OVA-specific Antibody Responses Research Articles

Ultrasmall Graphene Oxide Supported Gold Nanoparticles as Adjuvants Improve Humoral and Cellular Immunity in Mice

Adjuvants play an important role in vaccines. Alum and MF59 are two dominant kinds of adjuvants used in humans. Both of them, however, have limited capacity to generate the cellular immune response required for vaccines against cancers and viral diseases. It is desirable to develop new and efficient adjuvants with the aim of improving the cellular immune response against the antigen. Here, the feasibility and efficiency of ultrasmall graphene oxide supported gold nanoparticles (usGO‐Au) as a new immune adjuvant to improve immune responses are explored. usGO‐Au is obtained from reduction of chloroauric acid using usGO and then decorated with ovalbumin (OVA, a model antigen) through physical adsorption to construct usGO‐Au@OVA. As the results show, the as‐synthesized usGO‐Au@OVA can efficiently stimulate RAW264.7 cells to secrete tumor necrosis factor‐α (TNF‐α), a mediator for cellular immune response. In vivo studies demonstrate that usGO‐Au@OVA can also promote robust OVA specific antibody response, CD8+ T cells proliferation, and different cytokines secretion. The results indicate that using usGO‐Au as an adjuvant can stimulate potent humoral and cellular immune responses against antigens, which may promote better understanding of cellular immune response and facilitate potential applications for cancer and viral vaccines.

Evaluation of Stearylamine-Modified Liposomes for the Oral Vaccine Adjuvant

The usefulness of stearylamine (SA)-modified liposomes for the oral vaccine adjuvant in the induction of immune responses was evaluated. Mice were orally immunized withunmodified liposomes containing ovalbumin (OVA) (group I), OVA-containing monophosphoryl lipid A (MPL)-modified liposomes (group II), OVA-having SA-modified liposomes (group III) or OVA alone (group IV). After immunization, significant OVA-specific antibodies were detected in the serum and intestine from mice of groups I to III, but not in group IV. Especially, intestinal IgG and IgA antibody responses against OVA were significantly higher in mice of group III than in groups I and II. When sera were analyzed for isotype distribution,OVA-specific IgG1 antibody responses were noted in mice of groups I and II, whereas the induction of OVA-specific IgG1 and IgG3 antibody responses was observed in mice of group III. Moreover, substantial production of IFN-γ(Th1-type) and IL-4 (Th2 type) was demonstrated in spleen cells from mice of group III in vitro. These results suggest that the SA-modified liposomes would serve effectively as mucosal vaccine adjuvant for inducing humoral and cell-mediated immune responses.

Cholera toxin breakdowns oral tolerance via activation of canonical NF-κB

The mechanisms of mucosal immunogenicity and adjuvanticity of bacterial exotoxins remains unknown. In this study, we investigated the role of the transcription factor nuclear factor-κB (NF-κB) in cholera toxin (CT)-induced alteration of oral tolerance. Feeding CT abrogated ovalbumin (OVA)-induced oral tolerance, as evaluated by OVA-specific serum antibody responses, and CD4+ T cell proliferation. CT feeding activated canonical NF-κB (one heterodimer type, p50–p65) and mRNA expression of NF-κB-dependent proinflammatory cytokines in mesenteric lymph node (MLN) and Peyer’s patch (PP) cells. CT no longer showed abrogation of oral tolerance in mice pretreated with p50 small interfering RNAs (siRNAs). ADP-ribosylation inhibitors inhibited CT-induced NF-κB activation. These data suggest that CT induces canonical NF-κB activation in intestinal lymphoid cells, which plays a key role in mucosal immunogenicity and adjuvanticity.

Humoral immune responses elicited by the polyanhydride particle vaccine platform are facilitated by enhanced T follicular helper cell phenotype (P4511)

Abstract Previous studies have shown that the use of the polyanhydride nanoparticle (NP) vaccine platform provides innate immunomodulatory activity while also controlling antigen release kinetics that enhance antibody-mediated immunity. The goal of this project was to demonstrate that the increased antibody responses detected by utilizing NP-based vaccines induced of robust T helper cell responses to promote B cell activation and differentiation into antibody secreting cells. Using the ovalbumin (Ova) specific T cell (OT II) model, we evaluated the mechanisms associated with the expansion and activation of Ova-specific CD4+ T after immunization using three different polyanhydride NP formulations that contained encapsulated Ova. Significant expansion of CD4+ T cells occurred following immunization with Ova-loaded 20:80 CPH:SA or 20:80 CPTEG:CPH PNPs. Similar to Alum and MPLA adjuvanted responses, these NP regimen induced T follicular helper cells (CXCR5high PD-1high) that are important for initiating and maintaining germinal centers. Following a single administration, PNP vaccines induced robust Ova-specific IgG1 antibody titers. Upon secondary immunization, the NP vaccinated mice effectively responded with robust Ova-specific IgG1 and IgG2c antibody responses. The NP vaccinated mice presented with more antigen-specific Tfh (vα2+, vβ5+) CD4 T cells and more numerous germinal centers cells (B220+ CD23high PNA+) that supported more avid Ova-specific IgG1.

Fully embeddable chitosan microneedles as a sustained release depot for intradermal vaccination

This study introduces a microneedle transdermal delivery system, composed of embeddable chitosan microneedles and a poly(l-lactide-co-d,l-lactide) (PLA) supporting array, for complete and sustained delivery of encapsulated antigens to the skin. Chitosan microneedles were mounted to the top of a strong PLA supporting array, providing mechanical strength to fully insert the microneedles into the skin. When inserted into rat skin in vivo, chitosan microneedles successfully separated from the supporting array and were left within the skin for sustained drug delivery without requiring a transdermal patch. The microneedle penetration depth was approximately 600 μm (i.e. the total length of the microneedle), which is beneficial for targeted delivery of antigens to antigen-presenting cells in the epidermis and dermis. To evaluate the utility of chitosan microneedles for intradermal vaccination, ovalbumin (OVA; MW = 44.3 kDa) was used as a model antigen. When the OVA-loaded microneedles were embedded in rat skin in vivo, histological examination showed that the microneedles gradually degraded and prolonged OVA exposure at the insertion sites for up to 14 days. Compared to traditional intramuscular immunization, rats immunized by a single microneedle dose of OVA showed a significantly higher OVA-specific antibody response which lasted for at least 6 weeks. These results suggest that embeddable chitosan microneedles are a promising depot for extended delivery of encapsulated antigens to provide sustained immune stimulation and improve immunogenicity.

Efficiency of pH‐Sensitive Fusogenic Polymer‐Modified Liposomes as a Vaccine Carrier

The usefulness of pH-sensitive fusogenic polymer-(succinylated poly(glycidol)-(SucPG-) modified liposomes as a vaccine carrier in the induction of immune responses was evaluated. Mice were intraperitoneally immunized with ovalbumin- (OVA-) containing SucPG-modified liposomes. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, OVA-specific IgG1 antibody responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ-(Th1-type-) and IL-4-(Th2 type-) specific mRNA were detected. Moreover, substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from OVA-containing SucPG-modified liposomes in vitro. These results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses.

Flagellin produced in plants is a potent adjuvant for oral immunization

The aim of this study was to produce adjuvant with high biosafety, efficacy and low cost. Towards this goal, the plant Nicotiana benthamiana transient expression system was successfully used to express Salmonella typhimurium's flagellin (FljB). The yield of the expressed FljB was 280mg per kg of fresh weight (FW) leaves. The lyophilized plant powder containing plant expressing FljB was mixed with ovalbumin (OVA) and used for oral immunization of BALB/c mice. The ELISA analysis showed higher and accelerated OVA-specific serum antibody responses in mice given the mixture when compared to animals receiving OVA alone. Furthermore, FljB elicited a mixed Th1/Th2 response as shown by the presence of specific anti-OVA IgG1, IgG2a and IgG2b isotypes. OVA-specific IgAs were also detected in mice given the mixture. Cell-mediated immune response to OVA was induced by FljB as determined by a spleen lymphocyte specific proliferation test. No immune response was generated against FljB. In conclusion, our results showed for the first time the production of FljB in plants and the efficient use of the crude lyophilized extract as an adjuvant for oral immunization.

Cutaneous delivery of Poly(dG) conjugated protein antigens targets and activates DCs in vivo.(100.32)

Abstract Efficient and specific targeting of exogenous antigen to antigen presenting cells (APCs) in vivo is a critical challenge for vaccine design. APCs acquire antigens from a variety of sources and endocytic mechanisms can predetermine intracellular antigen routing, processing and presentation to CD4+ or CD8+ T cells. We have designed an APC targeting strategy based on simple and direct coupling of Poly(dG) oligodeoxynucleotides to protein antigens. In comparison to soluble OVA, we find that conjugates of Poly(dG) and OVA (OVA-Poly(dG)) are rapidly internalized by dendritic cells (DCs) and follow distinct intracellular processing pathways. Functionally, OVA-Poly(dG) is both presented to OVA-specific CD4+ T cells (OT-II cells) and efficiently cross-presented to OVA-specific CD8+ T cells (OT-I cells) by DCs in vitro. Importantly, immunization of naïve animals by direct injection of OVA-Poly(dG) results in efficient priming of potent antigen specific CTL responses. Further, OVA-Poly(dG) generate a specific memory CD8+ T cells response and promote OVA-specific antibody responses. Extending our findings to human skin, we show that I.D. injection of antigen-Poly(dG) conjugates results in efficient uptake by human skin migratory DCs, preferentially dermal CD14+DCs that also acquire potent T cell allostimulatory function. Our data suggests that Poly(dG) can both target antigens to human skin immune APCs and function as an adjuvant to induce potent CD4 and CD8 mediated T cell immunity.

Immunological adjuvant effect of Boswellia serrata (BOS 2000) on specific antibody and cellular response to ovalbumin in mice

In this study, the biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for its potential ability as adjuvants on the immune responses to ovalbumin (OVA) in mice. Balb/c mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing alum (200 μg) or BOS 2000 (10, 20, 40 and 80 μg) on Days 1 and 15. Two weeks later, OVA specific antibodies in serum; concanavalin A (Con A), OVA stimulated splenocyte proliferation, CD4/CD8/CD80/CD86 analysis in spleen cells and its estimation of cytokines (IL-2 and IFN gamma) from cell culture supernatant were measured. OVA specific IgG, IgG1 and IgG2a antibody levels in serum were significantly enhanced by BOS 2000 (80 μg) compared with OVA control group. Moreover, the adjuvant effect of BOS 2000 (80 μg) on the OVA-specific IgG, IgG1, and IgG2a antibody responses to OVA in mice were more significant than those of alum. BOS 2000 significantly enhanced the Con A and OVA induced splenocyte proliferation in the OVA immunized mice especially at a dose of 80 μg (p < 0.001). However, no significant differences were observed among the OVA group and OVA/alum group. At a dose of 80 μg (p < 0.001), there was a significant increase in the CD4/CD8 and CD80/CD86 analysis in spleen cells and cytokine (IL-2 and IFN-gamma) profile in the spleen cell culture supernatant was observed. In conclusion, BOS 2000 seems to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.

Relationship between the size of nanoparticles and their adjuvant activity: Data from a study with an improved experimental design

There is a growing interest in identifying the relationship between the size of nanoparticles and their adjuvant activity, but the results from recent studies remain controversial. To address the controversy, it was thought that one should pay attention to the nanoparticle formulations to make sure that the antigen-loaded nanoparticles to be compared are not only different in particle size, but more importantly, as identical to each other as possible in all other formulation properties. In the present study, using ovalbumin (OVA) as a model antigen conjugated onto nanoparticles engineered from lecithin/glyceryl monostearate-in-water emulsions, we prepared OVA-nanoparticles of 230 nm and 708 nm. Before evaluating the immune responses induced by them in a mouse model, we made sure that: (i) the sizes of the two OVA-nanoparticles did not extensively overlap, (ii) the nanoparticles have similar zeta potentials and comparable antigen-loading, and (iii) the nanoparticles did not aggregate when suspended in simulated biological media. We then showed that when subcutaneously injected into mice, the 230 nm OVA-conjugated nanoparticles induced stronger OVA-specific antibody and cellular immune responses than the 708 nm OVA-nanoparticles. Future studies attempting to correlate the size of nanoparticles and their adjuvant activities need to consider formulation parameters to ensure that the particles are different only in size and are stable before and after injection.

Immunosuppressive properties of aqueous extract of Plumbago zeylanica in Balb/c mice

In our investigation, we have screened herbal drug, which consists of a mixture of seven plants, used by the tribal population (in Paderu agency area of Visakhapatnam district of Andhra Pradesh state in India), for the identification of potential immunosuppressive property by testing the aqueous extracts of the plant parts by inducing ovalbumin (OVA) specific IgG antibody responses in a murine system. The aqueous root extract of Plumbago zeylanica (PZE) exhibited the significant suppression of OVA-specific IgG antibody response determined byEnzyme-Linked Immunosorbent Assay (ELISA). PZE also suppressed the anti-OVA antibody response in dose dependent manner. In addition ethyl acetate fraction of PZE eluted from silica gel (PZE-6) was found to exert a significant suppression of OVA stimulated T cell proliferation. Moreover, PZE is potent in exerting the suppressive effect on the down regulation of anti-OVA antibody and T cell responses and in all the three haplotypes of the mice studied, which indicates that the PZE exerted immunosuppression without linking to genetic variation. Key words: Plumbago zeylanica, immunosuppression, anti-OVA antibody response, enzyme-linked immunosorbent assay.

Co-administration of cholera toxin and apple polyphenol extract as a novel and safe mucosal adjuvant strategy

Although native cholera toxin (CT) is an extremely effective adjuvant, its toxicity prevents its use in humans. We report here that apple polyphenol extract (APE), obtained from unripe apples, reduces CT-induced morphological changes and cAMP accumulation. Based upon this finding, we have attempted to design a novel, effective and safe mucosal vaccine by using CT with several dosages of APE as nasal adjuvants. Mice nasally immunized with OVA plus CT and an optimal dosage of APE showed significantly reduced levels of inflammatory responses as well as total and OVA-specific IgE antibodies when compared with mice given without APE. However, levels of both mucosal and systemic OVA-specific antibody responses were maintained. Further, APE significantly down-regulated accumulation of CT in the olfactory nerves and epithelium. In summary, an optimal dosage of APE would take full advantage of mucosal adjuvanticity of native CT without any toxicity for application in humans.

The effects of salt on the physicochemical properties and immunogenicity of protein based vaccine formulated in cationic liposome

Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to protein based vaccines. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30mM) was added to the DOTAP (100nmol)/E7 (20μg) formulation, anti-tumor activity was generated in the immunized mice. Correlatively, 30mM NaCl in the DOTAP/E7 protein formulation increased the particle size from ∼350 to 550nm, decreased the protein loading capacity (from 95 to 90%), and finally increased the zeta potential (from 29 to 38mV). Next, a model protein antigen ovalbumin (OVA) was formulated in different doses of DOTAP liposomes. Similarly, the results showed that 20μg OVA formulated in 200nmol DOTAP with 30mM NaCl had the best OVA-specific antibody response, including both IgG1 and IgG2a, suggesting both Th1 and Th2 immune responses were generated by this formulation. In conclusion, we have expanded the application of cationic DOTAP liposome formulation to protein based vaccines and also identified that small amounts of salt could change the physicochemical properties of the vaccine formulation and enhance the activity of the DOTAP/protein based vaccine. The enhancement of immune responses by salt is possibly due to its interference of the electrostatic interaction between the cationic lipid and the protein antigen to facilitate the antigen release from the carrier and at the same time activate the antigen presenting cells.

A promising balanced Th1 and Th2 directing immunological adjuvant, saponins from the root of Platycodon grandiflorum

The haemolytic activities and adjuvant potentials of Platycodon grandiflorum saponin (PGS) and its fractions on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. PGS was subjected to silica gel column chromatography to afford four fractions, and two fractions PGSC and PGSD selected for testing for activities because of containing dominant saponin peaks. PGS, PGSC, and PGSD showed a slight haemolytic effect, with their HD 50 value being 37.91 ± 2.24, 21.30 ± 1.22, 37.58 ± 1.86 μg/ml against 0.5% rabbit red blood cell, respectively. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing Alum (200 μg), Quil A (10 μg), PGS (50, 100 or 200 μg), PGSC, or PGSD (25, 50 or 100 μg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PGS and PGSC significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in OVA-immunized mice at three doses ( P < 0.01 or P < 0.001). However, no significant differences ( P > 0.05) were observed among the OVA group, OVA/Alum group and OVA/PGSD group. OVA-specific IgG, IgG1, and IgG2b antibody levels in serum were significantly enhanced by PGS, PGSC, and PGSD compared with OVA control group ( P < 0.05, P < 0.01, or P < 0.001). Moreover, the adjuvant effects of PGSC (50 or 100 μg) on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice were more significant than those of Alum. In conclusion, PGS seem to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.

Haemolytic activities and adjuvant effect of Anemone raddeana saponins (ARS) on the immune responses to ovalbumin in mice

In this study, saponins (ARS) extracted from the rhizoma of Anemone raddeana were evaluated for their haemolytic activities and its potential ability as adjuvant on the cellular and humoral immune responses of ICR mice against ovalbumin. The haemolytic activity of ARS was determined using 0.5% rabbit red blood cell. ARS showed a slight haemolytic effect, with its haemolytic percents being 16.50 and 3.56% at the concentrations of 500 and 250 μg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing Alum (200 μg), QuilA (10 and 20 μg) or ARS (50, 100 or 200 μ g) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. ARS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 μg ( P < 0.01 or P < 0.05). The OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by ARS compared with OVA control group ( P < 0.01 or P < 0.05). Moreover, no significant differences ( P > 0.05) were observed between enhancing effect of ARS and QuilA on the OVA-specific IgG2b antibody responses to OVA in mice. The results suggest that ARS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice.

Amelioration of Skewed Th1/Th2 Balance in Tumor-Bearing and Asthma-Induced Mice by Oral Administration of Agaricus blazei Extracts

We showed in a previous study that hot-water extracts of Agaricus blazei (Agaricus extracts) had anti-tumor activity to Meth A fibrosarcoma, but it remains unclear whether the Agaricus extracts ameliorate the skewed balance of type-1 T helper (Th1) and type-2 T helper (Th2) cells. We examined whether Agaricus extracts effect the skewed Th1/Th2 balance in tumor-bearing and asthma-induced mice. When Meth A-bearing mice were given orally either Agaricus extracts or water once a day starting 5 days after tumor implantation, spleen T cells, prepared from tumor-bearing mice treated with Agaricus extracts, in response to anti-CD3 monoclonal antibody produced significantly higher levels of interferon γ (IFN-γ) than that of controls. The mRNA expression of IFN-γ-inducing protein 10 and the frequency of CD69+ or CD49d+ cells, among activated T cells infiltrated into tumors, significantly increased in Agaricus-treated mice, compared with those of tumor-controls. In asthma-induced mice, treatment with the Agaricus extracts caused significant downregulation of OVA-specific antibody responses of IgG1 and IgE but not of IgG2a, and significantly decreased total cell numbers, levels of interleukin 5, and eosinophil numbers in bronchial alveolar lavage fluids. IFN-γ production by anti-CD3-stimulated spleen cells, obtained from Agaricus-treated mice, significantly increased. Our results strongly suggest that oral administration of Agaricus extracts ameliorates the Th1/Th2 balance from the Th2-skewed conditions.

IgE Enhances Specific Antibody and T‐cell Responses in Mice Overexpressing CD23

IgE administered with its specific antigen in vivo induces enhanced proliferation of specific T cells as well as enhanced production of specific antibodies. Both effects are dependent on the low-affinity receptor for IgE (CD23) and the underlying mechanism is thought to be increased antigen presentation following uptake of IgE/antigen complexes via CD23(+) B cells. By contrast, CD23 negatively regulates antibody responses to antigens administered with alum, i.e. without IgE. This effect has been observed as low IgG1 and IgE responses in transgenic mice overexpressing CD23 (CD23Tg). The present study was designed to test whether IgE could enhance antibody and T-cell responses in CD23Tg animals or whether CD23's downregulatory effect precludes IgE-mediated enhancement. IgE-anti-TNP administered with OVA-TNP enhances the OVA-specific antibody responses in wild-type (wt) and CD23Tg mice equally well. Interestingly, the total magnitude of antibody responses to IgE + OVA-TNP and to uncomplexed OVA-TNP, as well as to sheep erythrocytes and keyhole limpet haemocyanine, were lower in the CD23Tg mice. IgE induced proliferation of OVA-specific CD4(+) T cells to the same degree in wt and CD23Tg mice. The effect on T cells was dependent on CD23(+) B cells as demonstrated in in vitro proliferation assays. In conclusion, CD23 does indeed have dual immunoregulatory effects in the same animal. The receptor mediates enhancement of antibody and T-cell responses to IgE-complexed antigen, most likely via increased presentation of complexed antigen, while it negatively regulates the total antibody response to a variety of antigens.

Immunological Adjuvant Effect of Ginsenoside Rh4 from the Roots of Panax notoginseng on Specific Antibody and Cellular Response to Ovalbumin in Mice

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.

Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice

Protopanaxadiol saponins (Rg3, Rd, Rc, Rb1 and Rb2) and protopanaxatriol saponins (Rg1, Re and Rg2) isolated from the root of Panax ginseng C.A. Meyer were evaluated for their adjuvant effects on the immune responses to ovalbumin (OVA) in mice. BALB/c mice were subcutaneously injected twice at a 3-week interval with 10 μg of ovalbumin or 10 μg of OVA plus 50 μg of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Rg1, Re or Rg2 or Quil A ( n = 5). Blood samples were collected for measuring specific total-IgG, IgG1 and IgG2a, and splenocytes were harvested for determining lymphocyte proliferation as well as IFN-γ and IL-5 production 2 weeks after the boosting. The results indicated that OVA-specific antibody responses were significantly higher in mice immunized with OVA co-administered with Rg1, Re, Rg2, Rg3 and Rb1 but not with Rd, Rc and Rb2 when compared with the control (immunized with OVA only). Significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as the production of both IL-5 and IFN-γ stimulated by OVA were also detected in mice immunized with OVA co-administered with Rg1 but not with Rb1, Re and Rg3. Of the ginsenosides studied, Rg1, Re, Rg2, Rg3 and Rb1 have more potent adjuvant properties than the others, indicating that they are the major constituents contributing to the adjuvant activities of total ginseng saponins. Varieties of ginsenosides in adjuvant activity might be attributed to the varieties of molecular conformations determined by the side sugar chains attaching to their dammarane skeleton.

Haemolytic Activity and Adjuvant Effect of Notoginsenoside K from the Roots of Panax notoginseng

Notoginsenoside K (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD50 value) being 318+/-13 microg/ml, on a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1, especially at a dose of 25 mug compared to an OVA control group (P<0.001). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (AlOH gel; P<0.01). These results suggest that 1 exhibits a slight haemolytic activity and a significant adjuvant effect on specific antibody and cellular response against OVA in mice.