Sie sind auf Seite 1von 9

Vaccine 29 (2011) 32843292

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

A synthetic TLR4 agonist formulated in an emulsion enhances humoral and Type


1 cellular immune responses against GMZ2 A GLURPMSP3 fusion protein
malaria vaccine candidate
Susana Lousada-Dietrich a,b,c , Prajakta S. Jogdand a,b,c , Sren Jepsen a , Vera V. Pinto b,c ,
Sisse B. Ditlev b,c , Michael Christiansen a , Severin Olesen Larsen a , Christopher B. Fox d ,
Vanitha S. Raman d , Randall F. Howard d , Thomas S. Vedvick d , Gregory Ireton d , Darrick Carter d ,
Steven G. Reed d , Michael Theisen a,b,c,
a

Department of Clinical Biochemistry and Immunology, State Serum Institute, Artillerivej 5, 2300 Copenhagen S, Denmark
Centre for Medical Parasitology at Department of International Health, Immunology and Microbiology, University of Copenhagen, Denmark
Department of Infectious Diseases, Copenhagen University Hospital, CSS, Oester Farimagsgade 5, PO Box 2099, 1014 Copenhagen K, Denmark
d
Infectious Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle, WA 98104, USA
b
c

a r t i c l e

i n f o

Article history:
Received 1 September 2010
Received in revised form 14 January 2011
Accepted 7 February 2011
Available online 22 February 2011
Keywords:
Malaria vaccine candidate
Toll-like receptor agonist
Long-lived plasma cell

a b s t r a c t
GMZ2 adjuvanted by aluminum hydroxide is a candidate malaria vaccine that has successfully passed
phase 1 clinical testing in adult German and Gabonese volunteers and Gabonese children under ve. Here
we report a preclinical study screening a series of adjuvant vehicles and Toll-like receptor (TLR) agonists
in CB6F1 mice to identify an improved formulation of GMZ2 suitable for further human clinical studies.
GMZ2 formulated in an oil-in-water emulsion plus the synthetic TLR4 agonist GLA elicits the highest (a)
vaccine-specic IgG2a and total IgG titers, (b) parasite-specic IFA titers, (c) levels of Type 1 cytokine
responses (IFN-), and (d) number of long-lived-plasma cells (LLPC) secreting antibodies against both
the GMZ2 fusion and its two components. Thus, GLA helps to elicit a vaccine-specic Type 1 antibody
prole together with high levels of LLPC, both of which are thought to be essential for the development
of long-term protective immunity against clinical malaria.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Immunization is one of the most effective tools for controlling infectious diseases, preventing millions of deaths annually [1].
Since Plasmodium falciparum malaria is a leading cause of death
and morbidity in children below ve years of age, major efforts
have been invested in developing a vaccine. One of the leading
candidates for an anti-falciparum vaccine is GMZ2, a fusion protein consisting of the N-terminal portion of the Glutamate Rich
Protein (GLURP) genetically fused to a C-terminal fragment of
Merozoite Surface Protein 3 (MSP3) [2]. GLURP27500 represents
the conserved non-repeat part of GLURP [3] and is a major epitope for B cells [4], and MSP3212380 represents the conserved
portion of the otherwise highly polymorphic MSP3 [5]. There are
good reasons for combining these two antigens. First, immunoepidemiological studies have demonstrated that antibodies of the

Corresponding author at: Department of Clinical Biochemistry and Immunology,


State Serum Institute, Artillerivej 5, 2300 Copenhagen S, Denmark.
Tel.: +45 20888302.
E-mail address: mth@ssi.dk (M. Theisen).
0264-410X/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2011.02.022

cytophilic subclasses (Immunoglobulin G1 [IgG1] and IgG3), against


the individual GLURP and MSP3 domains are signicantly elevated
in children who are protected from clinical malaria [617]. Second,
both antigens elicit in vitro growth-inhibitory antibodies during the
natural course of infection [4,1820] as well through experimental
immunizations [2,21,22]. Of the cytophilic IgG antibodies, several
studies have demonstrated that IgG3 is particularly important in
relation to both protection from clinical malaria [14,15] and in vitro
growth inhibition [23]. These IgG antibodies do not inhibit parasite
growth in vitro on their own, but act synergistically with human
monocytes through binding to Fc receptors [4,19]. The critical
involvement of Fc receptors in the Antibody-dependent Cellular Inhibition (ADCI) may explain why it is predominantly levels
of cytophilic (IgG1 and IgG3) antibodies that are associated with
protection against clinical malaria. The aim of immunization with
GMZ2 is to produce high levels of long-lasting cytophilic IgG antibodies, particularly those of the IgG3 subclass, against both the
GLURP and the MSP3 domains.
The current formulation of GMZ2 with Al(OH)3 [24] has a
satisfactory safety prole and is immunogenic. However, this formulation elicits a predominantly IgG1 antibody response against
GLURP and to a lesser degree against MSP3. Thus, it is highly desir-

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

able to develop a new formulation that enhances a Type 1 antibody


prole consisting of both IgG1 and IgG3 antibodies directed against
both the GLURP and MSP3 domains. New water-in-oil and oil-inwater emulsions are being developed and especially the latter are
highly stable and have proven to be potent adjuvants with few toxic
side effects [2527]. The importance of activating innate immunity to obtain strong adaptive immune responses and long-term
memory has also been the focus of vaccine research in recent years.
What were previously described as danger signals are today recognized as dened molecules with distinct chemical and genetic
properties. The activation of innate immunity by danger signals
is explained by a series of reactions strictly controlled by specic
receptors, so-called pattern-recognition receptors (PRRs), that bind
microbial membrane products or nucleic acids [28]. One family
of such receptors consists of the Toll-like receptors (TLR) composed of unique and distinct gene products of the TLR family that
bind such diverse microbial products as LPS, agellin, HSP60, CpG
DNA, dsRNA or peptidoglycans. Located on the plasma or vesicular membranes of antigen-presenting cells (APC), TLR provide
the molecular triggers by which APC present antigen and initiate
an adaptive immune response. Several studies have shown that
TLR ligands can be used to modulate vaccine-induced immune
responses [27,2932]. The success of a vaccine depends on its ability
to confer long-term immunity and, in the case of humoral immunity, both memory B cells and long-lived plasma cells (LLPCs) are
important. Both cell types originate from the germinal centres in
the lymphoid organs but, whereas memory B cells go into circulation, the LLPCs migrate to the bone marrow where, as terminally
differentiated cells, they constitutively secrete antibodies and provide a rst line of defence against re-infection [3335]. With the
aim of developing a much needed, highly efcient malaria vaccine
for children in endemic areas, we have investigated the effects of
the TLR 9 agonist CpG, the TLR 4 agonist GLA [27] and the TLR7/8
agonist R848 in CB6F1 mice on (i) the production of antigen-specic
IgG in terms of concentration, sub-class prole and reactivity with
recombinant and native parasite proteins, (ii) the Th1/Th2 cytokine
prole, and (iii) the induction of LLPCs. Two adjuvant vehicles
were used, namely Al(OH)3 and an oil-in-water stable emulsion
(SE). This study demonstrates that the SE-based formulations
induce stronger immune responses than Al(OH)3 -based formulations and that GLA enhances a Type 1 cellular immune response
with high levels of mouse IFN- and cytophilic IgG2a antibodies.
Importantly, the most immunogenic formulations elicit antibodies against both GLURP and MSP3 as determined by ELISA with
recombinant proteins and immunoblotting of solubilised parasite
proteins.
2. Materials and methods
2.1. Animals
Inbred female CB6F1 (BALB/C C57BL/6) mice were obtained
from Harlan (Harlan-Northern Europe, The Netherlands). All mice
were maintained under specic pathogen-free conditions in the
Biotech Research and Innovation Centre (University of Copenhagen) animal care facility and handled by authorised personnel.
All manipulations were conducted according to the Danish Ministry
of Justice regulations.
2.2. Antigen and adjuvants
Recombinant GMZ2, based on GLURP and MSP3 sequences
from the F32 parasite line, was expressed in Lactococcus lactis
and puried from culture supernatants following good manufacturing practice (GMP) at Henogen S.A. (Belgium). Recombinant

3285

GLURP25500 and MSP3212380 was produced in Escherichia coli [2].


Aluminum hydroxide (Al(OH)3 ) was from Statens Serum Institute
(Denmark). Mouse CpG (CpG 1826), R848 (resiquimod), a micellar
form of GLA (AQ001), a stable oil-in-water emulsion (SE; EM030),
and SE co-formulated with GLA (SE/GLA; EM031) were from IDRI
(Seattle, USA). GLA is a synthetic molecule with a structure reminiscent of the hexaacyl lipid found in monophosphoryl lipid A (MPL )
[27].
2.3. Immunization
Immunizations were performed by the s.c. route at the base of
the tail three times, three weeks apart with 5 g of GMZ2 adjuvanted with different formulations in a total volume of 100 l. For
the Al(OH)3 -based screening experiment, groups of 3 mice were
immunized with GMZ2 adsorbed to Al(OH)3 and supplemented
with either 25 g of CpG 1836, 20 g of R848, or 20 g of GLA
(AQ001). The TLR agonists were allowed to adsorb to Al(OH)3 for
30 min before GMZ2 was added to the mixtures followed by a further 30 min incubation. For the SE-based screening experiments,
groups of 3 to 4 mice were immunized with GMZ2 and SE supplemented with the TLR agonists described above except that SE/GLA
(EM031) was used. The different components of the SE-based formulations were mixed gently and allowed to rest on ice for 2 h
before injection.
2.4. Detection of vaccine-specic antibodies by ELISA
Total immunoglobulin G (IgG) and IgG subclasses were determined by ELISA using serum obtained 4 weeks after the last
immunization essentially as previously described [2]. Briey, plates
were coated overnight at 4 C with 0.5 g/ml of either GMZ2,
recombinant GLURP25500 or MSP3212380 in 0.05 M carbonate
buffer. Sera were diluted 1:1000 in phosphate-buffered saline
(PBS) supplemented with 0.5 M NaCl and 0.1% Tween20 (PBST)
plus 1% milk powder, and the plates were washed extensively
between each incubation step with PBST. Color was developed with
horseradish peroxidase-conjugated goat anti-mouse IgG (H + L),
anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Southern
Biotech) diluted 1:1000 in PBST plus 1% milk powder followed by
H2 O2 with o-phenylenediamine (DAKO). Absorbance values were
plotted as a function of the reciprocal dilution of serum samples.
The reciprocal serum dilution corresponding to 50% maximal binding (EC50) is referred to as the midpoint titer.
2.5. Recognition of P. falciparum proteins by IFAt and
immunoblotting
The indirect immunouorescent antibody test (IFAt) was performed using air-dried thin smears from red blood cell cultures
containing predominantly mature schizonts of P. falciparum (NF54).
Smears were xed with 4% paraformaldehyde and incubated
for 30 min at 37 C with diluted sera (an initial 1:100 dilution
in PBS + 1% bovine serum albumin and subsequent 5-fold serial
dilutions). After washing with PBS, antibodies were detected
using an Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500;
Molecular Probes/Invitrogen). A mounting media containing DAPI
(ProLong Gold antifade reagent with DAPI; Invitrogen) was used,
and cells at the same parasite stage were compared. Values are presented as endpoint titers and correspond to the reciprocal dilution
which presented a signal above the background. Immunoblotting
was performed as described previously [2] using goat anti-mouse
immunoglobulins conjugated to alkaline phosphatase (Promega,
Madison, Wis.) as the secondary antibody.

3286

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

2.6. Detection of specic long-lived plasma cells by ELISPOT


Antigen-specic long-lived plasma cells (LLPCs) were detected
in the bone marrow (BM) of the immunized mice four weeks after
the last immunization. BM cells were collected from the marrow of
mouse femurs and tibias by ushing them with RPMI supplemented
with 100 U of penicillin/ml, 100 U of streptomycin/ml and 2% fetal
calf serum (FCS). Single cell suspensions of the BM cells were
treated with ACK red blood cell lysis buffer (150 mM NH4 Cl, 10 mM
KHCO3 , 0.1 mM Na2 EDTA), and diluted to 107 cells/ml before plating. MultiscreenHTS -IP Filter Plates (Millipore) were pre-wet with
35% ethanol, washed with PBS, and coated overnight at 4 C with
10 g/ml of either the fusion protein GMZ2 or the individual GLURP
and MSP3 antigens in PBS. Coated plates were blocked for 2 h at
room temperature with RPMI supplemented with 100 U of penicillin/ml, 100 U of streptomycin/ml, 50 M 2-mercaptoethanol, and
10% FCS. Threefold serial dilutions of 106 BM cells were tested
in triplicates. Plates were incubated for 5 h at 37 C in 5% CO2 .
Plates were washed with PBS + 0.5% Tween20 and incubated for
1 h at room temperature with HRP-conjugated goat anti mouse
IgG (1:1000; Southern Biotech) in PBS + 0.5% Tween20 + 5% FCS.
Plates were washed ve times with PBS + 0.5% Tween20 followed
by three washes with PBS. Filters were developed using Vectastain
3-amino-9-ethylcarbazole (AEC) substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturers protocol.
The reactions were stopped by washing the plates with deionized
water, the plates dried in the dark, and the spots counted on an
automated ELISPOT Reader System and Software (AID, Strassberg,
Germany).
2.7. Antigen-specic cytokine production during ex vivo
stimulation
Cultures of spleens and inguinal lymph nodes were obtained
by homogenizing the organs in complete RPMI (RPMI 1640 supplemented with 50 M 2-mercapthoethanol, 1 mM glutamine, 1%
penicillinstreptomycin, 1% HEPES and 10% fetal calf serum, all
from Gibco; Invitrogen, Carlsbad, CA) and subsequently washed
twice and adjusted to a nal concentration of 2 105 cells/well in a
total volume of 200 l/well. Cells were plated in triplicate, and antigens were used at 5 g/ml. Culture supernatants were harvested
after 72 h of incubation for the measurement of IFN- and IL-5 by
ELISA as recommended by the manufacturer (BD Biosciences).
2.8. Statistical analysis
One-sided analysis of variance on the log-transformed values
was used to conrm that the data contain essential differences.
Wilcoxons test was then used to investigate the effects of GLA by
comparing Al(OH)3 with Al(OH)3 plus GLA and comparing SE with
SE plus GLA. p-Values are two-sided and quoted without adjustment for multiple testing since the signicance of the addition of
GLA was the primary problem under investigation. p-Values 0.05
were considered signicant.
3. Results
3.1. Screening of TLR agonists for enhancement of antibody
responses against GMZ2
In order to enhance the immunogenicity of GMZ2, we screened
six different TLR agonist formulations in combination with GMZ2.
Inbred CB6F1 (BALB/C C57BL/6) mice were immunized three
times with 5 g of GMZ2 administered in either Al(OH)3 or in an
oil-in-water stable emulsion (SE) supplemented with either CpG,

GLA, or R848, and antigen-specic antibody responses were measured by ELISA and IFA. The addition of the TLR 9 agonist CpG and
the TLR 4 agonist GLA to both Al(OH)3 - and SE-based formulations
lead to enhanced anti-GMZ2 total IgG responses as measured by
both ELISA (Fig. 1A and D) and IFA (Fig. 1G and H). Although the
number of mice per group in this screening experiment does not
allow for conducting statistical analysis of the data, it is evident
that the co-formulation of Al(OH)3 or SE with CpG or GLA elevated
the murine IgG2a responses to GMZ2, but had little effect on IgG1
responses to the immunizing antigen (Fig. 1B, C, E, and F), suggesting the TLR agonists induced B cells to undergo a subclass shift from
IgG1 to IgG2a production. In contrast, the TLR7/8 agonist R848 did
not enhance antibody responses against GMZ2 (Fig. 1). Moreover,
both CpG and GLA in either emulsion or Al(OH)3 formulations promote stronger IgG antibody responses against the MSP3 domain
of GMZ2 than any other formulation (data not shown). The two
formulations giving rise to the highest ELISA antibody titers also
gave the strongest IFA reactivity (Fig. 1G and H) indicating that
vaccine-induced antibodies recognize native parasite antigens as
well.
3.2. GLA enhances anti-GMZ2, anti-GLURP and anti-MSP3
antibody responses
Since GMZ2 in combination with GLA (in either emulsion or
Al(OH)3 formulations) elicit the strongest IgG responses observed,
we decided to study this immune modulator in detail. In agreement
with the screenings, GMZ2 in SE-based formulations is generally
more immunogenic than in Al(OH)3 -based formulations (Fig. 2A)
resulting in the strongest anti-MSP3 IgG responses (Fig. 2C). With
respect to the effect of the immune modulator, addition of GLA to
GMZ2 in Al(OH)3 resulted in signicantly higher levels of GLURPspecic IgG2a (Fig. 2E, Wilcoxons test, p = 0.0055), IgG2b (Fig. 2F,
Wilcoxons test, p = 0,0075) and the IgG2a/IgG1 ratio was signicantly higher (Wilcoxons test, p = 0.0087), but still much below a
ratio of 1. Likewise the addition of GLA in SE to GMZ2 also significantly increased the ratio of anti-GLURP IgG2a/IgG1 from 0.14 to
2.51 (Wilcoxons test, p = 0.0087), 2-fold more than with Al(OH)3 .
The anti-MSP3 IgG responses were also increased by the addition
of GLA to GMZ2 in both Al(OH)3 - and SE-based formulations; these
differences did not reach statistical signicance (Fig. 2C). Antibody
responses were also assessed by IFA on mature schizonts (Fig. 3).
This analysis demonstrates a statistically signicant effect of GLA on
the immunogenicity of GMZ2 in Al(OH)3 (Wilcoxons test, p = 0.014)
and in SE (Wilcoxons test, p = 0.0073). Interestingly, this analysis
suggests that Al(OH)3 and SE are equally efcient adjuvant vehicles
for generating parasite-specic antibodies. Due to limiting amounts
of sera available, we were unable to quantify titers of the antigenspecic IgG subclasses IgG1 and IgG2a in the IFA experiments and
their biological activities in ADCI.
3.3. Effects of GLA on anti-GMZ2, anti-GLURP and anti-MSP3 T
cell responses
To investigate the cellular mechanism underlying the effect of
GLA, T-cell responses were measured by ELISA four weeks after
the last immunization. IFN- and IL-5 concentrations were determined in supernatants of splenocytes re-stimulated with GMZ2 as
well as the component GLURP and MSP3 recombinant proteins.
Fig. 4 shows that GMZ2 in an SE-based formulation stimulated
a stronger CMI response than in an Al(OH)3 -based formulation.
The co-formulation of GLA with SE plus GMZ2 resulted in a further increase in the production of the Th1 cytokine IFN- and a
decreased production of the Th2 cytokine IL-5. A similar increase
was observed when splenocytes were re-stimulated with GLURP
(Fig. 4B and E) and, to a lesser extent, with MSP3 (Fig. 4C and F).

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

3287

Fig. 1. Effect of adjuvant vehicles (Al(OH)3 and SE) and immune modulators (CpG, GLA, and R848) on antibody responses against GMZ2. Groups of 34 CB6F1 mice were
immunized by the s.c. route three times at 3 week intervals with GMZ2 alone () or combined with Al(OH)3 or oil-in-water emulsion (SE) and the immune modulators CpG,
GLA or R848, as indicated. Four weeks after the last immunization, serum samples were tested for antibody responses on ELISA plates coated with GMZ2 (AF) or by IFA
(GH). ELISA titer was the inverse serum dilution giving an absorbance corresponding to 50% maximal binding (midpoint titer). The line represents the mean value.

T-cell responses measured in the draining lymph nodes yielded


similar results (data not shown). Thus, GLA promotes a Type 1 Tcell response characterized by the production of high levels of IFN-
relative to IL-5.

3.4. Effects of GLA on long-lived antibody-secreting plasma cell


responses
To determine whether GLA enhances the duration of antibody
responses against GMZ2, long-lived plasma cells (LLPCs) secreting
antibodies against GMZ2, GLURP and MSP3 were obtained from the
BM and counted by ELISPOT. Results are presented as Ag-specic
LLPCs per 106 BM cells. When compared to the Al(OH)3 -based
formulation, SE-based formulations induced a higher number
of LLPCs specic for both the full-length GMZ2 fusion protein
(Fig. 5A; Wilcoxons test, p = 0.0029) and the individual GLURP
(Fig. 5B;Wilcoxons test, p = 0.0003) and MSP3 (Fig. 5C; Wilcoxons
test, p = 0.070) domains. While the addition of GLA to GMZ2 in
Al(OH)3 did not increase the number of LLPCs against MSP3, GLA

did stimulate production of more LLPC to GMZ2 (Fig. 5A; Wilcoxons


test, p = 0.0022) and GLURP (Fig. 5B; Wilcoxons test, p = 0.0303).
While immunization with GMZ2 plus GLA in the SE does not appear
to further increase the relatively high LLPC numbers observed for
GMZ2 in SE formulation without GLA (Fig. 5), we anticipate that the
number of LLPC expressing GMZ2-specic IgG2a are elevated (and
the number of IgG1-generating LLPC are reduced) considerably in
the GMZ2 + SE/GLA mice compared to the GMZ2 + SE mice.

3.5. Reactivity of mouse anti-GMZ2 antisera with


parasite-derived GLURP and MSP3
The immunogenicity of GMZ2 was also investigated by
immunoblotting of parasite-derived proteins with plasma from
mice immunized with each of the four formulations. As illustrated
in Fig. 6, plasma from mice immunized with GMZ2 in SE and
SE/GLA recognized polypeptides of approximately 220,000 Da and
48,000 Da (lanes 2 and 3), the apparent molecular masses after
SDSPAGE that were previously reported for GLURP [36] and MSP3

3288

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

Fig. 2. Effect of GLA on antibody responses against GMZ2. Groups of 6 CB6F1 mice were immunized with GMZ2 alone () or combined with the adjuvants vehicle (Al(OH)3 or
SE) and GLA. Four weeks after the last immunization, sera were tested for antibody responses on ELISA plates coated with GMZ2, GLURP or MSP3. The midpoint titer for total
IgG against GMZ2 (A), GLURP (B), and MSP3(C), and IgG1 (D), IgG2a (E) and IgG2b (F) against GMZ2 are shown for individual mice () and the group mean value is indicated
by a horizontal bar. The mean ratios SD of IgG2a/IgG1 are shown in (G). The asterisks represent statistical signicance (*p < 0.05, **p < 0.01). The data are representative of
two independent studies.

[20], respectively. In contrast, GMZ2 in Al(OH)3 and Al(OH)3 plus


GLA recognized GLURP only (lanes 4 and 5) reecting the lower
immunogenicity of the MSP3 domain in Al(OH)3 -based formulations.
4. Discussion
The GMZ2 candidate vaccine is a chimeric protein consisting of
the conserved N-terminal portion of GLURP genetically fused to the
conserved C-terminal portion of MSP3 [2]. Studies in mice [2] and
humans [24] have demonstrated that GMZ2 generate stronger antibody responses against the individual GLURP and MSP3 domains
than the GLURP and MSP3 molecules used either alone or combined in a mixture. The difference was most pronounced for the
MSP3-specic antibody responses suggesting that T cell epitopes
located in the GLURP region provide help for B-cell epitopes in the
MSP3 region. However, these studies also demonstrate that GLURP
in general is more immunogenic than MSP3. The potential importance of increasing the overall immunogenicity of GMZ2, and the
MSP3 domain in particular, is suggested by efcacy studies performed in the Saimiri blood stage model [37]. Here, we found that

protection against an experimental P. falciparum blood stage infection was associated with the level of GMZ2-specic antibodies. In
the present study we have therefore screened a series of adjuvant
vehicles and immune modulators in mice to identify an improved
formulation of GMZ2 for further clinical studies. GMZ2 formulated
in an oil-in-water emulsion plus GLA effectively leads to the (a)
highest vaccine-specic cytophilic IgG2a, IgG2b and IgG titers, (b)
parasite-specic IFA titers as high as any observed in these studies, (c) the highest levels of the Type 1 cytokine IFN- after in vitro
stimulation, (d) the highest anti-MSP3-specic titers using GMZ2
as the immunizing antigen, and (e) larger numbers of LLPCs secreting antibodies to GMZ2 and its GLURP and MSP3 components than
observed with Al(OH)3 formulations tested.
GLA is a synthetic molecule that is structurally similar to the
hexaacyl lipid found in monophosphoryl lipid A (MPL ) and stimulates TLR4 [27]. Here we show that, of those adjuvants tested,
GMZ2 combined with GLA in a stable emulsion formulation elicited
the highest IgG2a/IgG1 ratio and the strongest IFN- response
compared to IL-5 response indicating that TLR4 signalling efciently stimulated a Th1-predominant response leading to antibody
switching to IgG2a. Murine IgG2a is a cytophilic subclass of IgG

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

Fig. 3. Recognition of P. falciparum-derived proteins after immunization with GLAbased formulations. Four weeks after the last immunization, sera from the mice
in Fig. 2 were tested for antibody responses by IFA. Endpoint titer was the inverse
serum dilution resulting in the specic staining of mature P. falciparum NF54 bloodstage schizonts. The cell-cycle stage was determined by DAPI staining. Endpoint
titers are shown for individual mice () and the group mean value is indicated (horizontal bar). The asterisks denote statistical signicance (*p < 0.05, **p < 0.01). The
data are representative of two independent experiments.

that binds with high afnity to the FcRI receptor on monocytes


and macrophages. IgG2a and IgG2b also bind to the FcRII and
FcRIII receptors [38,39]. The activation of Fc receptors can trigger antibody-dependent-cell-mediated inhibition (ADCI), which is
thought to play an important role in the control of P. falciparum multiplication in vivo [4042]. Afnity-puried IgG antibodies against
GLURP are highly effective in the in vitro ADCI assay, and two
independent studies have found that human IgG3 antibodies are

3289

particularly effective in this respect [4,23]. Although it is not known


with certainty whether human IgG3 is the orthologue of mouse
IgG2b, these two IgG subclasses share many characteristics, including effective anti-malaria responses. Vaccine-induced IgG2b was
found to be correlated with protection against blood-stage malaria
infection in mice [43], and epidemiological studies have demonstrated that human IgG3 is a strong predictor of protection from
clinical malaria [7,12,14,44,45]. It is well established that mouse IgG
antibodies against GMZ2 [2] and a MSP1MSP3 hybrid protein [46]
can collaborate with human monocytes to inhibit parasite growth
in vitro. Due to the limited amount of serum available we did not
investigate whether mouse IgG2a and IgG2b antibodies in particular had the same inhibitory capacity. Irrespective of the activity
of mouse antibodies, there is ample data to suggest that human
IgG3 is more effective in ADCI than IgG1 [4,23]. We have therefore been pursuing a formulation which elicits a qualitatively and
quantitatively different antibody response than the current formulation of GMZ2 in Al(OH)3 which elicits predominantly a human
IgG1 response.
GMZ2 in SE/GLA also elicits the highest levels of IgG and,
in particular, IgG2a against the MSP3 domain. This nding is
interesting and critical because the main IgG response that the
current Al(OH)3 formulation of GMZ2 elicits is against the GLURP
domain [24]. It is clearly important to enhance MSP3-specic
IgG responses since vaccine-induced anti-MSP3 antibodies show
strong growth-inhibitory activity on their own [47] and because
we have previously observed a complementary effect of the MSP3and GLURP-specic IgG3 antibodies in relation to malaria protection [15]. In those human studies, the individuals who did not
respond to one of the antigens showed a strong IgG3 response to
the other antigen, and these responses were consistently associated with protection against clinical malaria [15]. GMZ2 in SE/GLA
was also the most efcient formulation in terms of generating
long-lived antibody responses against both the GLURP and MSP3

Fig. 4. IFN- and IL-5 production in splenocytes. Splenocytes were isolated 4 weeks after the last immunization of the mice in Fig. 2, cultured individually, and re-stimulated
with 5 g/ml of GMZ2 (A and D), GLURP (B and E), or MSP3 (C and F). The IFN- and IL-5 responses were measured after 72 h of stimulation. The means of triplicate values and
the group mean value are shown for individual mice () and by a horizontal bar, respectively. Signicant differences are shown by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).
The data are representative of two independent studies.

3290

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

Fig. 6. Western blot reactivity of mouse anti-GMZ2 antisera with a P. falciparum


NF54 blood-stage schizont extract. Sera from mice immunized with GMZ2 in combination with the indicated adjuvants were diluted 200-fold and used as the primary
antibody. Lane 1, pre-bleed; lane 2, SE; lane 3, SE/GLA; lane 4, Al(OH)3 ; lane 5,
Al(OH)3 plus GLA. The positions of molecular markers are indicated on the left in
kilodaltons.

Fig. 5. Effect of GLA on specic long-lived antibody secreting plasma cells. The number of antigen-specic long-lived plasma cells (LLPC) present in the bone marrow
was enumerated 4 weeks after the last immunization of the mice in Fig. 2. The LLPC
secreting antibodies to GMZ2 (A), GLURP (B), and MSP3 (C) are shown for individual
mice () and the group mean value (horizontal bar). The asterisks denote statistical
signicance (*p < 0.05, **p < 0.01, ***p < 0.001). The data are representative of two
independent studies.

domains. Thus, GMZ2 in SE alone and in SE/GLA were the two formulations inducing high numbers of LLPCs secreting antibodies
against MSP3. This effect of SE/GLA is of particular importance as
LLPCs are thought to be instrumental for acquisition of immunity
against clinical malaria [48]. The effect of GLA was also evident
when the binding of vaccine-induced antibodies to the component parasite antigens was tested by IFA. In contrast to what was
observed by ELISA with recombinant proteins, GLA formulations
with both Al(OH)3 and SE signicantly enhanced IgG levels to the
native parasite proteins in IFA. In fact, the mean IFA endpoint titer
obtained with GMZ2 in Al(OH)3 /GLA is comparable to that obtained
with GMZ2 in SE/GLA (Fig. 5). This difference might indicate that
recombinant GMZ2 displays epitopes for antibodies that are not
accessible on the native parasite proteins and that GLA might
enhance production of antibodies that recognize epitopes shared
between recombinant GMZ2 and the native parasite-derived antigens. Irrespective of the explanation, the IFA results demonstrate
the benecial effect of GLA for induction of IgG antibodies against
native GLURP and MSP3.
Although IgG antibodies are thought to be the main effector molecule mediating protection against clinical malaria, blood
stage-relevant CMI responses may include not only T-cell help
for producing a robust antibody response, but also CD4+ effector mechanism(s). A recent study has found that, by repeatedly
exposing volunteers to infective mosquito bites while concurrently
giving them prophylactic anti-malaria treatment, the individuals were protected against a subsequent sporozoite challenge.
These immune individuals had no detectable antibodies against
blood stage antigens, but had solid cell-mediated immune (CMI)
responses [49]. In this model multifunctional effector memory
T cells producing IFN-, TNF, and IL-2 were positively correlated with protection [49]. The observation that IFN- responses
to both liver- and blood-stage antigens have been positively correlated to protection [50] is consistent with the potential importance
of IFN- to immunity. GMZ2 in SE/GLA induced high levels of
IFN- and low levels of IL-5 indicating that GMZ2 in SE/GLA
induces a type 1 T cell response. The elevated IFN levels are
dependent on the inclusion of GLA; GMZ2 in SE failed to elevate
IFN and class switching to IgG2a antibodies. These observations
are in agreement with the recent ndings that SE/GLA enhances
Type 1 cellular immune responses against the inuenza vaccine
Fluzone [27].
In conclusion, the results of these preclinical studies give good
reason for further accelerated product development aiming at clinical assessment of safety and efcacy of GMZ2 formulated in SE with
GLA.

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

Acknowledgements
This work was supported by contract LSHP-CT-2007-037506
from the European Commission, grants from EMVI, the Lundbeck Foundation, and Fonden til Lgevidenskabens fremme, and
grant #42387 (SGR) from Bill and the Melinda Gates Foundation.
The authors would like to thank Rune F. Jensen, Micha Jepsen,
Madeleine Dahlbck, and Anne Corftiz for technical assistance and
Irina Zharkikh and Tim Dutill for assistance in early in vivo studies
and training in LLPC analyses and formulation preparation, respectively.
Contributors: S. Lousada-Dietrich, P.S. Jogdand, V.V. Pinto, S.B.
Ditlev, M. Christiansen, S. Olesen Larsen, C.B. Fox, V.S. Raman, R.F.
Howard, T.S. Vedvick, G. Ireton, D. Carter, S.G. Reed, M. Theisen were
involved in the conception and design of the study, or acquisition
of data, or analysis and interpretation of data. S. Lousada-Dietrich,
S. Jepsen, M. Christiansen, V.S. Raman, R.F. Howard, G. Ireton, D.
Carter, M. Theisen were involved in drafting the article or revising
it critically for important intellectual content. S. Lousada-Dietrich,
P.S. Jogdand, S. Jepsen, V.V. Pinto, S.B. Ditlev, M. Christiansen, C.B.
Fox, V.S. Raman, R.F. Howard, G. Ireton, D. Carter, S.G. Reed, M.
Theisen were involved in nal approval of the version to be submitted.
References
[1] WHO. Available from: http://www.who.int.
[2] Theisen M, Soe S, Brunstedt K, Follmann F, Bredmose L, Israelsen H, et al. A Plasmodium falciparum GLURPMSP3 chimeric protein; expression in Lactococcus
lactis, immunogenicity and induction of biologically active antibodies. Vaccine
2004;22:118898.
[3] Stricker K, Vuust J, Jepsen S, Oeuvray C, Theisen M. Conservation and
heterogeneity of the Glutamate-rich protein (GLURP) among eld isolates and laboratory lines of Plasmodium falciparum. Mol Biochem Parasitol
2000;111:12330.
[4] Theisen M, Soe S, Jessing S, Okkels L, Danielsen S, Oeuvray C, et al. Identication
of a major linear B cell epitope of the Plasmodium falciparum Glutamate-rich
protein (GLURP), targeted by human antibodies mediating parasite killing. Vaccine 2000;19:20412.
[5] Huber W, Felger I, Matile H, Lipps HJ, Steiger S, Beck HP. Limited sequence
polymorphism in the Plasmodium falciparum merozoite surface protein 3. Mol
Biochem Parasitol 1997;87:2314.
[6] Dodoo D, Aikins A, Kusi KA, Lamptey H, Remarque E, Milligan P, et al. Cohort
study of the association of antibody levels to AMA1, MSP119, MSP3 and GLURP
with protection from clinical malaria in Ghanaian children. Malar J 2008;7:142.
[7] Dodoo D, Theisen M, Kurtzhals JA, Akanmori BD, Koram KA, Jepsen S,
et al. Naturally acquired antibodies to the glutamate-rich protein are associated with protection against Plasmodium falciparum malaria. J Infect Dis
2000;181:12025.
[8] Enevold A, Nkya WM, Theisen M, Vestergaard LS, Jensen AT, Staalsoe T, et al.
Potential impact of host immunity on malaria treatment outcome in Tanzanian
children infected with Plasmodium falciparum. Malar J 2007;6:153.
[9] Iriemenam NC, Khirelsied AH, Nasr A, ElGhazali G, Giha HA, Elhassan AETM,
et al. Antibody responses to a panel of Plasmodium falciparum malaria bloodstage antigens in relation to clinical disease outcome in Sudan. Vaccine
2009;27:6271.
[10] Lusingu JP, Vestergaard LS, Alifrangis M, Mmbando BP, Theisen M, Kitua AY,
et al. Cytophilic antibodies to Plasmodium falciparum glutamate rich protein
are associated with malaria protection in an area of holoendemic transmission.
Malar J 2005;4:48.
[11] Meraldi V, Nebie I, Tiono AB, Diallo D, Sanogo E, Theisen M, et al. Natural antibody response to Plasmodium falciparum Exp-1, MSP-3 and GLURP
long synthetic peptides and association with protection. Parasite Immunol
2004;26:26572.
[12] Nebie I, Diarra A, Ouedraogo A, Soulama I, Bougouma EC, Tiono AB, et al.
Humoral responses to Plasmodium falciparum blood-stage antigens and association with incidence of clinical malaria in children living in an area of
seasonal malaria transmission in Burkina Faso, West Africa. Infect Immun
2008;76:75966.
[13] Oeuvray C, Theisen M, Rogier C, Trape JF, Jepsen S, Druilhe P. Cytophilic
immunoglobulin responses to Plasmodium falciparum glutamate-rich protein
are correlated with protection against clinical malaria in Dielmo, Senegal. Infect
Immun 2000;68:261720.
[14] Roussilhon C, Oeuvray C, Muller-Graf C, Tall A, Rogier C, Trape JF, et al. Longterm clinical protection from falciparum malaria is strongly associated with
IgG3 antibodies to merozoite surface protein 3. PLoS Med 2007;4(11):e320.
[15] Soe S, Theisen M, Roussilhon C, Aye KS, Druilhe P. Association between protection against clinical malaria and antibodies to merozoite surface antigens in

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]
[29]

[30]

[31]

[32]

[33]
[34]
[35]

[36]

[37]

[38]
[39]

[40]

3291

an area of hyperendemicity in Myanmar: complementarity between responses


to merozoite surface protein 3 and the 220-kilodalton glutamate-rich protein.
Infect Immun 2004;72:24752.
Theisen M, Dodoo D, Balde AT, Soe SGC, Koram KA, et al. Selection of long
GLURP synthetic peptides for vaccine development: antigenicity, relationship with clinical protection and immunogenicity. Infect Immun 2001;69:
52239.
Courtin D, Oesterholt M, Huismans H, Kusi K, Milet J, Badaut C, et al. The
quantity and quality of African childrens IgG responses to merozoite surface
antigens reect protection against Plasmodium falciparum malaria. PLoS One
2009;4(10):e7590.
Singh S, Soe S, Mejia JP, Roussilhon C, Theisen M, Corradin G, et al. Identication
of a conserved region of Plasmodium falciparum MSP3 targeted by biologically
active antibodies to improve vaccine design. J Infect Dis 2004;190:10108.
Theisen M, Soe S, Oeuvray C, Thomas AW, Vuust J, Danielsen S, et al. The
glutamate-rich protein (GLURP) of Plasmodium falciparum is a target for
antibody-dependent monocyte-mediated inhibition of parasite growth in vitro.
Infect Immun 1998;66:117.
Oeuvray C, Bouharoun-Tayoun H, Gras-Masse H, Bottius E, Kaidoh T, Aikawa M,
et al. Merozoite surface protein-3: a malaria protein inducing antibodies that
promote Plasmodium falciparum killing by cooperation with blood monocytes.
Blood 1994;84:1594602.
Audran R, Cachat M, Lurati F, Soe S, Leroy O, Corradin G, et al. Phase I malaria
vaccine trial with a long synthetic peptide derived from the merozoite surface
protein 3 antigen. Infect Immun 2005;73:801726.
Hermsen CC, Verhage DF, Telgt DS, Teelen K, Bousema JT, Roestenberg M,
et al. Glutamate-rich protein (GLURP) induces antibodies that inhibit in vitro
growth of Plasmodium falciparum in a phase 1 malaria vaccine trial. Vaccine
2007;25:293040.
Tebo AE, Kremsner PG, Luty AJ. Plasmodium falciparum: a major role for IgG3 in
antibody-dependent monocyte-mediated cellular inhibition of parasite growth
in vitro. Exp Parasitol 2001;98:208.
Esen M, Kremsner PG, Schleucher R, Gassler M, Imoukhuede EB, Imbault N, et al.
Safety and immunogenicity of GMZ2 a MSP3GLURP fusion protein malaria
vaccine candidate. Vaccine 2009;27:68628.
Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, et al. Efcacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection
and disease in young African children: randomised controlled trial. Lancet
2004;364:141120.
Lell B, Agnandji S, von Glasenapp I, Haertle S, Oyakhiromen S, Issifou S, et al. A
randomized trial assessing the safety and immunogenicity of AS01 and AS02
adjuvanted RTS,S malaria vaccine candidates in children in Gabon. PLoS One
2009;4(10):e7611.
Baldwin SL, Shaverdian N, Goto Y, Duthie MS, Raman VS, Evers T, et al. Enhanced
humoral and Type 1 cellular immune responses with Fluzone adjuvanted with
a synthetic TLR4 agonist formulated in an emulsion. Vaccine 2009;27:595663.
Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol
2003;21:33576.
Garcon N, Chomez P, Van Mechelen M. GlaxoSmithKline adjuvant systems
in vaccines: concepts, achievements and perspectives. Expert Rev Vaccines
2007;6:72339.
Suzuki Y, Wakita D, Chamoto K, Narita Y, Tsuji T, Takeshima T, et al. Liposomeencapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type
1 innate immunity. Cancer Res 2004;64:875460.
Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C,
et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological
requirements. PLoS One 2008;3(9):e3116.
Abdulla S, Oberholzer R, Juma O, Kubhoja S, Machera F, Membi C, et al. Safety
and immunogenicity of RTS,S/AS02D malaria vaccine in infants. N Engl J Med
2008;359:253344.
McHeyzer-Williams MG, Ahmed R. B cell memory and the long-lived plasma
cell. Curr Opin Immunol 1999;11:1729.
Wrammert J, Ahmed R. Maintenance of serological memory. Biol Chem
2008;389:5379.
Radbruch A, Muehlinghaus G, Luger EO, Inamine A, Smith KG, Dorner T, et al.
Competence and competition: the challenge of becoming a long-lived plasma
cell. Nat Rev Immunol 2006;6:74150.
Theisen M, Vuust J, Gottschau A, Jepsen S, Hogh B. Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum
expressed in Escherichia coli. Clin Diagn Lab Immunol 1995;2:304.
Carvalho LJ, Alves FA, Bianco Jr C, Oliveira SG, Zanini GM, Soe S, et al.
Immunization of Saimiri sciureus monkeys with a recombinant hybrid protein derived from the Plasmodium falciparum antigen glutamate-rich protein
and merozoite surface protein 3 can induce partial protection with Freund and Montanide ISA720 adjuvants. Clin Diagn Lab Immunol 2005;12:
2428.
Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann
Hematol 1998;76:23148.
Sears DW, Osman N, Tate B, McKenzie IF, Hogarth PM. Molecular cloning
and expression of the mouse high afnity Fc receptor for IgG. J Immunol
1990;144:3718.
Bouharoun-Tayoun H, Attanath P, Sabchareon A, Chongsuphajaisiddhi T,
Druilhe P. Antibodies that protect humans against Plasmodium falciparum blood
stages do not on their own inhibit parasite growth and invasion in vitro, but
act in cooperation with monocytes. J Exp Med 1990;172:163341.

3292

S. Lousada-Dietrich et al. / Vaccine 29 (2011) 32843292

[41] Bouharoun-Tayoun H, Oeuvray C, Lunel F, Druilhe P. Mechanisms underlying


the monocyte-mediated antibody-dependent killing of Plasmodium falciparum
asexual blood stages. J Exp Med 1995;182:40918.
[42] Druilhe P, Sabchareon A, Bouharoun-Tayoun H, Oeuvray C, Perignon
JL. In vivo veritas: lessons from immunoglobulin-transfer experiments
in malaria patients. Ann Trop Med Parasitol 1997;91(Suppl. 1):37
53.
[43] Burns Jr JM, Dunn PD, Russo DM. Protective immunity against Plasmodium yoelii
malaria induced by immunization with particulate blood-stage antigens. Infect
Immun 1997;65:313845.
[44] Aribot G, Rogier C, Sarthou JL, Trape JF, Balde AT, Druilhe P, et al. Pattern of
immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, west
Africa). Am J Trop Med Hyg 1996;54:44957.
[45] Sarthou JL, Angel G, Aribot G, Rogier C, Dieye A, Toure Balde A, et al. Prognostic
value of anti-Plasmodium falciparum-specic immunoglobulin G3, cytokines,
and their soluble receptors in West African patients with severe malaria. Infect
Immun 1997;65:32716.

[46] Mazumdar S, Mukherjee P, Yazdani SS, Jain SK, Mohmmed A, Chauhan


VS. Plasmodium falciparum merozoite surface protein 1 (MSP-1)-MSP-3
chimeric protein: immunogenicity determined with human-compatible adjuvants and induction of protective immune response. Infect Immun 2010;78:
87283.
[47] Druilhe P, Spertini F, Soesoe D, Corradin G, Mejia P, Singh S, et al. A malaria
vaccine that elicits in humans antibodies able to kill Plasmodium falciparum.
PLoS Med 2005;2(11):e344.
[48] Weiss GE, Traore B, Kayentao K, Ongoiba A, Doumbo S, Doumtabe D, et al. The
Plasmodium falciparum-specic human memory B cell compartment expands
gradually with repeated malaria infections. PLoS Pathog 2010;6(5):e1000912.
[49] Roestenberg M, McCall M, Hopman J, Wiersma J, Luty AJ, van Gemert GJ, et al.
Protection against a malaria challenge by sporozoite inoculation. N Engl J Med
2009;361:46877.
[50] Luty AJ, Lell B, Schimdt-Ott R, Lehman LG, Luckner D, Greve B, et al.
Interferon-gamma responses are associated with resistance to reinfection
with Plasmodium falciparum in young African children. J Infect Dis 1999;179:
9808.

Das könnte Ihnen auch gefallen