The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration.

We have investigated the immunological consequences of feeding a protein antigen to previously immunized animals. BALB/c mice were systemically primed with ovalbumin (OVA) in complete Freund's adjuvant (CFA) and fed with high (10 mg/g body weight), medium (1 mg/g body weight) or low (1 microgram/g body weight) doses of OVA once (Day 1, 7 or 14) or sequentially for 5 days (Days 1-5, 7-11, 14-18). The specific IgG antibody response was suppressed only by early feeds of high-dose OVA (Days 1-5). Medium-dose OVA fed on Day 14 or low-dose OVA fed at any stage after immunization enhanced the IgG antibody response. In contradistinction, systemic delayed-type hypersensitivity responses (DTH) were usually suppressed by early feeds of high or medium doses of OVA but never after feeding low-dose OVA. The results suggest that systemic DTH and IgG antibody responses to oral antigen are subject to different control mechanisms in previously primed animals. Such responses depend on the immune status of the animal and are controlled by antigen dose, time and frequency of feeding. The immunological effects observed are also demonstrable following adoptive transfer of spleen cells collected 14 days after multiple feeds of high-dose OVA to immunized mice. Our findings suggest that oral hyposensitization after systemic immunization is regulated by (suppressor) spleen cells which are activated by gut-processed antigen.     

Depletion of suppressor T cells by 2'-deoxyguanosine abrogates tolerance in mice fed ovalbumin and permits the induction of intestinal delayed-type hypersensitivity.

We have re-examined the role of suppressor T cells (Ts) in regulating immune responses to fed proteins by investigating the effect of 2'-deoxyguanosine (dGuo) on systemic and intestinal immunity in mice fed ovalbumin (OVA). Administration of dGuo for 10 days abrogated the suppression of systemic delayed-type hypersensitivity (DTH) and antibody responses normally found after feeding OVA, and also prevented the generation of OVA-specific Ts. In parallel, mice given dGuo and fed OVA developed sensitization to OVA in the gut-associated lymphoid tissues (GALT) after oral challenge with OVA and had increased intraepithelial lymphocyte (IEL) counts and crypt cell production rates (CCPR) in the jejunal mucosa, indicating the presence of a local DTH response. These findings confirm the importance of Ts in preventing hypersensitivity to dietary protein antigens and suggest that enteropathies associated with food hypersensitivity are due to a defect in Ts activity.     

Suppression of an established DTH response to ovalbumin in mice by feeding antigen after immunization

Experiments were designed to examine whether systemic delayed-type hypersensitivity responses (DTH) to ovalbumin (OVA) can be suppressed when antigen is fed after immunization, and to investigate the immunological mechanisms involved. A single 25 mg feed of OVA given 7 or 14 days after immunization with OVA in complete Freund's adjuvant (CFA) suppressed the DTH response of BDF1 mice, but had no significant effect on the serum IgG antibody response. DTH suppression was greatest when antigen was fed soon after immunization, and became less pronounced as the time interval between feeding and immunization increased. The phenomenon was also demonstrated in mice of the BALB/c strain. Cell transfer experiments suggested that the post-immunization suppression was not due to a population of suppressor cells that have been described previously in association with classical oral tolerance for DTH. We conclude that there are separate and distinct mechanisms for the prevention of induction of DTH by antigen feeding in naive mice and the suppression of expression of DTH in sensitized animals.     

Divergent effects of bacterial lipopolysaccharide on immunity to orally administered protein and particulate antigens in mice.

We have investigated whether bacterial lipopolysaccharide (LPS) influences immune responses to dietary protein antigens in experimental animals. Simultaneous intravenous administration of LPS to normal mice fed ovalbumin (OVA) prevented the induction of tolerance for serum IgG antibody responses but did not alter the tolerance of systemic delayed-type hypersensitivity (DTH). In addition, exogenous LPS did not enhance the ability of spleen accessory cells to present OVA to primed T cells. LPS-unresponsive C3H/HeJ mice developed full tolerance of both humoral and cell-mediated immunity after feeding a range of doses of OVA that was equal in degree and persistence to that seen in normal, congenic C3H/HeOla mice and also had normal antigen-presenting cell (APC) activity for OVA. In contrast, C3H/HeJ mice were primed by feeding SRBC instead of developing the systemic tolerance found in normal C3H mice. Our results indicate the complexity of mechanisms that may regulate systemic immunity to orally administered antigens of different forms. Nevertheless, LPS does not modulate DTH responses to fed OVA and does not enhance APC activity, and we conclude that bacterial LPS may be unable to influence hypersensitivity to dietary proteins in man.     

Immunological responses to fed protein antigens in mice. IV. Effects of stimulating the reticuloendothelial system on oral tolerance and intestinal immunity to ovalbumin.

We have studied the role of the reticuloendothelial system (RES) in intestinal and systemic immunity in mice immunized orally with ovalbumin (OVA). Stimulation of the RES by oestradiol completely prevented the induction of systemic tolerance normally found in mice fed 25 mg OVA and this applied both to humoral immunity and delayed-type hypersensitivity (DTH). In addition, an active DTH response could be detected in the mucosa and mesenteric lymph nodes (MLN) of oestradiol-treated, OVA-fed mice on oral challenge with OVA. Oestradiol had no direct effect on lymphocyte function and we propose that RES activation may be one mechanism which predisposes to small intestinal disease associated with food hypersensitivity.     

Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.     

A genetically determined lack of oral tolerance to ovalbumin is due to failure of the immune system to respond to intestinally derived tolerogen

In this study we have examined whether differences between mouse strains in the induction of tolerance after feeding ovalbumin (OVA) are due to differences in intestinal processing of OVA or are determined by the systemic immune system. Compared with major histocompatibility complex (MHC)-congenic BALB/c mice, BALB/B mice develop much less tolerance of systemic delayed-type hypersensitivity (DTH) and humoral immunity after feeding OVA and this defect is also expressed partially in (BALB/B x BALB/c)F1 animals. Serum taken from either BALB/c or BALB/B mice fed OVA 1 h before produced significant suppression of systemic DTH responses in BALB/c, but not in BALB/B mice. Although OVA-fed BALB/B serum was slightly less tolerogenic than BALB/c serum, we conclude that the defective induction of oral tolerance in BALB/B mice is due primarily to a MHC-influenced defect with the immune system. These findings support the idea that clinical food-sensitive enteropathy reflects an immune response gene-controlled defect in tolerance to dietary proteins.     

The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin.

The induction of tolerance by feeding proteins may prevent potentially harmful delayed-type hypersensitivity (DTH) reactions to food antigens. Suppressor T cells (Ts) are present in mice with tolerance of systemic DTH after feeding ovalbumin (OVA) but, as other immunoregulatory mechanisms have also been described, the exact role of Ts in maintaining tolerance is not known. In this study, we have used the ability of native and denaturated OVA to cross-react at the level of helper/effector T cells, but not Ts, to re-examine the role of Ts in oral tolerance to OVA. Mice immunized with native OVA (nOVA) or denatured OVA (dOVA) in adjuvant had fully cross-reacting DTH to either nOVA or dOVA, but intravenous administration of antigen induced Ts which were specific for the appropriate form. Mice fed nOVA or dOVA had identical tolerance of systemic DTH to both forms of OVA, and feeding nOVA induced splenic Ts which suppressed the DTH response to both nOVA and dOVA. Splenic Ts could not be detected in mice fed dOVA. The results support the hypothesis that tolerance of systemic DTH in mice fed native proteins is due to Ts. Although, for the moment, there is no complementary evidence for a role for Ts in oral tolerance to denatured proteins, this study is consistent with the idea that Ts are the mechanism which normally prevent enteropathy due to DTH against dietary proteins. In addition, our study underlines the differences between orally and parenterally induced Ts and reinforces the view that fed proteins induce Ts after processing by the gut or its lymphoid accessory cells.     

Oral tolerance to ovalbumin in mice: studies of chemically modified and 'biologically filtered' antigen.

Suppression of systemic immunity after the feeding of antigen was investigated in mice by means of serum transfer experiments. Serum collected from mice 1 hr after a single intragastric dose of 25 mg OVA induced suppression of systemic DTH when injected intraperitoneally into recipient mice. This suppression was found to be restricted to the cell-mediated limb of immunity and was antigen-specific. A postulated function of the intestine, conversion of antigen into tolerogenic form by means of intestinal antigen processing, was studied by attempting to mimic intestinal alteration of OVA by chemical modification of the antigen. Parenteral injection of mice with either deaggregated or denatured OVA did not produce the typical pattern of unresponsiveness seen in animals given intestinally processed OVA. Intestinal processing was also shown to be distinct from systemic antigen processing. Mice injected with serum containing systemically 'filtered' OVA did not become tolerant to OVA in the manner of recipients of serum from OVA-fed mice.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.     

Examination of oral sensitization with ovalbumin in Brown Norway rats and three strains of mice

We studied the conditions needed to sensitize animals to the oral feeding of food allergens, without induction of tolerance, in order to investigate the allergenicity of orally ingested food proteins. Brown Norway (BN) rats were sensitized by daily OVA (ovalbumin)-gavage or by drinking OVA containing water ad libitum and the ASA (active systemic anaphylaxis) response, as the immediate hypersensitivity response to antigen stimulation after oral sensitization, was examined. The oral administration of OVA by gavage produced a higher OVA-specific IgE response and an increase in serum histamine after antigen challenge, as compared to those produced by drinking water. Next, we examined the effect of murine age, the oral feeding technique and the oral feeding dose on sensitization using BALB/c, B10A and ASK mice. Twenty-week-old mice showed the strongest OVA-specific IgE and IgG1 responses and ASA-associated serum histamine contents increased with gavage in the three different age groups of BALB/c mice. Administering 0.1 mg of OVA by gavage daily for 9 weeks appeared to induce a higher response than administering 1 mg of OVA, in terms of OVA-specific IgE and IgG1 antibody responses and ASA responses. Among the three strains of mice, B10A mice exhibited the highest response in terms of OVA-specific IgE and IgG1 antibody and ASA responses. These findings suggested BN rats and B10A mice were suitable models for oral sensitization with antigen protein and that oral sensitization in mice requires low dose, intermittent antigen intakes.     

Immunological responses to fed protein antigens in mice. I. Reversal of oral tolerance to ovalbumin by cyclophosphamide

Feeding ovalbumin over a wide range of doses is known to reduce subsequent systemic immune responses to parenteral immunization. In the present study, we have fed mice 2 mg and 25 mg ovalbumin (OVA) 2 weeks before systemic immunization and followed the resulting humoral antibody and cell-mediated immune (CMI) responses. The results indicate that while 25 mg OVA will reduce subsequent IgM, IgG and CMI responses to OVA, feeding 2 mg OVA will only suppress CMI responses and to a lesser extent the IgM response. Furthermore, the tolerant state induced by feeding 25 mg OVA was only partially prevented by 100 mg/kg cyclophosphamide (CY) while the suppressed CMI after feeding 2 mg OVA was completely blocked by CY pretreatment. These findings suggest that the humoral and cell-mediated limbs of the immune response may be controlled by different regulatory systems after feeding antigen, and that activation of these systems is dependent on the dose of oral antigen use. In addition, the results are in agreement with our previous finding that CY pretreatment will allow the development of CMI in the gut and gut-associated lymphoid tissue (GALT) after oral OVA and suggest that this phenomenon is related to breakdown of oral tolerance induction.     

T cell-mediated oral tolerance is intact in germ-free mice

Commensal enteric bacteria stimulate innate immune cells and increase numbers of lamina propria and mesenteric lymph node (MLN) T and B lymphocytes. However, the influence of luminal bacteria on acquired immune function is not understood fully. We investigated the effects of intestinal bacterial colonization on T cell tolerogenic responses to oral antigen compared to systemic immunization. Lymphocytes specific for ovalbumin-T cell receptor (OVA-TCR Tg(+)) were transplanted into germ-free (GF) or specific pathogen-free (SPF) BALB/c mice. Recipient mice were fed OVA or immunized subcutaneously with OVA peptide (323-339) in complete Freund's adjuvant (CFA). Although the efficiency of transfer was less in GF recipients, similar proportions of cells from draining peripheral lymph node (LN) or MLN were proliferating 3-4 days later in vivo in GF and SPF mice. In separate experiments, mice were fed tolerogenic doses of OVA and then challenged with an immunogenic dose of OVA 4 days later. Ten days after immunization, lymphocytes were restimulated with OVA in vitro to assess antigen-specific proliferative responses. At both high and low doses of OVA, cells from both SPF and GF mice fed OVA prior to immunization had decreased proliferation compared to cells from control SPF or GF mice. In addition, secretion of interferon (IFN)-gamma and interleukin (IL)-10 by OVA-TCR Tg(+) lymphocytes was reduced in both SPF and GF mice fed OVA compared to control SPF or GF mice. Unlike previous reports indicating defective humoral responses to oral antigen in GF mice, our results indicate that commensal enteric bacteria do not enhance the induction of acquired, antigen-specific T cell tolerance to oral OVA.     

Effect of sublingual administration with a native or denatured protein allergen and adjuvant CpG oligodeoxynucleotides or cholera toxin on systemic T(H)2 immune responses and mucosal immunity in mice.

BACKGROUND: Sublingual immunotherapy has been recently used for allergic diseases, but its mechanisms are still unclear. OBJECTIVE: To examine the effect of sublingual administration of a native or denatured allergen alone or plus adjuvant on systemic T(H)2 responses and mucosal immunity in mice. METHODS: Naive or sensitized BALB/c mice were sublingually vaccinated biweekly for 3 weeks with ovalbumin (OVA) or urea-denatured OVA (CM-OVA) only or plus adjuvant CpG oligodeoxynucleotides (CpG) or cholera toxin (CT). Two weeks later, their specific serum IgG, IgG1, IgG2a, IgE, and saliva secretory IgA (SIgA) antibody responses and the cytokine profiles of spleen and cervical lymph node cells were investigated. RESULTS: Specific SIgA antibody responses were induced by vaccination with CM-OVA plus CpG or CT. Whereas vaccination with CM-OVA and CpG enhanced T(H)1 responses but inhibited IgE production, vaccination with CT and CM-OVA or OVA increased cervical lymph node cell production of interleukin (IL) 4, IL-5, and IL-6 and serum IgG1 antibody responses. In previously sensitized mice, sublingual vaccination with OVA or CM-OVA plus CT or CpG stimulated mucosal SIgA antibody responses, but did not enhance ongoing IgE antibody responses. CONCLUSIONS: Sublingual vaccination with OVA or CM-OVA plus adjuvant CT or CpG all can induce systemic and mucosal immunity, but CM-OVA plus CpG had the best prophylactic and therapeutic effects on IgE antibody production. It is likely that sublingual vaccines may have a role for the prophylaxis and immunotherapy of allergic reactions.     

Salmonellosis in mice: Studies on oral immunization with live avirulent vaccines

The avirulentSalmonella typhimurium galE strain G30, following oral feeding to mice, developed a state of immunity to a secondary oral challenge with virulentSalmonella typhimurium. This immunity was concommitant with the development of intestinal and serum antibodies and delayed-type hypersensitivity (DTH) toSalmonella antigens. In contrast, repeated oral doses of a hybridE.coli vaccine, which expresses the O antigens ofSalmonella typhimurium, provided a lesser degree of immunity and, although able to stimulate the formation of serum antibodies, it elicited only a low level of intestinal antibodies and no measurable DTH.     

Oral tolerance in protein-deprived mice. I. Profound antibody tolerance but impaired DTH tolerance after antigen feeding.

We have examined the effects of protein deprivation on the induction of oral tolerance for systemic antibody and DTH responses to the protein antigen ovalbumin (OVA). Mice were fed 4% or 24% protein diets from weaning and given a single feed of OVA 2 weeks later (short-term deprivation) or after 10 weeks (long-term deprivation). Tolerance for serum antibody responses was more profound in protein-deprived animals than in 24% protein-fed control groups. Conversely, tolerance for DTH responses was impaired in protein-deprived mice. This was demonstrated both for short-term deprivation, where nutritional rehabilitation after OVA feeding was necessary to demonstrate this effect on DTH, and for long-term deprivation. Furthermore, the effect of short-term deprivation on tolerance for DTH responses was similar to that observed after cyclophosphamide pretreatment of OVA-fed mice. Protein deprivation has disparate effects on the humoral and cell-mediated limbs of oral tolerance, and our results support the hypothesis that this regime selectively depletes a population of suppressor T cells responsible for the fine control of DTH tolerance.     

Induction of mucosal and systemic immune responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide) microparticles.

We have examined the range of mucosal and systemic immune responses induced by oral or parenteral immunization with ovalbumin (OVA) entrapped in poly(D,L-lactide-co-glycolide) (PLG) microparticles. A single subcutaneous immunization with OVA-PLG primed significant OVA-specific IgG and delayed-type hypersensitivity (DTH) responses. The DTH responses were of similar magnitude to those obtained using immunostimulating complexes (ISCOMS) as a potent control adjuvant, although ISCOMS stimulated higher serum IgG responses. Both vectors also primed OVA-specific in vitro proliferative responses in draining lymph node cells following a single immunization and strong OVA-specific CTL responses were found after intraperitoneal (i.p.) immunization. ISCOMS were more efficient in inducing cytotoxic T lymphocytes (CTL), requiring much less antigen and only ISCOMS could stimulate primary OVA-specific CTL responses in the draining lymph nodes. Multiple oral immunizations with OVA in PLG microparticles or in ISCOMS resulted in OVA-specific CTL responses and again ISCOMS seemed more potent as fewer feeds were necessary. Lastly, multiple feeds of OVA in PLG microparticles generated significant OVA-specific intestinal IgA responses. This is the first demonstration that PLG microparticles can stimulate CTL responses in vivo and our results highlight their ability to prime a variety of systemic and mucosal immune responses which may be useful in future oral vaccine development.     

Oral tolerance in T cells is accompanied by induction of effector function in lymphoid organs after systemic immunization

The physiological ramifications of oral tolerance remain poorly understood. We report here that mice fed ovalbumin (OVA) exhibit oral tolerance to subsequent systemic immunization with OVA in adjuvant, and yet they clear systemic infection with a recombinant OVA-expressing strain of Salmonella enterica serovar Typhimurium better than unfed mice do. Mice fed a sonicated extract of S. enterica serovar Typhimurium are also protected against systemic bacterial challenge, and the protection is Th1 mediated, as feeding enhances clearance in interleukin-4-null (IL-4(-/-)) and IL-10(-/-) mice but not in gamma interferon-null (IFN-gamma(-/-)) mice. When T-cell priming in vivo is tracked temporally in T-cell receptor-transgenic mice fed a single low dose of OVA, CD4 T-cell activation and expansion are restricted largely to mucosal lymphoid organs. However, T cells from spleens and peripheral lymph nodes of fed mice proliferate and secrete IFN-gamma when restimulated with OVA in vitro, indicating the presence of primed T cells in systemic tissues following oral exposure to antigen. Nonetheless, oral tolerance can be observed in the fed mice as reduced recall responses following subsequent systemic immunization with OVA in adjuvant. Soluble OVA administered systemically has similar effects in vivo, and the "tolerance" seen in both cases can be partially reversed if the initial priming is made more immunogenic. Together, the results indicate that antigen exposure under poor adjuvantic conditions, whether oral or systemic, may lead to T-cell commitment to effector rather than proliferative capabilities, necessitating a reassessment of therapeutic modalities for induction of oral tolerance in allergic or autoimmune states.     

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma.

Adjuvants are a critical component of non-viable vaccine vectors, particularly for those to be used via mucosal routes. Although most adjuvants act by inducing local inflammatory responses, the molecular basis of many of these effects is unclear. Here we have investigated whether interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) are required for the induction of local and systemic immune responses by oral and parenteral administration of ovalbumin (OVA) in immune stimulating complexes (ISCOMS), a potent mucosal adjuvant vector. Our results show that after oral or systemic immunization with OVA ISCOMS, IL-4 knockout (IL4KO) and IFN-gamma receptor knockout (IFN-gamma RKO) mice develop an entirely normal range of immune responses including delayed-type hypersensitivity (DTH), serum immunoglobulin G (IgG) antibodies, T-cell proliferation and cytokine production, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) activity and intestinal IgA antibodies. These responses were of a similar magnitude to those found in the wild-type mice, indicating that the immunogenicity of ISCOMS is not influenced by the presence of IL-4 or IFN-gamma and emphasizing the potential of ISCOMS as widely applicable mucosal adjuvants.     

Ginsenoside Rg1 and aluminum hydroxide synergistically promote immune responses to ovalbumin in BALB/c mice

The combined adjuvant effect of ginsenoside Rg1 and aluminum hydroxide (alum) on immune responses to ovalbumin (OVA) in mice was investigated. BALB/c mice were subcutaneously (s.c.) inoculated twice with OVA alone or in combination with Rg1, alum, or Rg1 plus alum. Samples were collected 2 weeks after the boosting for the measurement of anti-OVA immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-5 (IL-5) produced in singular splenocyte cultures. Delayed-type hypersensitivity (DTH) responses were measured in mice immunized as described above. After 10 days, the mice were injected s.c. with OVA at the footpads. Thereafter, the thickness of the footpads was measured once daily for 5 days. The results indicated that alum enhanced mainly Th2 (IgG1 and IL-5) responses (P < 0.05), while Rg1 enhanced both Th1 (IgG1 and IL-5) and Th2 (IgG2a, IFN-γ, and DTH) responses (P < 0.05). The highest immune responses were found in the mice injected with OVA solution containing both alum and Rg1. In addition, the hemolytic activity of Rg1 was much lower than that of Quil A. Therefore, Rg1 deserves further studies in order to tailor desired immune responses when a mixed Th1/Th2 immune response is needed.