Introduction

Throughout the history of vaccine development, science has always focused on finding a safe and efficacious immune response. This could already be seen in the early stages of vaccine immunology in the late eighteenth century, from the first tests against smallpox by Edward Jenner (Riedel, 2005) followed by the research of Louis Pasteur in the nineteenth century and up until the present.

The first descriptions of the use of adjuvants dates from the 1920s (Schijns, 2001) and the list of substances with adjuvantal properties has been growing ever since then, alongside technological development in the field of vaccines (formulation of subunits, peptides, nucleic acid fragments, etc.). Modern immunology of the last few decades has opened the door to detailed studies of the immunological properties of these substances and the development of new and more potent adjuvants.

Key Aspects of Immunology

Some of the latest advances in modern immunology are the description of the systems of pathogen recognition receptors (PRR) such as Toll-like receptors (TLR) and the role of dendritic cells in immune response development (Iwasaky et al. 2004).

The development of vaccines has taken advantage of these advances to study and develop better adjuvants according to this knowledge. Therefore, the main job adjuvants seem to have is to establish a strong link between the non-specific immune response and specific immune response (Werling et al., 2003, Mazzoni et al., 2004, Garlapati et al., 2008).

In this brief description of the first reactions of the immune response at the inoculation site of the vaccine, let us say the first are the antigen presenting cells (APCs), which are mainly macrophages and dendritic cells (DCs). They are responsible for antigen capture and presentation to the rest of the immune system. The DCs can be found throughout the body in subcutaneous tissue or muscle, when vaccines are given by intramuscular injection and in mucosal tissues for vaccines given orally or through the respiratory tract. Once the antigens have been captured, the APCs will trigger the adaptive immune response.

Traditionally, innate and specific immunity were understood as independent pathways in the immune response. But in reality, both types of responses are very close at least during the first steps in developing an immune response. Thus, we now know that the classical strategies of the innate system for recognising pathogens have a great significance in the specific response generated (Werling et al. 2003). These strategies are based on the recognition of pathogen-associated molecular patterns (PAMPs) by mononuclear cells. Toll-like receptors (TLR) and other PRR systems of mononuclear cells are the main routes of PAMP recognition that these cells use to recognise the presence of the pathogen and stimulate dendritic cell maturation. Finally it has been reported that ginseng can stimulate mononuclear cells by means of TLR-4 (Nayaka et al., 2004).

Ginseng Saponins as an Adjuvant

With this immunological summary, the vaccinal immune response should be understood as the result of all components at once. In the case of HIPRAMUNE® G (the new adjuvant released by Laboratorios HIPRA for its vaccine, SUISENG®) an aluminium component (aluminium hydroxide) is included as is a plant extract (ginseng saponins) (Sun et al., 2008; Rivera et al., 2003).

Ginseng saponins (the ginsenosides) are the pharmacologically active ingredient of the extracts of this root. These saponins, unlike others of plant origin, have low toxicity and a haemolytic effect that makes them ideal for use as vaccine adjuvants.

Figure 1. Chemical structure of the ginsenosides (Sun et al., 2007)

Different immunological effects of ginseng root (Panax ginseng) have been described in scientific literature. If we start with the most primary specific response, some authors have reported a general increase in oxidative reactions and phagocytosis of polymorphonuclear leukocytes and macrophage cells (Oh et al., 2004). In addition, there has been a proliferation of immune cells in many studies due to the activity of ginsenosides. This has been described in mouse, cattle, poultry and rabbit cells (Joo et al., 2004; Sun et al., 2005).

If we focus further on the specific immune response to certain antigens, most of the articles published study the stimulation of specific antibodies due to the effects of ginseng. The increase in specific immunoglobulins has been demonstrated in many species, including mice, rats, rabbits, chickens, pigs and humans (Scaglione et al., 1996).

Some studies have succeeded in characterising the pattern of cytokines expressed after stimulation with ginseng derivates. Thus, Keranova et al. (1990) observed an increase in the expression of interleukin (IL)-1 in mouse macrophages. The same effect was described by Nayaka et al. in 2004, as was an increase in IL-6 the INF. Kim et al. (1998) described an increase in IL-2 in stimulated lymphocytes. In another study with mouse spleen cells, it was described how the F3 ginsenoside induced and increased the synthesis of IL-2 and INF. and a reduction in the expression of IL-4 and IL-10 (Yu et al., 2004). Larsen et al. (2004) described an enhancement of the synthesis of IL-12 in peripheral blood mononuclear cells of humans (PBMCs) stimulated in vitro and suggested that there could be a strong induction of the TH1 immune response. However, other research groups (Lee et al, 2004) found that ginsenosides could induce an increase in the synthesis of the IL-2 and IL-4 cytokines in CD4+ T lymphocytes.

With these observations and other results from the project, the authors suggested that ginseng could induce a type 2 (Th2) immune response. Another study, published the same year (Liou et al., 2004), showed that ginseng induced an increase in the expression of Th1 (related to IL-2 and IFN.) as well as Th2 (related to IL-4 and IL-10). These authors also observed an increase in IgG, IgM and IgA due to stimulation with ginseng. The paper concludes that the ginseng extract can regulate the production of antibodies by stimulating the synthesis of Th1 cytokines (IL-2, IFN.) and Th2 (IL4, IL10).

Hipramune G: Ginseng Saponins Plus Aluminium Hydroxide

In pursuit of the ideal adjuvant, the combination of different substances is a very broad field to explore. The final objective of the combinations is to be able to combine the benefits and properties of substances in a new and better formulation. In the case of ginseng saponins, the synergistic effect of the combination with aluminium hydroxide has been demonstrated in different species.

A study conducted by Sun et al. (2008) used an immunisation model with ovalbumin to assess different adjuvant formulations in vaccines. The authors compared formulations that included aluminium hydroxide, Ginsenoside Rg1, and the combination of these two substances. They characterised the immune response (Th1 and Th2) by measuring levels of IgG isotype in serum, the production of IFN. and IL-5. Their main conclusions were that ginsenosides promoted both the Th1 and Th2 response and that the combination of aluminium and ginsenosides regulated the Th1/Th2 response, thereby obtaining improved protection.

Rivera et al. (2003) also observed this effect in guinea pigs when aluminium hydroxide was combined with ginsenosides. Their results showed that the combination of ginseng and aluminium enhanced the production of antibodies compared with vaccines containing either substance on their own. Rivera et al, 2003b subsequently performed the same experiment in pigs and demonstrated the same effect as in guinea pigs. The study showed that animals vaccinated with the combination of aluminium hydroxide plus ginseng had higher levels of specific antibodies (IgGs) than other groups.

In regard to the mechanism of action, the study concluded by citing the hypothesis that this effect must be caused by the known depot effect of aluminium hydroxide along with the immunological properties of ginseng saponins.

Bibliography

Garlapati, S., M. Facci, M. Polewicz, S. Strom, L.A. Babiuk, G. Mutwiri, R.E. Hancock, M.R. Elliott, and V. Gerdts. 2008. Strategies to link innate and adaptive immunity when designing vaccine adjuvants. Vet.Immunol.Immunopathol.

Iwasaki, A. and R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat.Immunol. 5:987-995.

Joo S.S., Won T.J., Kim M.S. and Lee D.I. 2004. Hematopoietic effect of ginsenoside Rg3 in ICR mouse primary cultures and its application to a biological response modifier. Fitoterapia.

Kenarova et al. 1990. Immunomodulating Activity of Ginsenoside Rg1 from Panax Ginseng.

Kim K.H., Lee Y.S., Jung I.S., Park S.Y., Chung H.Y., Lee I.R. and Yun Y.S. 1998. Acidic polysaccharide from Panax ginseng, ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med.

Lee, E.J., E. Ko, J. Lee, S. Rho, S. Ko, M.K. Shin, B.I. Min, M.C. Hong, S.Y. Kim and H. Bae. 2004. Ginsenoside Rg1 enhances CD4 (+) T-cell activities and modulates Th1/Th2 differentiation. Int. Immunopharmacol. 4:235-244.

Liou, C.J., M.L. Li and J. Tseng. 2004. Intraperitoneal injection of ginseng extract enhances both immunoglobulin and cytokine production in mice. Am.J.Chin Med. 32:75-88.

Mazzoni, A. and D.M. Segal. 2004. Controlling the Toll road to dendritic cell polarization. J.Leukoc.Biol. 75:721-730.

Nakaya T.At., Kita M., Kuriyama H.,; Iwakura Y. and Imanishi J. 2004. Panax ginseng induces production of proinflammatory cytokines via toll-like receptor. J Interferon Cytokine Res.

Oh G.S., Pae H.O., Choi B.M., Seo E.A., Kim D.H., Shin M.K., Kim J.D., Kim J.B. and Chung H.T. 2004. 20(S)-Protopanaxatriol, one of ginsenoside metabolites, inhibits inducible nitric oxide synthase and cyclooxygenase-2 expressions through inactivation of nuclear factor-kappaB in RAW 264.7 macrophages stimulated with lipopolysaccharide. Cancer Letters.

Riedel, S. 2005. Edward Jenner and the history of smallpox and vaccination. Proc. (Bayl.Univ Med.Cent.). 18:21-25.

Rivera, E., A. Daggfeldt and S. Hu. 2003. Ginseng extract in aluminium hydroxide adjuvanted vaccines improves the antibody response of pigs to porcine parvovirus and Erysipelothrix rhusiopathiae. Vet.Immunol.Immunopathol. 91:19-27.

Rivera, E., S. Hu and C. Concha. 2003. Ginseng and aluminium hydroxide act synergistically as vaccine adjuvants. Vaccine. 21:1149-1157.

Rivera, E., P.F. Ekholm, M. Inganas, S. Paulie and K.O. Gronvik. 2005. The Rb1 fraction of ginseng elicits a balanced Th1 and Th2 immune response. Vaccine. 23:5411-5419.

Scaglione F., Cattaneo G., Alessandria M. and Cogo R. 1996. Efficacy and safety of the standardised Ginseng extract G115 for potentiating vaccination against the influenza syndrome and protection against the common cold. Drugs Exp Clin Res.

Schijns, V. 2001. Activation and programming of adaptive immune responses by vaccine adjuvants. Veterinary Sciences Tomorrow. 3: 1-7.

Sun et al. 2005. Relationship between haemolytic and adjuvant activity and structure of protopanaxadiol-type saponins from the roots of Panax notoginseng. Vaccine.

Sun, J., S. Hu and X. Song. 2007. Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice. Vaccine. 25:1114-1120.

Sun, J., X. Song, and S. Hu. 2008a. Ginsenoside Rg1 and aluminium hydroxide synergistically promote immune responses to ovalbumin in BALB/c mice. Clin.Vaccine Immunol. 15:303-307.

Sun, Y., H. Tong, M. Li, Y. Li, S. Guan, and J. Liu. 2008b. Immunological adjuvant effect of Japanese ginseng saponins (JGS) on specific antibody and cellular response to ovalbumin and its haemolytic activities. Vaccine.

Werling, D. and T.W. Jungi. 2003. TOLL-like receptors linking innate and adaptive immune response. Vet.Immunol.Immunopathol. 91:1-12.

Yu, J.L., D.Q. Dou, X.H. Chen, H.Z. Yang, N. Guo and G.F. Cheng. 2004. Immunoenhancing activity of protopanaxatriol-type ginsenoside-F3 in murine spleen cells. Acta Pharmacol.Sin. 25:1671-1676.

Larsen, M.W., C. Moser, N. Hoiby, Z. Song and A. Kharazmi. 2004. Ginseng modulates the immune response by induction of interleukin-12 production. APMIS. 112:369-373.