Enhancement of immune responses to influenza vaccine (H3N2) by ginsenoside Re.
ABSTRACT This study was designed to evaluate the adjuvant effect of ginsenoside Re isolated from the root of Panax ginseng on the immune responses elicited by split inactivated H3N2 influenza virus antigen in a mouse model. Forty-eight ICR mice were randomly distributed into six groups with 8 mice in each group. All animals were subcutaneously (s.c.) immunized twice on weeks 0 and 3 with 50 microg Re, inactivated H3N2 influenza virus antigen equivalent to 10 or 100 ng of hemogglutinin (HA) or inactivated H3N2 influenza virus antigen equivalent to 10 ng HA adjuvanted with Re (25, 50 or 100 microg). Two weeks after the boost, blood samples were collected for measurement of serum IgG, the IgG isotypes and HI titers. Splenocytes were separated for the detection of lymphocyte proliferation and production of IFN-gamma and IL-5 in vitro. Results showed that co-administration of Re significantly enhanced serum specific IgG, IgG1, IgG2a and IgG2b responses, HI titers, lymphocyte proliferation responses as well as IFN-gamma and IL-5 secretions, indicating that both Th1 and Th2 were activated. Considering the adjuvant effect demonstrated in this study, Re deserve further studies for improving the quality of vaccines where mixed Th1/Th2 immune responses are needed.
Article: Lethal synergism between influenza virus and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor.[show abstract] [hide abstract]
ABSTRACT: A lethal synergism exists between influenza virus and pneumococcus, which likely accounts for excess mortality from secondary bacterial pneumonia during influenza epidemics. Characterization of a mouse model of synergy revealed that influenza infection preceding pneumococcal challenge primed for pneumonia and led to 100% mortality. This effect was specific for viral infection preceding bacterial infection, because reversal of the order of administration led to protection from influenza and improved survival. The hypothesis that influenza up-regulates the platelet-activating factor receptor (PAFr) and thereby potentiates pneumococcal adherence and invasion in the lung was examined in the model. Groups of mice receiving CV-6209, a competitive antagonist of PAFr, had survival rates similar to those of control mice, and lung and blood bacterial titers increased during PAFr inhibition. These data suggest that PAFr-independent pathways are operative in the model, prompting further study of receptor interactions during pneumonia and bacteremia. The model of lethal synergism will be a useful tool for exploring this and other mechanisms underlying viral-bacterial interactions.The Journal of Infectious Diseases 09/2002; 186(3):341-50. · 6.41 Impact Factor
Article: Influenza A virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in mice.[show abstract] [hide abstract]
ABSTRACT: The apparent worldwide resurgence of invasive Streptococcus pyogenes infection in the last two decades remains unexplained. At present, animal models in which toxic shock-like syndrome or necrotizing fasciitis is induced after S. pyogenes infection are not well developed. We demonstrate here that infection with a nonlethal dose of influenza A virus 2 days before intranasal infection with a nonlethal dose of S. pyogenes strains led to a death rate of more than 90% in mice, 10% of which showed necrotizing fasciitis. Infection of lung alveolar epithelial cells by the influenza A virus resulted in viral hemagglutinin expression on the cell surface and promoted internalization of S. pyogenes. However, treatment with monoclonal antibodies to hemagglutinin markedly decreased this internalization. Our results indicate that prior infection with influenza A virus induces a lethal synergism, resulting in the induction of invasive S. pyogenes infection in mice.Journal of Virology 05/2003; 77(7):4104-12. · 5.40 Impact Factor
Article: Pathogenicity of influenza virus.Microbiological reviews 07/1980; 44(2):303-30.
Enhancement of immune responses to influenza vaccine (H3N2) by ginsenoside Re
Xiaoming Songa,c, Jian Chena, Kedsirin Sakwiwatkula, Ruili Lia, Songhua Hua,b,⁎
aDepartment of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
bKey Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, P. R. China, Hangzhou, Zhejiang 310029, China
cZhejiang Center of Laboratory Animals, Zhejiang Academy of Medical Sciences, Hangzhou 310013, China
a b s t r a c ta r t i c l e i n f o
Received 20 July 2009
Received in revised form 11 December 2009
Accepted 15 December 2009
This study was designed to evaluate the adjuvant effect of ginsenoside Re isolated from the root of Panax
ginseng on the immune responses elicited by split inactivated H3N2 influenza virus antigen in a mouse
model. Forty-eight ICR mice were randomly distributed into six groups with 8 mice in each group. All
animals were subcutaneously (s.c.) immunized twice on weeks 0 and 3 with 50 µg Re, inactivated H3N2
influenza virus antigen equivalent to 10 or 100 ng of hemogglutinin (HA) or inactivated H3N2 influenza
virus antigen equivalent to 10 ng HA adjuvanted with Re (25, 50 or 100 µg). Two weeks after the boost,
blood samples were collected for measurement of serum IgG, the IgG isotypes and HI titers. Splenocytes were
separated for the detection of lymphocyte proliferation and production of IFN-γ and IL-5 in vitro. Results
showed that co-administration of Re significantly enhanced serum specific IgG, IgG1, IgG2a and IgG2b
responses, HI titers, lymphocyte proliferation responses as well as IFN-γ and IL-5 secretions, indicating that
both Th1 and Th2 were activated. Considering the adjuvant effect demonstrated in this study, Re deserve
further studies for improving the quality of vaccines where mixed Th1/Th2 immune responses are needed.
Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
Influenza viruses belong to the Orthomyxoviridae family and are
the major cause of respiratory disease in humans. Previously,
outbreaks in humans due to influenza types A and B were responsible
for substantial mortality and morbidity: 200,000 hospitalizations and
36,000 deaths annually in the U.S. (www.cdc.gov), particularlyin high
risk groups, such as the elderly, infants, and those with chronic
underlying medical conditions . Influenza infections in the elderly
often lead to secondary bacterial infection that results in severe
symptoms and occasionally death [2–4]. Furthermore, the highly
pathogenic avian influenza virus H5N1 strain has caused outbreaks of
disease in domestic poultry in Asian countries, and the unprecedented
spread of the H5N1 influenza virus has been associated with several
human infections and deaths in Vietnam, Thailand, Indonesia, and
Influenza vaccines have effectively been used for prevention of
influenza infections. Nichol et al. [11,12] have reported that influenza
vaccines have decreased the number of cases requiring hospitalization
due to any type of respiratory condition. In a study using experimental
animals, influenza vaccine has effectively protected host animals from
severe influenza–bacteria superinfectious diseases . In spite of the
high efficacy of inactivated vaccines against influenza, the efficacy of
inactivated influenza vaccines used widely is reduced in elderly people
and infants [14–16], and these vaccines protect poorly against drift
variants of influenza virus [17,18]. At the same time, the use of some
immunogenicity and protective efficiency, but also increases their
ability to protect from drift variants of influenza virus. The potential
threat of an influenza pandemic has led to studies on the development
of new effective and safe adjuvants. An optimal adjuvant should not
only increase immunogenicity and effectiveness of a vaccine, but also
should be cheap to produce, non-toxic, biodegradable, biocompatible,
immunologically nert, stable, and with longshelf life. It should promote
not only humoral immunity, but also induce cellular immune response
without inducing IgE antibodies (i.e., be non-allergenic) [19,20].
Panax ginseng C. A. Meyer as a traditional medicine has been
utilized in China for at least 2000 years . The drug has been
believed to stimulate the natural resistance against infections .
Ginseng saponins (GS), or ginsenosides, are believed to be the
pharmacologically active substances in total ginseng extracts. Recent
studies on ginseng saponins have demonstrated that GS has adjuvant
activity capable of boosting both cellular (Th1) as well as humoral
triterpenoid glycosides of the dammarane series. At present, more
than 30 ginsenosides have been identified in P. ginseng . The
adjuvant activities of ginsenosides with different molecular structures
are dependent mainly on the sugar side chains attached to their
dammarane skeleton. More recent investigation has found that the
adjuvant activity of ginsenosides Rg1, Re, Rg2, Rg3 and Rb1 from the
root of Panax ginseng is more potent than that of Rd, Rc and Rb2 .
Chemical analysis has shown that Re is found not only in the root but
International Immunopharmacology 10 (2010) 351–356
⁎ Corresponding author. 268 Kaixuan Rd, Hangzhou, Zhejiang 310029, China. Tel.:
+86 0571 8697 1852; fax: +86 0571 8697 1690.
E-mail address: email@example.com (S. Hu).
1567-5769/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/intimp
also in the stem and leaf of P. ginseng . This discovery has greatly
decreased the cost of Re production. Present study was designed to
evaluate the adjuvant properties of ginsenoside Re by measuring
serum specific antibody responses, hemagglutination inhibition titers
(HI), lymphocyte proliferation as well as cytokine production by
splenocytes in mice immunized with inactivated H3N2 influenza
vaccines in combination with Re.
2. Materials and methods
Female ICR mice were purchased from Shanghai Laboratory
Animal Center (SLAC) Co. Ltd. (Shanghai, China), and housed in
polypropylene cages with sawdust bedding in hygienically controlled
environment. Feed and water were supplied ad libitum. All procedures
related to the animals and their care conformed to the internationally
accepted principles as found in the Guidelines for Keeping Experi-
mental Animals issued by the government of China.
2.2. Antigen and adjuvant
Split inactivated influenza virus A/Fujian/411/2002 (H3N2) strain
was kindly supplied by ZhejiangProvincial Center for Diseases Control
and Prevention, which contained 326 μg/ml of hemagglutinin (HA).
Ginsenoside Re extracted from the root of Panax ginseng C.A. Meyer
was white powder with purity of 98% and molecular weight at 947. Re
was first dissolved in dimethyl sulfoxide (DMSO), then diluted with
physiological saline solution (1000 μg/ml) and sterilized by passing
through a 0.22 µm filter. The endotoxin level in above solutions was
less than 0.5 endotoxin unit (EU)/ml by a gel-clot Limulus amebocyte
lysate assay (Bath no., Zhanjiang A&C Biological Ltd., Zhanjiang,
8 mice each. Each of the animals was subcutaneously (s.c.) immunized
twice at 3 week intervals with 200 µl of physiological saline solution
containing (1) 50 µg Re; (2) 10 ng HA; (3) 100 ng HA; (4) 10 ng HA+
25 µgRe;(5)10 ngHA+50 µgor(6)10 ngHA+100 µgRe.Twoweeks
HI titers using chicken red blood cells, and HA-specific IgG titers as well
as IgG isotype levels. Splenocytes were prepared for determination of
cellular proliferation and production of IFN-γ and IL-5.
2.4. Measurement of specific IgG and the IgG isotypes
Serum samples were analyzed for measurement of HA-specific IgG
titer and IgG isotype responses by indirect enzyme-linked immuno-
sorbentassay. Allthe wells of polyvinyl96-wellmicrotitreplates were
coatedwith100 μlof1 μg/mlHAdilutedin0.05 Mcarbonatebuffer,pH
9.6 and incubated overnight at 4 °C. After five washes with phosphate
buffer saline (PBS, pH 7.2) 0.05% Tween-20 (PBST), the wells were
blocked with PBS 5% skimmed milk and incubated at 37 °C for 2 h.
well and incubated at 37 °C for 45 min. Plates were then washed five
times in PBST.For IgG titer detection, 100 μl of goatanti-mouseIgG (1/
500) (Kirkegaard, Perry Lab., Maryland, USA), was added to all wells
and incubated at 37 °C for 45 min. Plates were washed again with
PBST. A hundred microliters of 3,3′,5,5-tetramethylbenzidine solution
(100 μg/ml of 0.1 M citrate–phosphate, pH 5.0) solution (100 µg/ml of
0.1 M citrate–phosphate, pH 5.0) was added to each well and
incubated for 15 min at room temperature. The reaction was stopped
by adding 50 µl of 2 M H2SO4to each well. The optical density of the
plate was read by an automatic ELISA plate reader at 450 nm. Values
above the cut-off background level (mean value of sera from saline-
immunized mice multiplied by a factor of 2.1) were considered
positive. Titers were depicted as reciprocal end-dilutions. For
subclasses, 100 μl of biotin conjugated goat anti-mouse IgG1 or
IgG2a or IgG2b or IgG3 (1:600 dilution, Santa Cruz Biotechnology
Inc., California, USA) was added to corresponding plate and then
incubated for 45 min at 37 °C. After washing, 100 μl of horseradish
peroxidase conjugated anti-biotin (BD Biosciences, Pharmingen, USA)
diluted 1:4000 in PBST was added to each well and incubated for
30 min at 37 °C. Incubations, washing and development were as
described above for detection of HA-specific total IgG. The optical
density of the plate was read at 450 nm.
2.5. Hemagglutination inhibition (HI) assay
Serum HI titers were determined according to the protocol
adapted from the Center for Disease Control laboratory-based
influenza surveillance manual . Serum samples were serially
diluted 2-fold into V-bottom 96-well microtiter plates. An equal
volume of virus, adjusted to 4 HA units of antigen was added to each
well. The plates were covered and incubated at room temperature for
30 min followed by the addition of freshly prepared 1% chicken
erythrocytes (RBCs) in PBS. The plates were mixed by agitation,
covered, and allowed to set for 60 min at 25 °C. The HI titer was
determined by thereciprocal ofthe lastdilutionwhich contained non-
agglutinated RBCs. Positive and negative serum controls were
included on each plate. Mean HI titers and standard deviation were
calculated for each group.
2.6. Lymphocyte proliferation assay
Spleen collected from the HA-immunized ICR mice under aseptic
conditions, in Hank's balanced salt solution (HBSS, Sigma), was
cell suspension. After centrifugation (380×g at 4 °C for 10 min), the
Fig. 1. Serum IgG titers elicited by inactivated H3N2 influenza virus antigen. Mice
(n=8) received twice subcutaneous injection at weeks 0 and 3 of 50 µg Re, inactivated
H3N2 influenza virus antigen (equivalent to 10 or 100 ng HA) or inactivated H3N2
influenza virus antigen (equivalent to 10 ng HA) adjuvanted with Re (25, 50 or 100 µg).
The mice were bled 2 weeks after the second immunization for analysis of IgG titers by
indirect ELISA. Values above the cut-off background level, mean value of sera from
saline-immunized mice (negative controls) multiplied by a factor of 2.1, were
considered positive. Values represent mean±S.D. Titers were depicted as reciprocal
end-dilutions. Significant differences with 10 ng HA groups were designated as
*P<0.05 and **P<0.01.
X. Song et al. / International Immunopharmacology 10 (2010) 351–356
pelleted cells were washed three times in PBS and resuspended in
complete medium (RPMI 1640 supplemented with 0.05 mM 2-
mercaptoethanol, 100 UI/ml penicillin, 100 μg/ml streptomycin and
10% heat inactivated FCS). Cell numbers were counted with a
exceeded 95%. Splenocyte proliferation was assayed as described
previously  with some modification. Briefly, splenocytes were
seeded into a 96-well flat-bottom microtiter plate (Nunc) at 5.0×106
cell/ml in 100 μl complete medium, thereafter concanavalin A (Con A,
final concentration 5 μg/ml), LPS (final concentration 7.5 μg/ml) or
medium were added giving a final volume of 200 μl. The plates were
incubated at 37 °C in a humid atmosphere with 5% CO2for 2 days. All
the tests were carried out in triplicate. The cell proliferation was
evaluatedusingMTTmethods.Briefly,50 μlofMTTsolution(2 mg/ml)
were added to each well 4 h before the end of incubation. The plates
were centrifuged (1400×g, 5 min) and the untransformed MTT was
solution (192 μl DMSO with 8 μl 1 N HCl) was added, and the
absorbance was evaluated in an ELISA reader at 570 nm with a
630 nm reference after 15 min. The stimulation index (SI) was
calculated based on the following formula: SI=the absorbance value
for mitogen cultures divided by the absorbance value for non-
2.7. Measurement of IFN-γ and IL-5 produced by splenocytes
Single cell suspensions were adjusted to a concentration of
2.5×106cells/ml in complete medium. To a 96-well flat-bottom
microtiter plate (Nunc), 100 μl of the cell suspension and equal
volume of Con A solution (final concentration 5 μg/ml) were added.
The plates were incubated at 37 ºC in a 5% CO2atmosphere for 48 h.
After that, the culture supernatants were collected for cytokine assay.
The concentrations of IFN-γ and IL-5 were determined by a
commercial capture ELISA kit (R & D Systems Inc., Minneapolis,
USA). Concentrations of cytokines were calculated from interpolation
of the cytokine standard curve.
2.8. Statistical analysis
Data are expressed as mean±S.D. Boniferroni method was used to
compare the parameters between groups . P-values of less than
0.05 were considered statistically significant.
3.1. Serum specific IgG and IgG isotypes
Serum specific IgG and the IgG subclasses were measured by an
indirect ELISA to evaluate the adjuvant effect of ginsenoside Re on the
humoral immune responses. Fig. 1 shows that 10 ng of HA induced
significantly lower HA-specific IgG titers (1:160) than 100 ng of HA
(1:1659) (P<0.05). However, IgG titer induced by co-administration
of HA (10 ng) with Re (50 μg) was 19 times higher (1:3044) than that
induced by the same dose of HA (10 ng) administered alone
(P<0.01) and even numerically higher than the IgG titer elicited by
100 ng of HA (P>0.05). While IgG titers were higher in mice
immunized with 10 ng of HA plus Re at a dose range from 25 to
100 μg than in the control, highest IgG titer was found in the group
adjuvanted with 50 μg of Re. As no OD values of the sera from Re-
injected mice were recorded above 2.1×mean value of the sera from
saline-injected mice (negative controls), IgG titer was actually
undetectable in Re-injected group.
Fig. 2 indicates that 100 ng of HA induced higher HA-specific IgG1
(P<0.05), IgG2a, IgG2b (P<0.05) and IgG3b responses than 10 ng of
HA. Supplement of Re (25, 50, 100 μg) in 10 ng of HA enhanced the
isotypes IgG1 (P<0.01), IgG2a (P<0.05), IgG2b (P<0.05) and IgG3b
(P<0.01) responses with significantly higher isotypes found in mice
immunized with 10 ng HA plus 50 μg Re.
3.2. HI titers
To investigate the effect of Re on serum HI titers, mice were
immunized twice s.c. and serum HI responses were determined. Fig. 3
indicates that 100 ng of HA induced numerically higher HA-specific HI
titer (1:624) than 10 ng of HA (1:156) (P>0.05). However, co-
administration of HA (10 ng) with Re (50 μg) induced 10.8 times
higher HI titer (1:1680) than the same dose of HA was administered
3.3. Lymphocyte proliferation
The effect of ginsenoside Re on splenocyte proliferative responses
to Con A and LPS stimulation, and the results are shown in Fig. 4.
Higher splenocyte responses were found in mice immunized with
100 ng HA to Con A (P<0.05) and LPS when compared to mice
immunized with 10 ng HA only. Supplement of Re (50 μg) in 10 ng HA
significantly enhanced splenocyte proliferative responses to both Con
A and LPS when compared to mice immunized with those with 10 ng
HA alone (P<0.01).
3.4. Production of IFN-γ and IL-5
After in vitro stimulation of splenocytes with Con A for 48 h,
splenocytes from the mice immunized with 100 ng HA secreted
significantly higher IL-5 (P<0.05) but numerically higher IFN-γ
(P>0.05) than the control (10 ng HA)(Fig. 5). However, both IFN-γ
(P<0.01) and IL-5 (P<0.05) were significantly higher in the cultures
of the splenocytes from the mice immunized with 10 ng HA plus Re
(50 μg) than those with 10 ng HA alone.
Adjuvant properties of ginsenoside Re have been demonstrated
for inactivated H3N2 influenza vaccines in a mouse model. Co-
administration of ginsenoside Re with inactivated influenza virus A/
Fujian/411/2002 (H3N2) induced significantly higher serum specific
IgG and the isotype responses, HI titers, splenocyte proliferation in
response to Con A and LPS as well as production of IL-5 and IFN-γ by
splenocytes than in mice administered the antigen alone.
The mouse model has been used to study the immunity of a host
against influenza infection. For examples, Prabakaran et al.  have
reported that intranasal vaccination of recombinant baculovirus
surface-displayed hemagglutinin (BacHA) or inactivated whole
H5N1 viral vaccine with a recombinant cholera toxin B subunit
(rCTB) as a mucosal adjuvant can provide 100% protection against
10MLD50of homologous and heterologous H5N1 strains. Cox et al.
 have observed that the mice immunized with a split virus vaccine
can effectively limit viral replication and this correlates high influenza
specific serum IgG concentrations. The IgG response elicited by
antigen is dose-dependent. In this study, the mice immunized with
100 ng HA antigen had significantly higher IgG and the IgG isotypes
than the mice immunized with 10 ng HA antigen. However,
supplement of ginsenoside Re (50 µg) in 10 ng HA antigen signifi-
cantly amplified IgG and the IgG isotype responses as indicated in
Figs. 1 and 2. Ginsenoside Re is one of the ginseng saponins identified
in P. ginseng. Enhanced immune responses by ginseng saponins have
also been found previously in other studies. For examples, Rivera et al.
 have found an increased specific HI titers in guinea pigs
immunized with co-administration of porcine parvovirus antigen
with ginseng saponin; Hu et al.  have reported an enhanced
specific IgG responses in both milk and peripheral blood of cattle
vaccinated with Staphylococcus aureus bacterin; Song et al.  have
X. Song et al. / International Immunopharmacology 10 (2010) 351–356
recently observed an enhanced IgG response in mice injected with
inactivated foot-and-mouth disease virus antigen in combination
with saponins isolated from ginseng stem and leaf.
Immunity to different infectious agents requires distinct types of
immune responses. Defense against intracellular pathogens tends to
involve Th1 type immune responses dominated by the production of
IFN-γ, IgG2a antibodies, delayed type hypersensitivity (DTH) and
cytotoxic T lymphocytes (CTL), while resistance to extracellular
pathogens is often associated with humoral responses dominated by
high levels of IgG1 and production of IL-4 and IL-5 . One of the
major challenges in vaccinology is the development of vaccine
formulations that will induce immune responses appropriate for the
particular pathogen since the wrong response could lead to increased
pathology and possibly enhanced spread of the pathogens. Thus,
adjuvants can be a valuable tool for tailoring the desired immune
responses. Polarized Th1 type immunity can be achieved by addition
of complete Freunds adjuvant (CFA) and CpG DNA to an antigen
[35,36]. In contrast, Th2 antibody responses can be enhanced by alum
or incomplete Freunds adjuvant (IFA), as indicated by more IgG1
relative to IgG2a [36,37]. Some adjuvants or their combinations can
promote mixed Th1/Th2 responses. For instance, purified Quillaja
saponin (QS 21), a combination of CFA+IFA induce IFN-γ (Th1 type)
Fig. 3. Serum hemagglutionation inhibition titers after mice (n=8/group) received
twice subcutaneous injection at weeks 0 and 3 of 50 µg Re, inactivated H3N2 influenza
virus antigen (equivalent to 10 and 100 ng HA) or inactivated H3N2 influenza virus
antigen (equivalent to 10 ng HA) adjuvanted with Re (25, 50 and 100 µg). The mice
were bled 2 weeks after the second immunization for analysis of hemagglutination
inhibition test. Values represent mean±S.D. Significant differences with 10 ng HA
groups were designated as *P<0.05 and **P<0.01.
Fig. 4. Mitogen-stimulated proliferation of splenocytes isolated from mice (n=8)
receiving twice subcutaneous injection at weeks 0 and 3 of 50 µg Re, inactivated H3N2
influenza virus antigen (equivalent to 10 and 100 ng HA) or inactivated H3N2 influenza
virus antigen (equivalent to 10 ng HA) adjuvanted with Re (25, 50 and 100 µg).
Splenocytes were prepared 2 weeks after the last immunization and cultured with Con A,
LPS or RPMI 1640. Splenocyte proliferation was measured by the MTT method as
described in the text, and shown as a stimulation index. The values were represented
mean±S.D. Significant differences with 10 ng HA groups were designated as *P<0.05
Fig. 2. Serum anti-HA IgG1, IgG2a, IgG2b and IgG3 levels after mice (n=8/group) received twice subcutaneous injection at weeks 0 and 3 of 50 µg Re, inactivated H3N2 influenza
virus antigen (equivalent to 10 and 100 ng HA) or inactivated H3N2 influenza virus antigen (equivalent to 10 ng HA) adjuvanted with Re (25, 50 and 100 µg). The mice were bled
2 weeks after the second immunization for IgG isotype analysis by indirect ELISA. Values represent mean±S.D. Significant differences with 10 ng HA groups were designated as
*P<0.05 and **P<0.01.
X. Song et al. / International Immunopharmacology 10 (2010) 351–356
and IL-4 (Th2 type) responses , while a liposomal formulation of
leishmania antigens or ginseng stem and leaf saponin (GSLS)/oil
emulsion results in mixed Th1/Th2 (both serum IgG1 and IgG2a)
responses . There are two types of influenza vaccines currently in
use: inactivated whole virus vaccines and disrupted viral antigens
containing viral surface glycoproteins . As the whole virus vaccine
causes significantly higher adverse reactions than does the hemag-
glutinin split vaccine , only the HA split vaccine is used in China.
The split vaccine exhibits 70–90% efficacy in reducing the incidence of
clinical illness but fails to prevent influenza virus infection. Subcuta-
neous vaccination with the HA vaccine can induce the production of
virus-neutralizing antibody in the serum but not a cell-mediated
immune response, including CTL activity [40,41]. According to recent
observation by Bungener et al. , Th1 immune response plays an
important role in the immunity against influenza virus infection. Our
present study has demonstrated that addition of Re in influenza virus
HA antigen not only can enhance IgG1 but also IgG2a responses. Such
enhanced IgG1/IgG2 responses may be attributed the increased
production of IL-5 and IFN-γ as indicated in Fig. 5, suggesting that
purpose of a vaccination is to activate both Th1 and Th2 immune
response, the supplement of Re in vaccine is indicated.
Results of lymphocyte proliferation assay depend on the mitogen
used. Both Con A- and LPS-induced proliferative responses were
enhanced in the mice receiving co-administration of influenza virus
antigen and Re. Enhanced lymphocyte response to LPS indicates that B
lymphocytes wereactivated,indicatingthatboth T andB lymphocytes
were stimulated .
In conclusion, co-administration of ginsenoside Re with inacti-
vated H3N2 influenza virus antigen in mice significantly amplified
serum specific IgG and the IgG isotype responses, HI titers,
lymphocyte proliferation as well as IL-5 and IFN-γ secretions,
suggesting that both Th1 and Th2 immune responses were activated.
Considering the adjuvant effect of Re demonstrated in this study, and
a GS preparation containing Re has been licensed for injection in
humans, Re deserve further studies for improving the quality of
vaccines where mixed Th1/Th2 immune responses are needed.
This study was supported by the National Scientific Foundation of
China (30771592) and the Ministry of Science and Technology of
 Wright PF, Neuman G, Kawaoka Y. Orthomyxoviruses. In: Knipe DM, Howley PM,
Griffin DE, Martin MA, Ramb RA, Roizman B, et al, editors. Fields virology 5th ed.
Philadelphia Lippincott-Raven; 2007. p. 1691–740.
 McCullers JA, Rehg JE. Lethal synergism between influenza virus and Streptococcus
pneumoniae: characterization of a mouse model and the role of platelet-activating
factor receptor. J Infect Dis 2002;186(3):341–50.
 Okamoto S, Kawabata S, Nakagawa I, Okuno Y, Goto T, Sano K, et al. Influenza A
virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in
mice. J Virol 2003;77(7):4104–12.
 Sweet C, Smith H. Pathogenicity of influenza virus. Microbiol Rev 1980;44
 Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF, Senne DA, et al.
Human influenza A H5N1 virus related to a highly pathogenic avian influenza
virus. Lancet 1998;351(9101):472–7.
 de Jong MD, Bach VC, Phan TQ, Vo MH, Tran TT, Nguyen BH, et al. Fatal avian
influenza A (H5N1) in a child presenting with diarrhea followed by coma. N Engl J
 Maines TR, Lu XH, Erb SM, Edwards L, Guarner J, Greer PW, et al. Avian influenza
(H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence
in mammals. J Virol 2005;79(18):11788–800.
 Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, et al. Characterization of an
avian influenza A (H5N1) virus isolated from a child with a fatalrespiratory illness.
 Tran TH, Nguyen TL, Nguyen TD, Luong TS, Pham PM. Nguyen vVC, et al. Avian
influenza A (H5N1) in 10 patients in Vietnam. N Engl J Med 2004;350
Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med
 Nichol KL. The efficacy, effectiveness and cost-effectiveness of inactivated
influenza virus vaccines. Vaccine 2003;21(16):1769–75.
 Nichol KL, Wuorenma J, von Sternberg T. Benefits of influenza vaccination for low-,
intermediate-, and high-risk senior citizens. Arch Intern Med 1998;158
 Okamoto S, Kawabata S, Fujitaka H, Uehira T, Okuno Y, Hamada S. Vaccination
with formalin-inactivated influenza vaccine protects mice against lethal influenza
Streptococcus pyogenes superinfection. Vaccine 2004;22(21–22):2887–93.
 Vu T, Farish S, Jenkins M, Kelly H. A meta-analysis of effectiveness of influenza
vaccine in persons aged 65 years and over living in the community. Vaccine
 Maeda T, Shintani Y, Nakano K, Terashima K, Yamada Y. Failure of inactivated
influenza A vaccine to protect healthy children aged 6–24 months. Pediatr Int
 Diseases AAoPCoI. Recommendations for influenza immunization of children.
 Belshe RB, Gruber WC, Mendelman PM, Cho I, Reisinger K, Block SL, et al. Efficacy
of vaccination with live attenuated, cold-adapted, trivalent, intranasal influenza
(n=8) received twice subcutaneous injection at weeks 0 and 3 of 50 µg Re, inactivated
H3N2 influenza virus antigen (equivalent to 10 ng HA) adjuvanted with or without Re
(50 µg).Splenocyteswereprepared2 weeksafterthelastimmunizationandculturedwith
ConA(5 μg/ml) for48 h.The supernatantswereharvestedfordeterminationofIFN-γ and
IL-5 by a capture ELISA. The values are presented as mean±S.D. Significant differences
with 10 ng HA group were designated as *P<0.05 and **P<0.01.
X. Song et al. / International Immunopharmacology 10 (2010) 351–356