Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.