SummaryAntigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Thl and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4 + T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4 + T cells. Interestingly, both PP and SP CD4 + T cell cultures showed increased numbers of IL-4-and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no intefferon-3~ (IFN-'y) or IL-2 (Thl-type) SFCs were noted. Cytokine-spedfic Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-3, or IL-2 mRNA, were present in CD4 + T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2-and IFN-3,-producing Thl-type cells as well as IL-4-and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT (125I-CT) or CT-B subunit (125I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with 125I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas 125I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice. After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders. High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern. Oral LT given with TT induced TT-specific response patterns identical to LT-B. Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response. Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types.

In the oleaginous yeast Yarrowia lipolytica, de novo lipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase in Y. lipolytica. Strains with a Y. lipolytica snf1 (Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into a Y. lipolytica strain engineered to produce omega-3 eicosapentaenoic acid (EPA), Ylsnf1 deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Yarrowia lipolytica is one of the most extensively studied "nonconventional" yeasts with importance in multiple industrial applications (1) and has been engineered for the commercial production of omega-3 eicosapentaenoic acid (EPA) (2). Although Y. lipolytica is an oleaginous yeast capable of accumulating large amounts of lipid in the cell, lipid accumulation schemes using hydrophobic carbon sources as substrates have been reported in many cases (3-5). For de novo lipid synthesis using glucose as a carbon source, nitrogen limitation or a high carbon-to-nitrogen (C/N) ratio is the most commonly employed condition to increase intracellular lipid accumulation. However, in wild-type cells, lipids accumulate to Ͻ20% of dry cell weight (DCW) (6, 7), and it usually takes 3 to 10 days to accumulate the maximum level of lipids (7). Thus, an understanding of the regulation of lipid synthesis and accumulation will allow us to engineer this organism for increased rates of lipid accumulation, thereby significantly reducing the cost of manufacture for lipids and valuable lipid-derived compounds.Biochemical studies of various enzymes involved in de novo lipid synthesis have provided an explanation for how oleaginous microbes accumulate lipids under conditions of nitrogen limitation (reviewed in references 4 and 8). Briefly, nitrogen exhaustion in the medium results in stimulation of AMP deaminase, which breaks down AMP to IMP and ammonium in order to salvage nitrogen. The decrease in the AMP concentration inhibits isocitrate dehydrogenase, and accumulated isocitrate is equilibrated by aconitase with citrate, which then exits the mitochondria for acetyl coenzyme A (acetyl-CoA) generation by cytosolic ATPcitrate lyase (ACL). ACL activity is thought to be critical for lipid synthesis, and cytoplasmic malic enzyme was also shown to be important for lipid synthesis by supplying NADPH for fatty acid synthesis in certain oleaginous microorganisms. Although homologs of these enzymes were found in Y. lipolytica (9), there are not many biochemical studies of these enzymes in this yeast. It also remains to be examined if this mechanism is applicable to other nutritional limitations that induce lipid accumulation, such as phosphate, magnesium, or sulfur limitation.We are interested in discovering and controll...

Our past studies have shown that the mucosal adjuvant cholera toxin (CT) induces T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (S-IgA) antibodies (Abs). In this study, recombinant murine IL-12 (rmIL-12) was given either parenterally or orally to mice orally immunized with tetanus toxoid (TT) and CT to determine whether this cytokine could redirect the CT-induced Th2-type responses and what effect this shift would have on S-IgA Ab responses. Intraperitoneal administration of rmIL-12 shifted TT-specific responses toward Th1-type and resulted in CD4+ T cells producing IFN-γ and IL-2 with markedly reduced levels of Th2-type cytokines. This cytokine profile was accompanied by increased delayed-type hypersensitivity (DTH) and shifts in serum IgG1 to IgG2a and IgG3 anti-TT Ab responses. Further, serum IgE and S-IgA Ab responses were markedly reduced by parenteral IL-12. When IL-12 complexed to liposomes was given orally both shifts to IgG2a and IgG3 and low IgE Abs again occurred concomitant with enhanced serum IFN-γ and DTH responses. Interestingly, oral rmIL-12 did not result in significant levels of serum IL-12 nor altered S-IgA Ab responses and resulted in higher levels of some Th2-type cytokines both in vitro and in vivo when compared with parenteral IL-12. Our results show that the shifts in systemic immune responses with intact S-IgA Abs which occur after oral delivery of IL-12-liposomes are due to cytokine effects in the Peyer's patches and suggest new strategies for the targeted manipulation of Th1- and Th2-type responses to mucosal vaccines.

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Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 jg of TT given orally with 10 ,ug of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-IT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or * Corresponding author.