Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55588—Adjuvants of undefined constitution

Definitions

• Vaccine adjuvants comprising ginseng plant extract and added aluminium salt.
• the present invention relates to a new group of adjuvants capable of enhancing the immunogenic effect of antigens used in immunization of mammals.
• the invention also relates to a method of producing an immunogenic composition comprising the adjuvant according to the invention.
• Immunization to protect against communicable disease is one of the most successful and cost-effective practices of modern medicine. Smallpox has been completely eliminated by vaccination, and the incidence of many other dreaded diseases such as polio and diphtheria has been drastically reduced through immunization programs.
• vaccines especially those based on the use of inactivated viruses, vary in effectiveness.
• adjuvants are frequently used.
• adjuvants are often aluminum compounds, in the form of hydroxides or phosphates.
• these aluminium compounds result in vaccines, the immunopotentiation action of which is explained by the resulting vaccine depots, from which the release of antigen for adsorption is retarded.
• the aggregation and settling tendencies of solid aluminium compounds, as well as the behaviour of their residue is a disadvantage of the use of these compounds.
• further problems arise with vaccines adjuvanted with Al-salts due to a number of other reported limitations, e.g. a) the antibody response is often of short duration, b) they induce poor cell mediated immunity (CMI) and c) they can support the production of IgE antibodies which may lead to hypersensitive reactions.
• CRI cell mediated immunity
• the present invention solves the problems defined above by providing a novel composition comprising efficient and safe ingredients capable of exerting specific and highly efficient adjuvant properties to a vaccine administered before, after or simultaneous with said ingredients.
• the composition of the invention is capable of enhancing the immunogenic effect of a vaccine, comprises an extract of a ginseng plant and an aluminium salt, and is obtainable by a method comprising the steps of:
• the composition according to the invention is an extract from a Ginseng root, and preferably Panax ginseng root.
• a ginseng extract which comprises tri-terpenoid glycosides, including the Rbj structure disclosed below, is combined in the composition of the invention with an aluminium salt, such as Al(OH) 3 .
• an aluminium salt such as Al(OH) 3 .
• the Rbl structure is preferably included at a vaccine dose of approximately 166 ⁇ g.
• the extract comprises one or more, most preferably all, of the ginsenosides Rgl, Re, Rbl, Re, Rb2 and Rd, as well as any of the other 28 known ginsenoside compunds.
• the present invention relates to a kit comprising the composition for use as adjuvant or co-adjuvant of an immunogenic composition as well as to the use of such a composition in the manufacture of a pharmaceutical preparation for enhancing the immunogenic effect of a vaccine. Further, the invention also relates to a pharmaceutical preparation manufactured by the present use, which pharmaceutical preparation further comprises a pharmaceutically acceptable carrier.
• FIG 1 shows the antibody response induced by different Porcine Parvovirus (PPV) vaccines as measured by Hi-tests (Example 1 of the present invention).
• PSV Porcine Parvovirus
• Figure 2 shows the effect of the pre-treatment with ginseng on the antibody response of guinea pigs to PPV virus (Example 3 of the present invention), A being animals immunized with Al(OH) 3 adjuvanted PPV- vaccines while B is with PPV only.
• Figure 3 shows a determination of the antibody response to Erysipelothrix rhusiopathiae as assayed by ELISA.
• Figure 4 shows a determination of the antibody response to Erysipelothrix
• Rhusiopathiae as assayed by immunoblotting.
• adjuvant means any substance or compound capable of promoting an increased immune response in a mammalian species, e.g. by modifying the activities of cells that are concerned with generating and maintaining the immune response or by modifying the presentation of antigen to the immune system.
• vaccine means any compound or preparation of antigens designed to stimulate a normal primary immune response, resulting in proliferation of the memory cells and the ability to exhibit a secondary memory or anamnestic response upon subsequent exposure to the same antigens.
• the present invention relates to a composition, which comprises ingredients capable of enhancing the immunogenic effect of a vaccine, and which composition is obtainable by a method comprising the steps of: (a) providing an extract of a ginseng plant, and (b) adding an aluminium salt to the extract.
• the composition according to the invention comprises an extract from ginseng root, preferably Panax ginseng root, e.g. in amounts of about 1-20 mg, such as about 1-15 mg, e.g. about 1-10 mg and more specifically about 2 mg, ginseng for each vaccine dose.
• Ginseng-dry extracts have been analysed and established to contain a number of active substances, e.g.
• the extract will comprise at least one tri-terpenoid glycoside, preferably with the structure of Rb] as defined by formula (I) below:
• the ginsenosides extracted from the Panax ginseng C.A. Meyer-root are tri-terpenoid glycosides of the dammaran series that according to the present invention have proved to exhibit a strong adjuvant effect, while being safe and without the above discussed disadvantages.
• the present ginsenosides apart from inducing an antibody response, compared to previously tested saponins, the present ginsenosides have other advantageous properties, which make them promising adjuvants, e.g.:
• the present ginsenosides trigger the immune system (see Example 3, Fig. 2) in such a manner that posterior antigen injection leads to an increased specific-antibody response, indicating that the treatment of low responders with ginseng may provide them with a better immunization.
• the present ginsenosides are safe for the stomach and resist the gastric secretions. Therefore, as discussed below, they are contemplated as suitable adjuvants for oral vaccines.
• the present invention concerns an extract comprising ginsenosides and an aluminium salt, as well as other ingredients, which is shown to have a stronger adjuvant activity, than the pure ginsenosides when tested alone (table 6).
• the present inventors have for the first time disclosed advantageous use of an extract of the ginsenosides as a vaccine adjuvant, even though ginseng has previously been shown to exhibit advantageous properties in relation to health aspects.
• the extract obtained from the Panax ginseng C.A. Meyer-root and named ginseng has been used in the Chinese medicine for different purposes e.g. as a stimulant of the central nervous system, increasing mental concentration or as an anti-stress, and as a general tonic improving the natural resistance against infections. More recently, studies demonstrate that the ginseng support-cell proliferation.
• Ginseng also promotes the phagocytic activity of human alveolar macrophages as well as the oxidative and phagocytic activities of the bovine polymorphonuclear leukocytes. However, as mentioned above, it has never been shown before to exhibit adjuvant or coadjuvant properties when used with vaccines against pathogenic microorganism.
• the composition according to the present invention will further comprise an aluminium salt, such as Al(OH) 3 .
• an aluminium salt such as Al(OH) 3 .
• any other salt of aluminium such as aluminium phosphate or aluminium sulphate, or alums, such as ammonium alum or potassium alum, may equally well be used in the present context, as long as it's chemical properties are sufficiently similar to the Al(OH) 3 used herein.
• HI- titre for the animals injected with PPV antigen only was 320 ⁇ 0.
• mean titre value was 2026 ⁇ 1206 for the sera from the animals injected with the same vaccine but adjuvanted with 4 mg of ginsenoside, while the antibody titre induced by a vaccine containing 50% Al(OH) 3 -gel was 2986 ⁇ 1596.
• the ginsenoside and Al(OH) 3 acted synergistically and further improved the antibody response to the PPV antigen to 6826 ⁇ 2413.
• vaccines containing ginsenoside as adjuvant induced the production of virus-neutralising antibodies; this was demonstrated by neutralisation assays using PK-15 cells and live PPV virus. Furthermore, it was demonstrated that the immunomodulatory effect of the ginsenoside is dose-dependent and that the ginsenosides are safe adjuvants when injected subcutaneously in guinea pigs, mice and minks or intramuscularly in pigs.
• the ginsenoside have a very low haemolytic activity on the red blood cells of swine, cattle, chicken or guinea pigs.
• the present composition will comprise Rb] within a range of 5-500 ⁇ g, such as about 40- 250 ⁇ g, e.g. about 60-175 ⁇ g and preferably about 80 ⁇ g to about 100 ⁇ g /vaccine dosage.
• Rb the tri-terpenoid glycoside
• the present Rb] may originate e.g. from chemical synthesis, even though it was first discovered by using an extract of ginseng.
• the quantity of aluminium in the present composition is not limited to the ones shown in the examples. Rather, since Al(OH) 3 has been used for some time, ideal concentrations for PPV- vaccines has been established and put in use (Joo, H.S., Molitor, T.W. and Leman, A.D. Antibody responses of guinea-pigs, rabbits and pigs to inactivated porcine parvovirus vaccines, Veterinary microbiol., 1989, 9, 27-33; Rivera, E., Sjosten, C.G., Bergman.R.
• the novel advantageous synergistic effect of ginseng specifically of tri-terpenoid glycosides, such as Rb l5 combined with an aluminium salt provides an improved adjuvant effect without need of increasing the amount of Al(OH) 3 , thus avoiding the disadvantages of large quantities of aluminium discussed above.
• ginseng as co-adjuvant it appears to be possible to minimise the total amount of Al-salts in vaccines, thereby minimising the risk e.g. for allergies.
• ginseng induces interferon production and stimulates the activity of cytotoxic T-lymphocytes
• the use of ginseng as a co- adjuvant in the Al-salts adjuvanted vaccines is contemplated to lead to better immunizations in many aspects.
• the invention relates to a kit comprising the ginseng and aluminium salt ingredients formulated into one single adjuvant composition, optionally together with a pharmaceutically acceptable carrier, such as sterile water or saline.
• a pharmaceutically acceptable carrier such as sterile water or saline.
• the present kit may further be prepared with sufficient amounts for one or more booster administrations, thus adapted for single administrations or a series of administrations.
• it does not appear to be critical whether the present adjuvant is injected before, after or simultaneously with the vaccine, even though conventionally the adjuvant and the antigen are simultaneously administered.
• ginseng improves the antibody response to the PPV antigen regardless of the administration procedure used (see Example nos.l, 2 and 3).
• the ingredients of the present kit may be administered parenterally, e.g. subcutaneously or intramuscularly, even though other methods, such as injections also are contemplated, as well as oral administration.
• parenterally e.g. subcutaneously or intramuscularly
• other methods such as injections also are contemplated, as well as oral administration.
• the skilled in this field will decide for each instance the suitable way of delivery, and for a brief review of methods of drug delivery, see e.g. Langer, Science 249:1527-1533 (1990).
• the present kit will also include written instructions regarding the use thereof, optionally also means for administration, etc., in a suitable container.
• the present invention relates to an extract comprising tri-terpenoid glycosides, preferably comprising the structure of Rb l5 for use as an adjuvant for use to enhance the immunogenic properties of a vaccine as well as to the use of a tri- terpenoid glycoside, preferably comprising the structure of Rb l 5 in the manufacture of a pharmaceutical composition effective as adjuvant, or in the manufacture of a complete vaccine comprising such adjuvant.
• a pharmaceutical composition effective as adjuvant, or in the manufacture of a complete vaccine comprising such adjuvant.
• the invention also includes a tri- terpenoid glycoside, preferably comprising the structure of Rb], together with an aluminium salt for use as a medicament aimed at treating conditions characterized by a weakened immune system.
• the invention also relates to a method of immunization, wherein an extract from ginseng root, preferably Panax ginseng root, is administered before, simultaneously with or after the administration of an immunogenic substance to enhance the effect thereof.
• said extract comprises a tri-terpenoid glycoside of the dammoran series, preferably comprising the structure of Rb] of formula (I) as defined above.
• said extract is administered as a co-adjuvant with an aluminium salt, such as Al(OH) 3 , wherein the ginseng is administered so as to provide an amount of about 5-500 ⁇ g, preferably from about 80 ⁇ g to 100 ⁇ g, of Rb].
• the invention relates to a pharmaceutical preparation for use in a method as defined above, wherein ginseng, such as the tri-terpenoid glycoside, has been combined with a pharmaceutically acceptable carrier, and/or supplemented with further excipients or additives.
• the present preparation further comprises an aluminium salt, such as Al(OH) 3 , while the tri-terpenoid glycoside is Rb] of the above defined formula (I) in amounts as defined above.
• the present kit includes the vaccine in addition to the above discussed ingredients formulated into one single composition.
• the formulation may comprise a hydrating agent (e.g., liposomes), a penetration enhancer, or both.
• the formulation may comprise AQUAPHOR (an emulsion of petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin), emulsions (e.g., aqueous creams), oil-in-water emulsions (e.g., oily creams), anhydrous lipids and oil-in-water emulsions, anhydrous lipids and water-in- oil emulsions, fats, waxes, oil, silicones, and humectants (e.g., glycerol).
• AQUAPHOR an emulsion of petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin
• emulsions e.g., aqueous creams
• oil-in-water emulsions e.g., oily creams
• anhydrous lipids and oil-in-water emulsions
• Figure 1 shows the antibody response induced by different PPV vaccines as measured by Hl-tests (Example 1, below).
• Vac.no.1 contained PPV virus only
• vac.no.2 contained virus and aluminium hydroxide
• vac.no.3 was a double adjuvanted vaccine containing aluminium hydroxide and ginseng
• vac.no.4 and vac.no.5 contained 4 or 2 mg of Chinese ginseng respectively
• vac.no.6 contained 2 mg of alian ginseng.
• Figure 2 shows the effect of the pre-treatment with ginseng on the antibody response of guinea pigs to PPV virus (Example 3, below).
• Groups of animals were immunized with Al(OH) 3 adjuvanted vaccines (A) or with PPV only (B).
• Each bar represent the group titre mean value.
• Figure 3 shows a determination of the antibody response to Erysipelothrix rhusiopathiae as assayed by ELISA. The results are presented as the group mean absorbance measured at 450 nm.
• Figure 4 shows a determination of the antibody response to Erysipelothrix
• Rhusiopathiae as assayed by immunoblotting. Sera from individual pigs, lane 2 - 9, were tested 3 weeks post booster. Lane 1 and 10; prestained molecular weight markers.
• the present experimental section has been divided into two parts, part I and part II.
• part I it is demonstrated that ginseng is a safe adjuvant and that ginseng and aluminium hydroxide act synergistically resulting in a potent adjuvant combination which induces the production of higher antibody titres than do either adjuvant when used alone.
• part II it is demonstrated that the immunogenic potency of aluminium- hydroxide adjuvanted vaccines against PPV and Erysipelothrix rhusiopathiae infections can be improved by using ginseng as a vaccine co-adjuvant.
• the experimental section has been disposed as follows: Disposition
• GS-De ginsenosides contained in the extract prepared from the Panax ginseng C.A. Meyer-root have adjuvant properties as demonstrated by a) injecting guinea pigs with a mixture of the GS-De and the inactivated porcine parvovirus (PPV) antigen as a conventional vaccine, b) injecting the PPV antigen and the GS-De simultaneously but separately at different places on the animal or c) injecting only the GS-De one or two weeks prior to immunization with the PPV antigen.
• PPV porcine parvovirus
• Guinea pigs (GPs) were chosen for testing the vaccines because GPs are recommended as suitable animals for testing the immunogenicity of vaccines against porcine parvovirus infection in the pig (Joo et al., 1989, supra).
• GPs Guinea pigs
• most available vaccines for veterinary use include PPV vaccines
• contain aluminium-hydroxide gel as adjuvant (Morein, B., L ⁇ vgren-Bengtsson, K. and Cox, J. General principles of vaccinology, Modern adjuvants. Functional aspects. In Concepts in Vaccine Development, Ed.
• Ginseng Extracts Two ginseng dry extracts were evaluated as adjuvant for PPV vaccines; both extracts were prepared from the Panax ginseng C.A. Meyer-root and contained similar amounts of the protopanaxatriol ginsenosides Re and Rgl .
• the ginseng extracted in China was kindly provided by the Pharmaceutical Co. Ltd. (Chiatai Qingchubao, China); in this study it is referred to as CH-GS.
• the ginseng extracted in Italy was provided free of charge by the Indena SPA company (Italy) and is referred to as IT- GS.
• the PK-15 cell line was used to propagate the PPV virus, to control virus inactivation and in the serum neutralisation assays.
• the cells and the virus were cultured as described previously (Rivera et al., 1986, supra).
• a harvest of PPV virus was clarified by filtration through a 0.25 ⁇ m filter. Thereafter, the filtered virus material, containing 2048 haemagglutinating units (HAU) per 0.05ml, was inactivated with formalin at a final concentration of 0.2%
• Virus inactivation was carried out at 37°C for three weeks. The absence of residual live virus was demonstrated by serially passing (4 passages) the formalinised virus material in PK-15 cells. After the fourth passage, the cells were fixed by immersing the specimens in a 20% acetone bath (Rivera et al., 1986, supra) and stained using mouse anti-PPV-monoclonal antibodies (Rivera, E., Gronvik, K.O and Karlsson, K.A. A new method for rapidly removing contaminating micro-organisms from porcine parvovirus or pseudorabies virus master-seed suspensions.
• Vaccine 1993,11, 363-365
• HRPO-conjugated goat anti-mouse antibodies Bio Rad Laboratories, Richmond, CA, USA.
• the inactivated virus was diluted with physiological sodium chloride solution (NaCl-sol.) until it contained 256 HAU per 0.05 ml. After adjusting the pH to 7.2, the diluted virus (dil-virus) was used in the vaccine preparations.
• adjuvants were assayed: a) 3%-Al(OH) 3 gel (Alhydrogel, Superiors A/S; Denmark), b) Chinese ginseng, i.e. CH-GS, c) Italian ginseng, i.e. IT-GS, d) a mixture of Al(OH) 3 and CH-GS, e) NaCl-solution instead of adjuvant.
• Vaccine (vac.) No. 1 contained dil-virus and NaCl-solution instead of adjuvant (aqueous vaccine).
• Vac. No. 2 contained 3%-Al(OH) 3 as adjuvant.
• Vac. No. 3 contained double adjuvant and was a mixture of dil-virus containing 2 mg CH-GS/ml and 3%-Al(OH) 3 .
• Vaccines No. 4 and No. 5 were mixtures of dil-virus and NaCl- solutions containing (per ml) 4 or 2 mg. CH-GS, respectively.
• Vac. No. 6 included IT-GS as adjuvant at a concentration of 2mg.per ml NaCl- solution.
• the first experiment was carried out to ascertain whether ginseng has a adjuvant effect or not, to compare its effect with the one obtained using Al(OH) 3 and to investigate if ginseng and Al(OH) 3 possibly act synergistically.
• 24 GPs were divided into six groups and they were injected subcutaneously with 2 ml of one of the vaccines described above. The animals received two vaccine doses at 3 week intervals. Blood samples were taken 2 weeks after the second injection.
• This experiment was designed to determine the number of injections needed for an optimal immunization using ginseng adjuvanted vaccines and to ascertain whether or not the antigen and ginseng bind together and build a complex.
• 22 GPs were divided into four groups of five GPs each and one group of two GPs (negative control animals).
• the animals included in group 1 were vaccinated once with a mixture of 1 ml dil-virus and 1 ml NaCl-solution containing 1 mg of CH-GS.
• the animals included in group 2 were vaccinated twice at three week intervals using the same vaccine as for group 1.
• the GPs in group 3 were injected once with 1 ml dil-virus and with one ml NaCl-solution containing 1 mg of CH-GS. Both preparations were injected simultaneously but each of them at different sites on the animals.
• the GP in group 4 were immunized in the same way as group 3, but on two occasions with a three week interval.
• the animals included in group 5 received two injections of NaCl-solution. Blood samples were taken as follows: 3 weeks after the first vaccination, and both one and three weeks after the booster dose.
• the aim of this experiment was to investigate if ginseng activates the immune system in an unspecified manner, triggering it for an enhanced antibody response upon antigen injection or immunization using a conventional vaccine.
• the animals were either treated with 2 mg of CH-GS or not treated at all.
• the ginseng was given diluted in NaCl, subcutaneously, on two or three occasions. Then, the animals were immunized twice at a three week interval, using the Al(OH) 3 -adjuvanted vaccine or dil-virus without adjuvant. In this experiment, 25 GPs were divided into 5 groups.
• the GPs in group 1 received in total three doses of ginseng and two doses Al(OH) 3 adjuvanted vaccine.
• the two first doses of ginseng were given with a weeks interval, 2 and 1 week before primary vaccination.
• the third dose of ginseng was given one week before re- vaccination.
• the GPs in group 2 received two doses of CH-GS, and two doses of Al(OH) 3 adjuvanted vaccine. Ginseng was given two weeks before the first vaccination and one week before re-vaccination.
• the animals in group 3 were not treated with ginseng, but they were vaccinated twice with the Al(OH) 3 -adjuvanted vaccine.
• the animals in group 4 were treated with ginseng in the same way as the animals included in group 1 except that they were immunized twice using 1 ml dil-virus without any adjuvant.
• the animals in group 5 were not treated with ginseng but they were vaccinated twice with the vaccine used for group 4. Blood samples were taken three weeks after primary vaccination and both one and three weeks after the booster dose.
• the antibody response to PPV virus was determined by means of haemagglutination inhibition (HI) and serum neutralisation (SN) tests.
• the HI test was carried out in V- type microplates using a 0.5%> suspension of GP red-blood cells and 16 HAU of PPV virus.
• the SN-test was carried out in flat bottomed microplates using freshly trypsinised cells and TCID 100/0.1 ml PPV virus.
• Virus infected cells were detected by means of anti-PPV monoclonal antibodies (Rivera et al., 1993, supra) and peroxidase-conjugated goat anti-mouse antibodies (Bio Rad Laboratories, Richmond, CA, USA.).
• the substrate used was 3-Amino-9-ethylcarbazole (AEC).
• the safety of the CH-GS was proven by injecting it subcutaneously into mice, guinea pigs and minks or intramuscularly in pigs. After injections, the mice and guinea pigs were observed daily for a week; the period of observation for minks and pigs was six weeks. Deviations from their normal behaviour or reactions at the injection site were recorded.
• the 25 NMRI-mice were divided into groups of five animals each and were inoculated either with 100, 50, 20 or 10 micrograms of CH-GS which had been re-suspended in 0.5 ml NaCl-sol. Control mice received 0.5 ml of NaCl-sol. The weight of the mice was controlled at day 0 and at day 4 and 7 post injection.
• the 8 guinea pigs were divided into groups of two animals each and were injected either with 4, 2 or 1 mg of CH-GS re-suspended in 2ml NaCl-sol.; the two control animals received NaCl-sol.
• the 20 minks were divided into groups of five animals each and were injected subcutaneously with 500, 100 or 50 micrograms ginseng in one ml NaCl-sol.
• the 9 pigs were injected twice with 2 mg CH-GS diluted in 2 ml of NaCl- sol. The injections were given intramuscularly in the neck, at a 3 week interval.
• the haemolytic activity of the CH-GS was assayed by incubating the ginseng with red blood cells (RBC's) from guinea pig, swine, cattle and chicken.
• RBC's red blood cells
• the assay was carried out as follows: RBC's from the species listed above were washed 3 times using NaCl-sol and low speed centrifugation (800 rpm, 10 min). Thereafter, the RBC's pellets were re-suspended at a concentration of 0.5%> in 0.01 M PBS, pH 7.2 containing 0.05% v/v of bovine serum albumin.
• the RBC's preparations were mixed with equal volumes of NaCl-solutions containing CH-GS at concentrations ranging from 100 to 2000 micrograms/ml preparation.
• the RBC's preparations were also incubated together with an ammonium chloride (NH 4 C1) solution to obtain haemolysis-positive controls.
• the mixtures were incubated at 37°C for one hour. Thereafter, the samples were centrifuged as described above. Next, the optical density of the supernatants was measured at 414 run.
• the vaccine containing double adjuvant Al(OH) 3 and CH-GS was also centrifuged as described above and the supernatant was tested for the presence of free PPV antigen by means of a haemagglutination test.
• the Al(OH) 3 adjuvanted vaccine (vac.no.2). was found to be more potent than the vaccine adjuvanted with 4 mg. CH-GS. However, the use of ginseng and Al(OH) 3 in the same vaccine further potentiated the antibody response to the PPV antigen from 2986 +1596 to 6826 ⁇ 2413,inducing the highest antibody titres recorded in this experiment (Fig.l).
• Fig.2 it is shown that treatment with ginseng prior to vaccination with PPV- antigen lead to increased specific antibody response.
• the triggering effect of ginseng on the immune system can more clearly be seen by comparing the results recorded for the animals included in the groups 3 and 4 and which were injected with PPV- virus without adjuvant (Fig.2).
• the mean antibody titre for the treated animals was 2816 vs 352 for the non-treated ones. Ginseng also improve the antibody response of the animals injected with the Al(OH) 3 -adjuvanted vaccine.
• the quantitative antibody response to PPV-virus assayed by Hi-tests is reported in the Figs. 1 and 2 and in Table 1.
• the qualitative antibody response assayed by the SN-tests demonstrated that the antibodies produced after vaccinations with ginseng adjuvanted vaccines are virus-neutralising-antibodies as demonstrated by the absence of virus positive cells (data not shown).
• mice used for testing the safety of ginseng remained clinically healthy during the entire period of the experiments. Neither local reactions nor alterations in the animals behaviour were observed.
• the mean body-weight recorded for the mice injected with the highest dose of ginseng was 18.78 g and 20.34 g at the beginning and the end of the experiment respectively.
• the mean body-weight recorded for the control mice was 18.34 g and 20.6 g respectively.
• the weight of the minks, GP or pigs was not controlled. However, apparently the pigs developed normally since the slaughterhouse recorded their weight to be similar to that of other animals of the same age.
• ginseng did not cause haemolysis of bovine RBC's but does have a small effect on swine and chicken RBC's.
• the highest haemolytic activity was recorded in the assays carried out with the RBC's of GP as shown in Table 3 below. However, it was also noted that the GP RBC's showed the highest degree of spontaneous haemolysis.
• Table 3 The haemolytic activity of various doses of CH-GS on erythrocytes from different species. The results are presented as the absorbance mean-value obtained from two replicates.
• the quantity of free ginseng detected in the supernatant obtained from the sample incubated with Al(OH)3 gel was found to be greatly reduced from 2 mg to 0.375 mg/ml. By comparison the sample incubated with NaCl-sol. Showed 1.23 mg ginseng/ml.
• the antibody response to porcine parvovirus was assayed by haemagglutination inhibition tests whereas the immune response to Erysipelothrix rhusiopathiae was evaluated by the mouse potency test and ELISA.
• the ginseng used in this study was a dry extract prepared from the Panax ginseng C.A. Meyer-root and was provided free of charge by the Pharmaceutical Co. Ltd. (Chiatai Qingchubao, People's Republic of China). Before use, the ginseng was re- suspended in a physiological NaCl solution (saline) and added to the vaccines at a final concentration of 2 mg ginseng per 2 ml dose of vaccine.
• a physiological NaCl solution saline
• the vaccines included two antigens: binary ethylenimin-inactivated porcine parvovirus (PPV) and formalin- inactivated Erysipelothrix rhusiopathiae (E. rhusiopathiae) serotype 2.
• PSV binary ethylenimin-inactivated porcine parvovirus
• E. rhusiopathiae formalin- inactivated Erysipelothrix rhusiopathiae serotype 2
• vaccine- 1 vacc.l
• vaccine-2 vacc.2
• HAU haemagglutination units
• E. rhusiopathiae serotype 2 bacteria corresponding to 50 protective international units (LU.) per dose.
• Both vacc.l and vacc.2 contained 50%, v/v aluminium hydroxide.
• Samples of the vaccines described above were mixed together with a ginseng-solution to a final concentration of 2 mg ginseng per 2 ml dose of vaccine.
• the vaccines were named vacc.l -GINSENG and vacc.2-GINSENG respectively. Apart of the vaccines listed above, the study also included the test of a standard Erysipelas vaccine containing 58 LU.
• the animals included in the evaluation of the vaccines were five-month-old, specific pathogen- free pigs (No.l4).They were moved to our experimental facilities, two weeks before vaccination and were kept isolated during the experiment. The pigs were divided into four vaccination groups of three animals each, and one control group of two animals. The pigs were injected intramuscularly with 2 ml of one of the vaccines listed above; the injections were given twice with a three week interval between them. Blood samples were taken before vaccinations and 2, 3 and 4 weeks after the second injection.
• the animals included in group 1 were immunized with vacc.l, the ones in group 2 with vacc.l -GINSENG, the pigs in group 3 with vacc.2 and the ones in group 4 with vacc.2-GINSENG.
• mice potency test was carried out according to the guidelines given by the European Pharmacopoeia for batch evaluation of the licensed erysipelas vaccines for swine, as follows: 270 female NMRI-mice from our own breeding house were randomly selected and moved to the test facilities four days before vaccination. The mean body weight of the mice was approximately 20 grams.
• mice were divided into 15 vaccination groups with 16 mice in each group and into 3 control groups with 10 mice each.
• One vaccination group was used for each vaccine dilution.
• the standard vaccine was diluted with saline to a concentration of 8, 4, 2, 1 or 0.5 I.UJml.
• the two licensed test vaccines were also diluted with saline to give 1 :10, 1 :20, 1 :40, 1 :80 and 1 :120 vaccine solutions.
• the mice were injected subcutaneously with 0.5 ml diluted vaccine using 16 mice for each vaccine solution.
• the mice included in the three control groups were injected in the same manner with 0.5 ml saline.
• mice Twenty-one days after vaccination, all immunized mice including those from control group number 1 , were inoculated intraperitoneally with 0.5 ml of E. rhusiopathiae (strain R9, serotype 1) bacteria suspension (challenge suspension) containing 1 LD/100. The challenge suspension was further diluted 10 and 100 folds. Thereafter, the suspensions were used for injecting the mice in control groups 2 and 3, respectively. All inoculated animals were observed daily for 8 days, and the number of survivors recorded.
• E. rhusiopathiae strain R9, serotype 1
• the challenge suspension was further diluted 10 and 100 folds. Thereafter, the suspensions were used for injecting the mice in control groups 2 and 3, respectively. All inoculated animals were observed daily for 8 days, and the number of survivors recorded.
• Example 2 Haemagglutination inhibition assay (antibody response) The antibody response to PPV- virus of the vaccinated pigs was assayed by haemagglutination inhibition (HI) tests. The Hi-test was carried out in V-bottomed microplates using a 0.5%> suspension of guinea-pig red-blood cells and 16 HAU/50 ⁇ l of PPV- virus (Rivera E, Sj ⁇ land L, Karlsson KA. A solid phase fluorescent immunoassay for the rapid detection of virus or antibodies in fetuses infected with porcine parvovirus. Arch Virol 1986; 88:19-26).
• the E rhusiopathiae (strain R9, serotype 1) antigen used for sensibilising ELISA- Nunc MaxiSorp microplates was prepared as previously described for a Pseudomonas-ELISA (Rivera E, Jackert-Jernberger M, Mejerland T, Karlsson KA. Evaluation of protein A and protein G as indicator system in a ELISA for detecting antibodies in mink to Pseudomonas aeruginosa. Vet Microb 1994;42:265-271). In short, 100 ⁇ l/well of bacterial antigen containing 2 ⁇ g protein per ml coating buffer was incubated at 37°C for 48 hours.
• test samples 100 ⁇ l diluted 1 : 100, were pipetted into each of two wells. The plates were incubated at 37°C for 30 min.. Thereafter, the plates were carefully washed. Antigen-antibody complexes were detected by means of a peroxidase-conjugated protein-G (Bio Rad Laboratories, Richmond, CA, USA) diluted 1 : 10,000. The plates containing the conjugated protein- G (100 ⁇ l/well) were incubated and washed again as described above.
• the plates were incubated at room temperature for 15 min. with the TMB-substrate (200 ⁇ l/well). The reaction was stopped with 50 ⁇ l/well of 2 M H 2 S0 4 . Next, the optical density of the reactions was measured at 450 nm. using a spectrophotometer.
• the E. rhusiopathiae (strain R9, serotype 1) used for preparing bacterial antigens for immunoblot analyses was first cultured on blood agar plates and then overnight at 37°C using a modified Feist medium, a serum- free medium shown to enhance the bacteria production of the 64-66 kD glycoprotein, the antigen involved in immunity to swine- erysipelas (Groschup MH, Timoney JF. Modified Feist broth as a serum- free alternative for enhanced production of protective antigen of Erysipelothrix rhusiopathiae. J Clin Microbiol. 1990; 28(11):2573-2575).
• the bacteria were harvested by centrifuging and thereafter, the cells were washed twice by centrifuging and using distilled water. After that, the bacterial-protein was extracted with EDTA as previously described (Groschup MH, Cussler K, Weiss R, Timoney JF. Characterisation of a protective antigen of Erysipelothrix rhusiopathiae. Epidemiol Infect 1991;107:637-649). Next, the protein extract was concentrated using a centrifugal-concentrator Filtron 50K (MakrosepTM, Pall Gelman Sciences, Lund, Sweden) diluted and heated 10 min.
• the membranes were first incubated in a 5% dry milk solution followed by a 2 h incubation with sera from vaccinated pigs diluted 1 : 100 in phosphate buffered saline containing 1% dry milk and 0.1% Tween 20 (serum diluent).
• the preparations were washed three times and incubated for 2 h with HRPO-conjugated rabbit anti-swine immunoglobulins (Dako A/B, Glostrup, Denmark) diluted 1 :3000 in serum diluent. After a new wash cycle, the reaction was developed using FAST-DAB with metal enhancer (Sigma, USA).
• the vaccinated pigs were observed daily for a period of eleven weeks. Reactions at the injection site or changes in the animals behaviour were recorded.
• Example 1 Mouse potency test (immune response) The results presented in Table 4 below demonstrate that vacc.l was more immunogenic than vacc.2. and induced a better protection. Eight days after infections, the number of survivors recorded for each of the groups immunized with vacc.l was higher than the number of survivors recorded for the groups of mice injected with the corresponding dilution of the vacc.2. Furthermore, it was found that vacc.l was even more potent than the standard vaccine, as shown in Table 4. The number of survivors in controls groups 1 , 2 and 3 was 0, 1 and 6, respectively (data not shown).
• Example 1 The mouse potency test. Per cent of survivors, eight days after infection with live Erysipelothrix rhusiopathiae serotype 1.
• the group mean antibody titres ⁇ SD at week
• the ELISA demonstrated that even in this case vacc.l was more potent than vacc.2 (Fig. 3).
• the ELISA results are in accordance with those obtained from the mouse potency test (Table 3) where vacc.1 protected a larger number of mice than did vacc.2.
• the serum from the pigs immunized with vacc.l- GINSENG or vacc.2-GINSENG recorded higher antibodies titres than the serum from the pigs injected with the ginseng-free vaccines (Fig. 3).
• FIG. 3 also illustrates that the antibody titres induced by the vaccines containing only Al(OH) 3 (vacc.1 and 2) decrease with time, while the titres recorded for the vaccines containing Al(OH) 3 and ginseng remain almost constant.
• the results from the Hi-test and ELISA demonstrated that ginseng and Al(OH) 3 acted synergistically, thereby improving the antibody response to both PPV and E. rhusiopathiae.
• the immunoblot assay revealed that the sera obtained from the animals immunized with the vaccines containing ginseng, react more strongly with the proteins in the 64-66 kD area shown to contain protective glykoproteins of E. rhusiopathiae (Groschup et al., 1991, supra), than do the sera from animals injected with the same vaccine without the addition of ginseng. In general, sera from the "ginseng-pigs" bind strongly with all E. rhusiopathiae proteins.
• the data obtained from the ELISA, immunoblot analyses and the mouse potency test further confirm the serological cross- reaction between E. rhusiopathiae serotype 1 and 2, since the vaccines included erysipelas antigen serotype 2 and the evaluation of the vaccines was carried out using serotype 1.
• the antibody titres in the serum samples were measured by means of a haemagglutination inhibition test.
• the data are presented (table 6) as the group (nr. 5) titre mean value recorded at the time for re- vaccination (booster) and two weeks post booster.
• Vaccine adjuvant Booster 2 weeks post booster a) Rbl 3 ⁇ 4.0 352 ⁇ 235 b) Rgl 8 ⁇ 7.4 304 ⁇ 192 c) Rbl + Rgl 6 ⁇ 8.0 256 ⁇ 78 d) Ginseng extract 10+8.9 832 ⁇ 384 e) Ginseng extract + ca. 10+3 6826 ⁇ 2413
• the ginsenoside content of the extract used in the other experiments was determined (table 7) by HPLC according to Journal of Chromatography, 1990, 504: 139-149. Further, another extract, produced according to the same procedures as for sample 1, was analysed according to the same HPLC-method.
• sample 1 shows that the extract contains several different ginsenosides, which add up to 36.44 % (sample 1) of the total content of the extract used in the experiments of this application.
• Sample 2 shows another extract, showing full adjuvant properties (not shown), but having a lower concentration of ginsenosides.
• rhusiopathiae- ⁇ L ⁇ SA used for evaluating the adjuvant effect of ginseng is a quantitative rather than qualitative assay, it appears from the immunoblotting analysis of the antibodies that ginseng also potentiates the antibody response to the E. rhusiopathiae-protec ⁇ ve antigen.
• the results according to the present invention may be extended to suggest that the immunogenicity of other vaccines adjuvanted with aluminium hydroxide can also be improved by using ginseng as a vaccine co-adjuvant.
• ginseng offers a good adjuvant alternative for viral vaccines, which, as in the case of live attenuated vaccines, can also vary in potency (e.g. distemper) from lot to lot. This difference in potency results most likely from part of the virus becoming inactivated during lyophilization. Freeze-dried vaccines containing inactivated or a mixture of live and inactivated virus antigen may still be immunogenic if the vaccines are resuspended in a solvent containing ginseng.
• potency e.g. distemper
• the results from Part II show: (a) That the synergistic effect of ginseng and aluminium hydroxide on the antibody response to PPV demonstrated in laboratory animals is even true for swine, (b) That in the pig, ginseng also enhance the antibody response to bacterial antigens e.g. E. Rhusiopathiae. (c) That ginseng provides a novel alternative for improving the immunogenic potency of alumimum hydroxide adjuvanted vaccines or vaccines in general, which is simple, safe and cheap.

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