Beruflich Dokumente
Kultur Dokumente
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
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
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Infectious Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle, WA 98104, USA
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a r t i c l e
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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
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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).
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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.
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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.
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
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.
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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.
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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.
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.
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