DNA immunization, although attractive, is poor for inducing mucosal immunity, thus limiting its protective value against most infectious agents. To surmount this shortcoming, we devised a method for mucosal transgene vaccination by using an M cell ligand to direct the DNA vaccine to mucosal inductive tissues and the respiratory epithelium. This ligand, reovirus protein 1, when conjugated to polylysine (PL), can bind the apical surface of M cells from nasal-associated lymphoid tissues. Intranasal immunizations with protein 1-PL-DNA complexes produced antigen-specific serum IgG and prolonged mucosal IgA, as well as enhanced cellmediated immunity, made evident by elevated pulmonary cytotoxic T lymphocyte responses. Therefore, targeted transgene vaccination represents an approach for enabling DNA vaccination of the mucosa.

An experimental vaccine for enterotoxigenic Escherichia coli (ETEC) composed of a live, attenuated Salmonella vector-expressing enterotoxigenic E. coli fimbriae, colonization factor Ag I (CFA/I), stimulated a biphasic Th cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-α, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with Salmonella-CFA/I construct 1 or 4 wk before induction of experimental autoimmune encephalomyelitis using an encephalitogenic proteolipid protein peptide, PLP139–151. Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-γ-secreting Th cells and was replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, experimental autoimmune encephalomyelitis, via immune deviation.

Pestalotiopsis microspora, isolate NE-32, is an endophyte of the Himalayan yew (Taxus wallichiana) that produces taxol, an important chemotherapeutic drug used in the treatment of breast and ovarian cancers. Conditions were determined to induce the perfect stage (teleomorph) of this organism in the laboratory as a critical first step to study inheritance of taxol biosynthetic genes. The perfect stage of Pestalotiopsis microspora NE-32 forms in a period of 3-6 weeks on water agarose with dried yew needles at 16-20 SC with 12 h of light per day. Morphological analysis of the teleomorph and sequencing of the 18S rDNA indicates that Pestalosphaeria hansenii is the perfect stage of Pestalotiopsis microspora. Only certain plants (e.g. yews, some pines, pecan, oat and some barley cultivars) allow the production of perithecia. Exhaustive methylene chloride extraction of yew (Taxus cuspidata) needles removes their capacity to induce production of perithecia. The methylene chloride extract is able to induce formation of perithecia by strain NE-32 in a bioassay system utilizing the sterilized sheaths of the Cholla cactus (Opuntia bigelovii) spine, indicating that a chemical compound(s) in yew stimulates the formation of the perfect stage. This hydrophobic plant compound(s) has been designated the perithecial-stimulating factor (PSF). The data suggest that plant products may play a role in regulating the biology of endophytic microbes.

To facilitate invasion, reovirus has evolved to attach to M cells, a specialized epithelium residing within the follicle-associated epithelium that covers mucosal inductive tissues. Thus, we questioned adapting reovirus protein σ1 to ferry DNA vaccines to the mucosa to immunize against HIV. Three expression plasmids encoding HIV(Ba-L) gp160, cytoplasmic gp140, and secreted gp140 were tested in mice as protein σ1-poly-l-lysine-DNA complexes (formulated vaccine) via the intranasal route. Evaluation of cell-mediated immunity showed that the formulated gp160 DNA vaccine was more effective for stimulating envelope (Env)-specific CTL responses in lungs, lower respiratory lymph nodes (LN), cervical LN, submaxillary gland LN, and spleens. Three doses of vaccine were required for CTL responses, and intranasal naked DNA immunizations were ineffective. The greatest CTL activity was observed between weeks 8 and 10 for gp160-vaccinated mice, and activity remained detectable by week 16. These Env-specific CTL responses were perforin dependent in peripheral tissues, but mostly Fas dependent in the lungs. These Env-specific CTLs also produced IFN-γ. Mice vaccinated with the formulated gp160 DNA vaccine showed potent antiviral immunity against vaccinia virus-env replication in ovaries. Thus, compared with live vectors, protein σ1-mediated DNA delivery represents an alternative mucosal formulation for inducing cellular immunity against HIV-1.

No abstract