developed clinical manifestations of equine viral arteritis (EVA). Virus was isolated from nasal swabs and mononuclear cells collected from both stallions +info)
DNA immunization to prevent autoimmune diabetes.
(43/2315)
Mice expressing lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a transgene in their beta cells develop insulin-dependent diabetes mellitus (IDDM) only after LCMV infection. Inoculation of plasmid DNA encoding the insulin B chain reduced the incidence of IDDM by 50% in this model. The insulin B-chain DNA vaccination was effective through induction of regulatory CD4 lymphocytes that react with the insulin B chain, secrete IL-4, and locally reduce activity of LCMV-NP-autoreactive cytotoxic T lymphocytes in the pancreatic draining lymph node. In contrast, similar vaccination with plasmids expressing the LCMV viral ("self") protein did not prevent IDDM, because no such regulatory cells were induced. Thus, DNA immunization with plasmids expressing self-antigens might constitute a novel and attractive therapeutic approach to prevent autoimmune diseases, if the antigens are carefully preelected for an ability to induce regulatory lymphocytes in vivo. (+info)
Protective immune responses induced by vaccination with an expression genomic library of Leishmania major.
(44/2315)
To develop an effective vaccine against the intracellular protozoan parasite Leishmania spp., we investigated the feasibility of expression library immunization (ELI) in the mouse. Genomic expression libraries of L. major were constructed and used to immunize mice. One of the three libraries (L1, with 10(5) clones) induced a significant protective immune response and delayed the onset of lesion development in highly susceptible BALB/c mice after i.m. immunization, compared with control mice immunized with the empty vector (EV). L1 was then divided into five sublibraries of approximately 2 x 10(4) clones each. Mice immunized with one of the sublibraries (SL1A) developed an even stronger protective effect than that induced by L1. SL1A was further divided into 20 sublibraries (SL2) of approximately 10(3) clones each. One of the SL2 libraries (SL2G) induced a strong protective effect against L. major infection. In direct comparative studies, the protective effect of the sublibraries was in the order of SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with SL2G produced more IFN-gamma and NO, compared with cells from control mice injected with EV. Serum from the vaccinated mice also contained more parasite-specific IgG2a Ab, compared with controls. Therefore, these data demonstrate that ELI is feasible against this complex intracellular parasitic infection, by preferentially inducing the development of Th1 responses. Furthermore, by sequential division of the libraries, this approach may be used to enrich and identify protective genes for effective gene vaccination against other parasitic infections. (+info)
Predominance of CD4 Th1 and CD8 Tc1 cells revealed by characterization of the cellular immune response generated by immunization with a DNA vaccine containing a Trypanosoma cruzi gene.
(45/2315)
Immunization with a plasmid DNA containing the gene encoding the catalytic domain of trans-sialidase (TS) elicits protective immune responses against experimental Trypanosoma cruzi infection. As several studies provided strong evidence that during infection CD4 Th1 and CD8 T cytotoxic type 1 (Tc1) cells are important factors in host resistance, the present study was designed to evaluate which T-cell types were activated in DNA-vaccinated BALB/c mice. We found that bulk cells from DNA-immunized mice had CD4 and CD8 T cells that produced gamma interferon (IFN-gamma) but not interleukin-4 (IL-4) or IL-10. To characterize the TS-specific T cells at the clonal level, we generated CD4 and CD8 clones. We obtained cytotoxic CD4 clones of the Th1 type that secreted large amounts of IFN-gamma but not IL-4 or IL-10. Unexpectedly, we obtained other CD4 clones with a Th2 phenotype, secreting IL-4 and IL-10 but not IFN-gamma. All CD8 clones were cytotoxic and produced IFN-gamma. IL-4 and IL-10 were not secreted by these cells. Using synthetic peptides, we determined a CD8 epitope recognized by several clones as being represented by amino acids IYNVGQVSI. The antiparasitic activity of a CD4 Th1 and a CD8 Tc1 clone was assessed in vitro. CD4 or CD8 T cells significantly inhibited T. cruzi development in infected macrophages or fibroblasts, respectively. We concluded that DNA vaccine efficiently generates potentially protective CD4 Th1 and CD8 Tc1 cells specific for a T. cruzi antigen, therefore reinforcing the possibility of using this strategy for developing a preventive or therapeutic vaccine against Chagas' disease. (+info)
Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein.
(46/2315)
Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium. (+info)
Interleukin-12 cDNA skin transfection potentiates human papillomavirus E6 DNA vaccine-induced antitumor immune response.
(47/2315)
Human papillomaviruses are associated with >90% of all cases of uterine cervical tumors. The E6 and E7 oncoproteins of human papillomavirus are potentially ideal targets of immune therapy for cervical cancer, because their expression is necessary for cellular transformation. Although both E6 and E7 proteins contain numerous predicted cytotoxic T lymphocyte (CTL) epitopes that are capable of binding to human leukocyte antigens, the majority of earlier in vivo tumor rejection studies have focused on E7. We show here that gene gun-mediated skin transfection of plasmid vector encoding the nontransforming, amino-terminal half of E6 resulted in the induction of E6-specific CTL activity and tumor rejection in a murine model. The use of recombinant murine interleukin-12 (rmIL-12) as a vaccine adjuvant has been shown to result in both an enhancement and suppression of immune responses, depending upon the doses of rmIL-12 and the experimental systems used. We demonstrate here that local expression of transgenic mIL-12 at the E6 DNA vaccination site potentiated E6-specific CTL responses and increased vaccine-induced antitumor therapeutic efficacy. Our results indicate that transfection of the mIL-12 gene at the vaccination site may represent an attractive adjuvant for cancer gene immunotherapy. (+info)
DNA vaccination against the ovarian carcinoma-associated antigen folate receptor alpha (FRalpha) induces cytotoxic T lymphocyte and antibody responses in mice.
(48/2315)
Human folate receptor alpha (FRalpha) is a folate-binding protein that is selectively overexpressed in ovarian carcinoma and has been regarded as a suitable target antigen for immunotherapy purposes. To study the possible use of this antigen in DNA vaccination, FRalpha cDNA was ligated into the VR1012 (Vical) expression vector under the transcriptional control of the cytomegalovirus promoter. A total of 100 microg of purified plasmid DNA was injected intramuscularly in BALB/c mice three times at 14-day intervals. At 10 days after the second injection, the sera of the animals (100%) displayed significant antibody titers (by indirect immunofluorescence and fluorescence-activated cell sorter analysis) against syngeneic C26 cells transduced with FRalpha, but not against unmodified C26 cells. Immunoglobulin G2a was the predominant isotype. In addition, specific cytotoxic T lymphocyte activity against FRalpha-transduced C26 cells could be detected in splenocytes from all immunized animals. Coinjection of a plasmid containing interleukin-2 cDNA increased both antibody titers and cytotoxic T lymphocyte activity. Challenge by subcutaneous injection with FRalpha-transduced C26 cells (performed 10 days after the third injection) showed a statistically significant delay in tumor growth. Vaccination with the FRalpha and interleukin-2 cDNA mixture, which was performed after an intravenous injection of FRalpha-transduced cells, enhanced the mean survival time and reduced the number of lung metastases, thus suggesting that such vaccination is effective even against preexisting tumor cells. (+info)