Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein.

The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

DNA immunization in vivo down-regulates nuclear all-trans retinoic acid receptors in mouse spleen cells.

Nuclear retinoid receptors - retinoic acid inducible transcription factors - participate in pathways influencing many components of the immune system. In the present study in vivo effects of DNA-based immunization of mice on binding parameters of all-trans retinoic acid receptors (RARs) in spleen cell nuclei was investigated. A eucaryotic expression vector encoding the gene for the model enzyme beta-galactosidase of Escherichia coli (pCMV-beta) was used for intradermal injection. Furthermore, immunostimulatory CpG motifs, which stimulate the expression of various cytokines and may serve as a 'danger signal' for the mammalian immune system, were coinjected as oligodeoxynucleotides. The results demonstrate that the concentration of RARs was significantly reduced in the late phase of the primary immune response (21 days after injection of plasmid DNA-indicated by high affinity IgG antibodies and IFN-gamma expression). Coinjection of CpG motifs did not change the course of the humoral response but enhanced and accelerated the proliferative response and expression of IFN-gamma, which correlated with the reduced RARs concentration.

Isoforms of the major allergen of birch pollen induce different immune responses after genetic immunization.

BACKGROUND: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. METHODS: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV-Beta) and Bet v 1d (pCMV-Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN-gamma and IL-4 levels were measured by ELISA. Allergen-specific proliferation was determined by incorporation of [(3)H]-thymidine. RESULTS: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th-1-type response. The antisera against both isoforms were highly cross-reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN-gamma and IL-4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV-Beta, pCMV-Betd caused no significant allergen-specific proliferation and induced only marginal levels of the key cytokines. CONCLUSIONS: Based on the assumption that the induction of a strong Th-1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.

Immune responses after immunization with plasmid DNA encoding Bet v 1, the major allergen of birch pollen.

BACKGROUND: Immunization with plasmid DNA encoding various antigens is a promising method in vaccine research. Recent studies also indicate that DNA-based immunization might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In this study we have characterized the immune responses induced by recombinant Bet v 1a and plasmid DNA encoding for Bet v 1a, the major allergen of birch pollen in a mouse system. METHODS: Balb/c mice were injected intraperitoneally with recombinant Bet v 1a and intradermally with plasmid DNA encoding for the gene of Bet v 1a (pCMV-Bet). In addition, the effect of immunostimulatory DNA sequences was investigated by appending CpG motifs to the gene of Bet v 1a, coinjecting CpG-oligodeoxynucleotides together with the pCMV-Bet construct, or both. IgE and IgG antibody responses, as well as IgG subclasses, were measured by ELISA in sera after each immunization. IFN-gamma and IL-4 levels were also measured by ELISA in sera and supernatants of allergen-stimulated spleen cells. RESULTS: The primary humoral response to a single treatment with pCMV-Bet was very weak, but the reaction could be boosted to higher levels by 2 additional injections. On the other hand, proliferation assays of spleen cells and measurements of cytokine levels already indicated a cellular response after the first injection of plasmid DNA. After 2 immunizations with pCMV-Bet, the ratio of IgG1 to IgG2a pointed to a TH1 subclass profile. IgE was not detectable in any group at any time during the immune reaction. Accordingly, IL-4 levels were markedly reduced in the serum, as well as in the supernatants, of stimulated spleen cells. Animals immunized with pCMV-Bet containing appended CpG motifs at the 3' end of the Bet v 1a gene and/or with the CpG-ODN GCTAGACGTTAGCGT plus pCMV-Bet displayed reduced humoral responses against Bet v 1a when compared with animals injected with pCMV-Bet alone. The levels of IFN-gamma measured after allergen stimulation of isolated spleen cells were significantly higher in animals immunized with pCMV-Bet plus CpG motifs than with pCMV-Bet alone. Immunization with recombinant Bet v 1a protein elicited a strong TH2 -type response, including IgE production, a high titer of IgG1, and IL-4 production in both serum and supernatants of proliferation cultures. CONCLUSION: In contrast to immunization with protein, DNA immunization induces a strong TH1 -type response against a relevant inhalant allergen. Our data support the concept of developing a novel type of allergen immunotherapy based on plasmid DNA immunization.