Immunogenicity of self-associated aggregates and chemically cross-linked conjugates of the 42 kDa Plasmodium falciparum merozoite surface protein-1.
ABSTRACT Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP1(42)) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP1(42) conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP1(42) self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP1(42) specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis.
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ABSTRACT: Background. Acquired immunity to malaria develops with increasing age and repeated infections. Understanding immune correlates of protection from malaria infection would facilitate vaccine development and identification of biomarkers that reflect changes in susceptibility resulting from ongoing malaria control efforts.Methods. The relationship between IgG antibody and IFN-γ and IL-10 responses to the 42 kD C-terminal fragment of Plasmodium falciparum Merozoite Surface Protein 1 (MSP142) and risk-of-(re)infection were examined following drug-mediated clearance of parasitemia in 94 adults and 95 children in a holoendemic area of western Kenya.Results. Positive IFN-γ ELISA and ELISPOT responses to MSP-142 3D7 were associated with delayed time-to-(re)infection whereas high-titer IgG antibodies to MSP-142 3D7 or FVO alleles were not independently predictive of risk-of-(re)infection. When IFN-γ and IL-10 responses were both present, the protective effect of IFN-γ was abrogated. A Cox proportional hazard model including IFN-γ, IL-10, MSP142 3D7 IgG antibody responses, hemoglobin S genotype, age and infection status at baseline, showed time to blood-stage infection correlated positively with IFNγ+ responses and negatively with IL-10+ responses, younger age, and asymptomatic parasitemia.Conclusion. Evaluating combined allele-specific cellular and humoral immunity elicited by malaria provides a more informative measure of protection relative to either measure alone.The Journal of Infectious Diseases 03/2013; · 5.85 Impact Factor
Immunogenicity of Self-Associated Aggregates and
Chemically Cross-Linked Conjugates of the 42 kDa
Plasmodium falciparum Merozoite Surface Protein-1
Feng Qian1,2, Karine Reiter1, Yanling Zhang1, Richard L. Shimp, Jr.1, Vu Nguyen1, Joan A. Aebig1,
Kelly M. Rausch1, Daming Zhu1, Lynn Lambert1, Gregory E. D. Mullen1,4, Laura B. Martin1,5,
Carole A. Long1,3, Louis H. Miller1,3, David L. Narum1*
1Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States
of America, 2Department of Rheumatology and Immunology, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China,
3Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of
America, 4Division of Imaging Sciences, School of Medicine, King’s College London, London, United Kingdom, 5Novartis Vaccines Institute for Global Health S.r.l. (NVGH),
Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or
monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine
candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when
evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative
approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is
worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum
merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-
linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa
exotoxin A. MSP142conjugates were prepared and characterized biochemically and biophysically to determine their molar
mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142self-associated aggregates, cross-
linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide
adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation.
Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142specific
antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition
of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the
formation of protein complexes must be established on an empirical basis.
Citation: Qian F, Reiter K, Zhang Y, Shimp RL Jr, Nguyen V, et al. (2012) Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates
of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1. PLoS ONE 7(6): e36996. doi:10.1371/journal.pone.0036996
Editor: Kevin K.A. Tetteh, London School of Hygiene and Tropical Medicine, United Kingdom
Received January 6, 2012; Accepted April 11, 2012; Published June 4, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was supported by the Intramural Research Program of the National Institutes of Health. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Laura B. Martin is an employee of Novartis Vaccines Institute for Global Health S.r.l. There are no patents, products in development, or
marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Chemical conjugation is widely used to make haptens such as
peptides and polysaccharides immunogenic. This is particularly
significant for the development of several important human
vaccines against polysaccharide moieties such as Hemophilus
influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis and
Salmonella enterica serovar Typhi [1,2,3,4]. Chemical conjugation
can also be used on some proteins that are poor immunogens in
order to enhance their immunogenicity. Conjugation effectively
enhance the immunogenicity of the Plasmodium falciparum Pfs25, a
transmission blocking malaria vaccine candidate, when recombi-
nant Pfs25 was conjugated either to carrier proteins such as the
outer-membrane protein complex of Neisseria meningitidis or
ExoProtein A of Pseudomonas aeruginosa, (a detoxified form of
exotoxin A from P. aeruginosa) or to itself (self-conjugation) [5,6,7].
Significant enhancement of Pfs25-specific antibody responses
induced in both mice and rhesus monkeys was achieved. Similar
results were observed when two other malaria antigens of P.
falciparum, Pfs28 and AMA1, were conjugated to the ExoProtein A
[7,8]. In addition to enhancing immunogenicity, conjugation may
overcome the restriction of host genetic backgrounds of vaccinees.
While the single MSP119 of P. yoelii failed to induce specific
antibody responses in mice expressing H-2smajor histocompat-
ibility complex haplotype, its conjugate coupled to diphtheria
toxoid induced functional antibody responses in these mice .
The P. falciparum merozoite surface protein 1 (MSP1) is
considered an important candidate for a vaccine approach
targeting clinical disease or more specifically erythrocytic stage
parasites. MSP1 is synthesized during blood stage development as
PLoS ONE | www.plosone.org1June 2012 | Volume 7 | Issue 6 | e36996
a precursor with a molecular mass of , 200 kDa, and later
undergoes post-translational proteolytic processing. The proteo-
lytic processing produces a C-terminal 42 kDa fragment (MSP142)
which is subsequently processed to 33 kDa and 19 kDa [10,11].
Although inhibitory antibodies of MSP142are principally directed
toward the 19 kDa fragment , the T cell epitopes on the
33 kDa MSP1 fragment enhance the immunogenicity and
protective efficacy of the recombinant MSP142 in non-human
primates  and humans .
Several formulated MSP142-based recombinant proteins of P.
falciparum have been tested in Aotus monkeys [13,15,16,17,18] as
well as in humans [19,20,21]. Protection against a lethal parasite
challenge in Aotus monkeys has been reported, and is generally
associated with a high level of MSP142specific antibody titers
using Freund’s adjuvant [17,18]. In contrast, in a phase 1 human
trial a recombinant MSP142/AlhydrogelTMvaccine formulation
induced only a weak antigen-specific antibody response .
Various efforts have been made to enhance the immunogenicity
and/or improve the efficacy of MSP142–based vaccines, including
the addition of toll–like receptor (TLR) agonists to the formula-
tions [20,21,22], and the construction of chimeric proteins that
replace the MSP133fragment either with other malarial antigens
[23,24] or adjuvanting protein fragments [25,26,27,28]. Only the
use of TLR agonists in vaccine formulations has subsequently been
evaluated in human clinical trials, showing enhanced antibody
In this study, we evaluated whether the immunogenicity of
MSP142in mice is enhanced when presented as 1) a self-associated
aggregated protein or 2) chemically conjugated to a carrier protein
formulated on Alhydrogel with or without CPG 7909, a synthetic
B type CpG-ODN (unmethylated oligodeoxynucleotide contain-
ing cytosine-guanosine (CpG) dinucleotide motifs). In contrast to
our previously reported findings for Pfs25 [5,6,7], neither self-
association nor chemical conjugation to ExoProtein A (EPA)
enhanced the immunogenicity of recombinant MSP142in mice.
Materials and Methods
Rodent studies were carried out in compliance with the
National Institutes of Health guidelines and an animal care and
use committee-approved protocol.
MSP142Antigens and Carrier Protein
MSP142-FUP and MSP142-FVO are two allelic forms of
recombinant P. falciparum MSP142, with an E-KNG or Q-KNG
MSP119 phenotype, respectively. The recombinant MSP142
proteins were expressed in Escherichia coli, refolded, purified and
characterized as previously described [16,29]. The MSP133
fragment of MSP142-FUP contains a single unpaired cysteine
residue, which is absent in the MSP133fragment of the FVO allele
that provided an unpaired sulfhydryl group for the conjugation of
the MSP142-FUP to a carrier protein modified by maleimide
groups (see below). The aggregated MSP142-FVO protein was
produced following the reported purification process, except the
S30 reverse-phase chromatography step was replaced with a
hydrophobic interaction chromatography (HIC) step following the
refold by rapid dilution. The HIC step used a Phenyl 650 M
(Tosoh Biosciences, Montgomeryville, PA) column equilibrated in
50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1.2 M NaCl, 1 M Urea
at 200 cm/h. Sodium chloride crystals were added to the rapid
dilution refolded protein to make a 1.2 M final concentration and
loaded onto the Phenyl 650 M at 200 cm/h after which unbound
proteins were washed from the column using equilibration
solution. The MSP142-FVO protein eluted from the column with
20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 M Urea was pooled
and polished using a Superdex 200 size exclusion column (GE
Healthcare, Piscataway, NJ) equilibrated with PBS with 0.02%
polysorbate 80, pH 7.4. The EPA used as a carrier protein in this
study, was produced in an E. coli expression system, as previously
Two chemical linkers, N-[e-maleimidocaproyloxy]sulfosuccini-
mide ester (Sulfo-EMCS) and succinimidyl-[(N-maleimidopropio-
namido)-diethyleneglycol] ester (NHS-PEO2-Maleimide) (Pierce
Inc., Rockford, IL), containing hydrocarbon and polyethylene
glycol spacers, respectively, were used to modify EPA. These two
chemical linkers are heterobifunctional cross-linkers with an N-
hydroxysuccinimide (NHS) ester and a maleimide group at each of
their termini. The maleimide-reaction pH was fixed at 7.2 and the
EPA concentration was fixed at 2 mg/mL. Three other param-
eters: reaction time, reaction temperature and chemical linker
concentration were optimized by employing a model using a
three-level Box-Behnken design and the JMP statistical software
(SAS Institute, Inc., Cary, NC). The final parameters used for
these conditions were a reaction time of 60 min, a reaction
temperature at 22uC and linker concentration of 2 mM.
The EPA was buffer exchanged to PBS-E (16 PBS, 5 mM
EDTA, pH 7.2) using 5 kDa MWCO spin filter (Millipore,
Billerica, MA). The sulfo-EMCS dissolved in PBS-E and NHS-
PEO2-Maleimide dissolved in Dimethylsulfoxide were added to
the EPA, respectively. The mixtures were incubated under the
defined conditions with gentle shaking. At the end of the reaction,
stop solution (1 M Tris-HCl pH 7.4) was added to a final
concentration of 20 mM and then the buffer was immediately
exchanged to PBS-E. The maleimide modified EPA (maleimide-
EPA) was characterized by reversed-phase HPLC and by
The purified MSP142-FUP protein was reprocessed prior to
conjugation using a preparative SEC column (16660 mm,
Superdex 200) equilibrated with PBS-EU (16PBS, 1 mM EDTA,
5 M urea, pH 7.2) in order to remove the 0.2% polysorbate 80
present in the protein solution and expose the single unpaired
cysteine residue on the MSP133fragment for conjugation. The
conjugation conditions were 22uC for 1 hour with gentle shaking
in PBS-EU. MSP142-FUP and EPA-maleimide were mixed based
on an equal number of moles of free sulfhydryl and maleimide
groups. The MSP142-FUP-EPA conjugate was loaded on a SEC
column (16/60 Superdex 200) equilibrated with PBS-A (16PBS,
0.5 M arginine, pH 7.2). The peak elution fractions containing
MSP142-FUP-EPA conjugates were selected and pooled based on
a Coomassie blue stained SDS-PAGE gel.
Characterization of Recombinant Protein Intermediates
A. SDS-PAGE and Western blotting.
of MSP142-FUP were characterized by Coomassie blue stained
SDS-PAGE under non-reduced condition . Western-blots
were performed as described previously using antigen specific
monoclonal antibodies AD223 and 13E3-53 .
B. Maleimide measurement.
measured using Ellman’s reaction (indirect) as per manufacturer’s
instructions (Pierce Inc., Rockford, IL). Briefly, the maleimide-
EPA samples were titrated with the solution of Cysteine
The EPA conjugates
Maleimide groups were
Immunogenicity of Recombinant MSP142Conjugates
PLoS ONE | www.plosone.org2 June 2012 | Volume 7 | Issue 6 | e36996
Hydrochloride Monohydrate. After the addition of Ellman’s
reagent to each reaction, the absorbance was read at 405 nm.
Based on the cysteine consumed in the reaction, the concentration
of maleimide in the maleimide-EPA sample was determined
relative to the standard curve of the cysteine with increasing
concentrations. The number of maleimide groups added onto the
EPA was obtained by dividing the moles of maleimide by the
moles of EPA.
C. Composition analysis of MSP142-FUP-EPA conjugate
by amino acid analysis.
Amino acid analysis was performed
by the W.M. Keck Facility at Yale University. These results were
used to calculate the molar ratios of MSP142-FUP to EPA (average
conjugation ratio)  and the concentrations of MSP142-FUP
D. Reversed Phase-HPLC.
terized, pre- and post-modification with maleimide groups, on an
analytical 2.16250 mm C4 column (GraceVydac, Hesperia CA)
connected to a Waters 2695 HPLC system (Waters, Milford, MA).
The column was equilibrated in 95% acetonitrile +0.1% TFA
(trifluoroacetic acid) and the proteins eluted in a gradient of 40%
to 58% acetonitrile +0.1% TFA over 36 minutes at a flow rate of
SEC-MALS-HPLC [7,29] was per-
formed on a Waters 2695 HPLC system, with an in-line Wyatt
Dawn EOS light scattering detector, a quasi-elastic light scattering
detector (QELS) and an Optilab refractive index detector (Santa
Barbara, CA). The MSP142-FVO self-associated aggregates or
MSP142-FUP-EPA conjugate were analyzed on a G4000SWxl size
exclusion column (Tosoh Biosciences, Montgomeryville, PA)
equilibrated with the following solution: 1.04 mM KH2PO4,
2.97 mM Na2HPO4, 308 mM NaCl, and 0.02% azide, pH 7.4 at
a flow rate of 0.5 mL/min.
F. Endotoxin level measurement.
various antigens were measured using Limulus amoebocyte lysate in
a 96-well plate with chromogenic reagents and PyroSoft software
(Associates of Cape Cod Inc., East Falmouth, MA) before
administration. The endotoxin values were all less than 41 EU/
mg of recombinant MSP142.
Recombinant EPA was charac-
Endotoxin levels of the
Animal Studies and Serological Assays
Antigens were formulated on 1600 mg/mL Alhydrogel (Brenn-
tag Biosector, Denmark) with or without the addition of 20 mg/
dose CPG 7909 (Coley Pharmaceutical Group, Wellesley, MA).
For the formulation containing CPG 7909, the antigens were first
formulated on Alhydrogel followed by the addition of CPG 7909.
The adsorption of the antigens or CPG 7909 to Alhydrogel was
examined by silver stained SDS-PAGE [32,33]. BALB/c mice
(Charles River Laboratories, Frederick, MD) were used and
randomly assigned into experimental groups each containing 10
mice. The mice were immunized intramuscularly two times on
days 0 and 28 with the delivery volume of 50 mL for each
immunization. Mouse sera were collected two weeks after the
second immunization (on day 42 in the first study) or two, four and
six weeks (on days 42, 56 and 70 in the second study) after the
second immunization for assessment of antigen specific antibody
titers by ELISA (see below).
Two independent mouse studies were carried out in compliance
with the National Institutes of Health guidelines and an animal
care and use committee-approved protocol. In the first study, the
antibody levels induced by the Alhydrogel formulations of
MSP142-FVO dimer and MSP142-FVO aggregate were compared
at the doses of 1, 3 and 10 mg. In the second study, the antibody
levels induced by the Alhydrogel formulations or Alhydrogel plus
CPG 7909 formulations of MSP142-FUP monomer and two types
of MSP142-FUP-EPA conjugates were compared at the doses of 5
and 15 mg.
Enzyme-linked immunosorbent assay (ELISA) was performed
on each individual mouse serum to measure the antibody titer
following a standardized protocol [32,34]. A Mann-Whitney U
Test was performed in the first mouse study to test for significant
differences of antibody titers between the two groups receiving
different kinds of antigens at each dose level. If the P value was less
than 0.05, the differences were considered significant. In the
second study, Kruskal-Wallis One-Way ANOVA was performed
among the groups at each dose level on any of three sera collection
days. If the P value was less than 0.025, a post hoc analysis of
Student-Newman-Keuls was performed. If the P value of Student-
Newman-Keuls was less than 0.05, the differences were considered
significant. For the IgG subclass analysis, ELISA was performed
on the pooled sera of each group, which were pooled based on the
equivalent ELISA unit of each individual serum [32,35].
Preparation and Characterization of MSP142-FVO Self-
associated Aggregate and MSP142-FUP-EPA Conjugates
A MSP142–FVO self-associated aggregate was produced while
developing a modified purification process that aimed to replace
usage of a Source 30 pilot-scale reversed phase column with a
hydrophobic interaction column in order to avoid the use of
organic solutions during pilot-scale manufacturing. The molar
mass of the MSP142-FVO aggregate in an aqueous solution was
determined by SEC-MALS-HPLC to be approximately 1.7 MDa
for 96% of the total peak area as compared to a purified form
following a standard procedure, which yielded 72% dimers and
28% multimers with a molar mass of approximately 80–100 kDa
and 0.700–1MDa, respectively (Figure 1A). Analysis of the same
protein lots by Coomassie blue stained SDS-PAGE analysis under
reduced and non-reduced conditions showed that a predominant
band at approximately 42 kDa was observed, which is consistent
with the expected mass of 42,173 Da. Based on protein mobility in
the presence of SDS, the solution state of the self-associated
aggregate appeared to be primarily due to hydrophobic or ionic
interactions, and not due to disulfide bond formation between
different forms of MSP142-FVO (Fig. 1B).
To determine the best conditions for linker addition, a Surface
Response Mode (RSM) was used to pinpoint the maximum or
minimum condition for three factors considered key for process
development: temperature, pH and linker concentration. To
evaluate these variables together, a Box-Behnken’s RSM was used
to determine the optimum condition for the maleimide modifica-
tion reaction based on the following conditions: temperature (22–
26uC), Sulfo-EMC concentration (1.0–4.0 mM) and reaction time
(30–90 minutes). All three factors were statistically significant,
p,0.05, and played an important role in the number of
modifications observed based on the Ellman’s Reaction. However,
due to process development constraints, and tolerability of
temperature and pH to marked linker substitution, the following
conditions were used: reaction temperature 22uC, linker concen-
tration of 2 mM, and reaction time 60 min (Figure 2A). To test the
validity of the Box-Behnken’s RSM, the residual plot was
investigated. Figure 2B shows the Ellman’s reaction was randomly
dispersed around the horizontal axis with no trend, which is
consistent of a linear regression model. Furthermore, the fitted
model accounted for (R-squared) 96% of the variation in Ellman’s
reaction. We were interested in only main factors in the RSM and
did not account for squared terms, interactions or synergistic
effects. Further process optimization, using the Box-Behnken
Immunogenicity of Recombinant MSP142Conjugates
PLoS ONE | www.plosone.org3 June 2012 | Volume 7 | Issue 6 | e36996
RSM, could address higher order terms, and surface response
Batches of chemical cross-linked MSP142-FUP-EPA conjugates
were prepared using two different maleimide cross-linkers Sulfo-
EMCS and NHS-PEO2-maleimide. The composition of each
linker indicates that the solubility properties may be different, even
though this did not appear to impact the solubility of the
conjugates (data not shown). Recombinant EPAAPAand EPAPEO,
modified by Sulfo-EMCS and NHS-PEO2-maleimide, respective-
ly, were prepared and the addition of the bifunctional linkers was
monitored by RP-HPLC analysis. The retention times as well as
the peak shape of the modified EPA shifted with the addition of
the maleimide groups onto the protein (Figure 3A). The extent of
the shift in retention time appeared dependent on the number and
physical properties of the chemical modifiers (Figure 3A and data
not shown). The number of maleimide groups added on EPA was
assessed by titrating with cysteine and measuring the absorbance
at 405 nm (Figure 3B). The titration curves of two batches of
EPAAPAand one batch of EPAPEOwere similar (Figure 3B). Based
on the titration curves and the standard curve of cysteine, the
number of maleimide groups added onto the EPA was calculated
to be 5.0 and 4.7 for the two batches of EPAAPAand 5.0 for the
The EPAAPA and EPAPEO carriers were conjugated with
MSP142-FUP and the resultant conjugates were purified by
preparative SEC. The SEC elution fractions were analyzed by
Coomassie blue stained SDS-PAGE and relevant fractions were
pooled (Figure 4A). Analysis of the mobility of the conjugates by
Coomassie blue stained SDS-PAGE indicated that the conjuga-
tion products were chemically cross-linked complex protein-
protein mixtures with ratios ranging from 1:1 MSP142-FUP per
carrier to approximately 6:1. The predominant conjugated forms
Figure 1. SEC-HPLC-MALS and Coomassie blue stained SDS-PAGE gel analysis of aggregated MSP142-FVO. (A) Size exclusion
chromatography with multi-angle light scattering and (B) SDS-PAGE analysis run on a 4–20% gradient Tris–glycine polyacrylamide gel under non-
reducing (NR) and reducing (R) conditions.
Figure 2. Multi-parameter analysis of linker substitution. Response curves for the analysis of temperature, modifier concentration and time
on the linker substitution of EPA using a Box-Behnken model (A). Residual plot of the Ellmans’ reaction showing there is no systematic pattern (B). A
linear regression model was used for analysis given the following assumptions: relationships between dependent and independent variables are
linear, no serial correlation, the response variables are normally distributed, and have the same variance.
Immunogenicity of Recombinant MSP142Conjugates
PLoS ONE | www.plosone.org4 June 2012 | Volume 7 | Issue 6 | e36996
appeared at a ratio of 3:1 and 4:1 (see Figure 4A asterisks). A
minor quantity of unreacted MSP142-FUP was observed in the
SEC pool. The average conjugation ratio for MSP142-FUP-
EPAAPAand MSP142-FUP-EPAPEOby amino acid analysis was
3.9 and 3.8, respectively. Thus the results obtained by mobility
on SDS-PAGE and amino acid analysis were consistent for the
ratio of MSP142-FUP and EPA by mass ratios and molar ratios.
Considering that the unreacted MSP142-FUP was not completely
removed from either conjugate, the average conjugation ratios
should be slightly lower than the reported values. Analysis by
SEC- MALS-HPLC showed the presence of three populations or
peaks for each conjugate with the second peak representing the
major conjugated form with the major peak consisting of 80% of
total protein for the MSP142-FUP-EPAAPAand 81% of the total
protein for the MSP142-FUP-EPAPEO(Figure 4B). The weighted
average masses of the MSP142-FUP-EPAAPAwere 1.1 6107, 1.6
6 106and 1.4 6 105Da, respectively, whereas the weighted
average masses for the MSP142-FUP-EPAPEO of three peaks
Figure 3. Characterization of chemically modified EPA. Analysis of un-modified EPA, EPAAPAbatch 1, EPAAPAbatch 2 and EPAPEOby RP-HPLC
(A), and titration analysis of EPAAPAbatch 1, EPAAPAbatch 2 and EPAPEO(B) are shown.
Figure 4. Characterization of MSP142-FUP-EPA conjugates. Panel (A) Coomassie blue stained SDS-PAGE gel analysis of maleimide-EPA (lanes 1
and 5); monomeric MSP142-FUP (lanes 2 and 6), un-purified conjugation mixture of MSP142-FUP-EPAAPA(lane 3), bulk purified MSP142-FUP-EPAAPA
(lane 4); un-purified conjugation mixture of MSP142-FUP-EPAPEO(lane 7) and bulk purified MSP142-FUP-EPAPEO(lane 8). The asterisks indicate the
conjugates with 3:1 and 4:1 ratio. Panel (B) SEC-HPLC-MALS analysis. Solid and dashed lines represent absorbance at 280 nm and molecular mass for
MSP142-FUP-EPAPEOand MSP142-FUP-EPAAPA, respectively. The capital letters A, B and C indicate the profile peaks of each conjugate.
Immunogenicity of Recombinant MSP142Conjugates
PLoS ONE | www.plosone.org5 June 2012 | Volume 7 | Issue 6 | e36996
were 1.16107, 1.36106and 1.16105Da (Figure 4B, A–C,
respectively). Since the predominant weighted average mass for
each conjugate by SEC-MALS-HPLC was greater than the
ratios determined by Coomassie blue stained SDS-PAGE, this
indicates that a non-covalent association exists in the presence of
the 0.5 M arginine, which was used to stabilize the solubility of
the conjugates. The conjugates in this form were stable to freeze-
thaw by analytical SEC-MALS-HPLC and were filterable
through a 0.22 mm filter (data not shown). Two conformation-
dependent monoclonal antibodies, AD223 and 13E3-53 ,
were used to assess the structural integrity of the conjugated
MSP142-FUP proteins by Western blot analysis. Each protein
band observed by Coomassie blue stained SDS-PAGE appeared
to be recognized by both monoclonal antibodies (data not
shown), demonstrating that the conformational structure of the
19 kDa fragment of MSP142-FUP remained intact during the
process of conjugation.
Assessment of Immunogenicity of Aggregated,
Chemically Conjugated or Dimeric Recombinant MSP142
To evaluate whether the MSP142-FVO self-associated aggregate
or MSP142-FUP-EPA conjugates could enhance antigen specific
antibody responses compared to the predominately monomeric or
dimeric forms, two mouse studies were performed with the
immunogens formulated on Alhydrogel, and in the case of the
MSP142-FUP-EPA conjugate with or without CPG 7909. The
antibody titers of each mouse serum were measured by ELISA. In
the first study, the antibody titers induced by aggregated or
dimeric MSP142-FVO formulated on Alhydrogel were compared
at the doses of 1, 3 and 10 mg. No significant differences were
observed at any of these dose levels although there is a trend of a
higher response for the aggregated form of MSP142-FVO
compared to the dimer (Table 1). In the second study, the
antibody titers induced by the two different MSP142-FUP-EPA
conjugates and unconjugated MSP142-FUP formulated on Alhy-
drogel with or without CPG 7909 were compared at the doses of 5
and 15 mg. When the comparison was performed between the
conjugated and unconjugated immunogens, again no significant
differences were observed at these dose levels on any of three
serum collection days (Table 2). However, at the 5 mg dose, the
response when formulated without CPG 7909 and the MSP142-
FUP monomer consistently had a higher response when formu-
lated with CPG 7909. When the comparison was performed
between formulation with and without CPG 7909, significant
differences were observed. Antibody levels induced by the
formulations with CPG 7909 were significantly higher than those
induced by the formulations without CPG 7909 at both doses of 5
and 15 mg on all three serum collection days (Table 2). On day 42,
the differences in antibody titers reached 31, 83-fold higher at the
dose of 5 mg and 13, 18-fold higher at the dose of 15 mg.
IgG subclass analysis was performed on the pooled sera of the
mouse groups to characterize the type of antibody responses
induced by the formulations with and without CPG 7909. The
Alhydrogel formulations of both conjugated and unconjugated
MSP142-FUP without CPG 7909 predominately induced an IgG1
response (data not shown). With the addition of CPG 7909 to the
Alhydrogel formulations, an IgG2a subclass level was greatly
increased. Both IgG1 and IgG2a were the predominant IgG
subclasses in these immune sera. The IgG1 to IgG2a ratios from
consistently had a higher
Table 1. Anti-MSP142-FVO antibody titers in mice.
Anti-MSP142-FVO unitsa(Geometric mean ± SEM)
Dose 1 mg Dose 3 mg Dose 10 mg
MSP142-FVO dimer 71562890 629764940 2366765022
MSP142-FVO aggregate 244464950 4973683575793667457
aThe antibody titers were compared at any of three doses levels with Mann-Whitney U Test. No significant differences were presented as the P values were all more than
Table 2. Anti-MSP142-FUP antibody titers in mice.
Anti-MSP142-FUP units (Geometric mean ± SEM)a
Dose 5 mg Dose 15 mg
Day 42Day 56 Day 70 Day 42Day 56 Day 70
MSP142-FUP monomer130962109 157062538 124161882 758864477949364050 836964306
177062701210263281182762369568562116 562762630 427562335
100961716128861665 106262023806463029774463732 488161692
MSP142-FUP monomer CPG 7909 109075621780892776200084423764929 107850613162 1184016139385279465243
MSP142-FUP-EPAAPACPG 79095432761065045206695322134165183 10391961729259705688293304264018
MSP142-FUP-EPAPEOCPG 790973995615475333636107012474266113 108278615234757676117893476663467
aThe antibody titers were compared at two dose levels on any of three serum collection days with Kruskal-Wallis One-Way ANOVA. As the P values were all less than
0.025, the post hoc analysis of Student-Newman-Keuls was performed. The differences between the groups of conjugated and unconjugated immunogens were not
significant (MSP142-FUP-EPAAPAor MSP142-FUP-EPAPEOvs. MSP142-FUP monomer and MSP142-FUP-EPAAPACPG 7909 or MSP142-FUP-EPAPEOCPG 7909 vs. MSP142-FUP
monomer CPG 7909) (P.0.05), whereas the differences between the groups with and without CPG 7909 were significant (MSP142-FUP monomer CPG 7909 vs. MSP142-
FUP monomer, MSP142-FUP-EPAAPACPG 7909 vs. MSP142-FUP-EPAAPAand MSP142-FUP-EPAPEO.
Immunogenicity of Recombinant MSP142Conjugates
PLoS ONE | www.plosone.org6 June 2012 | Volume 7 | Issue 6 | e36996
day 42 to day 70 were different between the groups of conjugated
and unconjugated MSP142-FUP. The ratios decreased in the sera
elicited by the conjugated MSP142-FUP, indicating that in those
sera the IgG1 waned more quickly than the IgG2a (Table 3).
An effective malaria vaccine is urgently needed to augment
existing control measures for individuals living in malarial endemic
areas. Unfortunately, several investigative malaria vaccines tested
in phase 1 or 2 trials have not induced antibody levels that have
warranted further development [19,36,37,38]. Self-assembled
virus like particles  including the leading malaria vaccine
RTS,S that contains the circumsporozoite protein fused with the
hepatitis B surface antigen are capable of inducing protective
responses in humans. RTS,S has protected about 50% of
vaccinees for a duration of 12–18 months in Phase 2 trials
[40,41], and in a recent Phase 3 trial . In preclinical studies, a
non-human primate immunogenicity study demonstrated that
recombinant Pfs25H protein, a mimic of the sexual stage specific
protein Pfs25, conjugated to the outer membrane complex of N.
meningitidis increased antibody titers to a greater degree than their
monomeric forms and increased the apparent duration of the
antigen specific antibodies . Similar observations of an increase
in antibody titers have been made when recombinant Pfs25H was
conjugated to a different carrier protein i.e., EPA or to itself [6,7].
Unfortunately, based on the results reported here, whether
recombinant MSP142was presented as a uniform non-covalently
associated aggregate or chemically cross-linked to EPA no
significant increase in antibody responses were observed in mice
(Tables 1 and 2). The basis for the differences in antibody
responses observed for recombinant Pfs25 conjugates and those
described here is unclear. Both Pfs25 and MSP142native proteins
contain 4 or 2 epidermal growth factor like domains, respectively
[10,43]. Native Pfs25 is believed to be deficient of T cell epitopes,
while MSP142contains the 33 kDa fragment which is believed to
already provide T cell help. The presence of the MSP133fragment
may negate the benefit of chemical cross-linking to EPA.
CpG ODN has been demonstrated to be an effective adjuvant
for vaccines against a variety of pathogens including malarial
antigens [20,32,35,44,45,46,47,48]. When CpG ODN was added
to an aluminum hydroxide formulation, the combination of both
adjuvants altered the subclass profile of the IgG response from a
predominant IgG1 (Th2) to a more balanced IgG1 and IgG2a,
reflecting both Th1 and Th2 patterns of immune responses and
leading to an increased total antibody level [32,35]. Moreover, the
protective effect of the antibodies against the challenge of malaria
parasites in mice was improved by the addition of CpG ODN to
the Montanide formulation of P. yoelii MSP119. While the
aggregated and conjugated MSP142failed to enhance the antibody
responses, the addition of CPG7909 to the Alhydrogel formulation
of the MSP142significantly enhanced the specific antibody levels
in mice with balanced IgG subclasses, which is consistent with the
result observed in our previous phase 1 human trial .
In summary, neither recombinant MSP142 self-associated
aggregates nor chemical cross-linked conjugates enhanced the
immunogenicity of the MSP142 compared to monomeric or
oligomeric forms of the antigen. The addition of CPG 7909 into
the Alhydrogel formulation of the MSP142-EPA conjugates
significantly enhanced the specific antibody levels in mice as
compared to the formulation using Alhydrogel as a single
adjuvant. Even though the MSP142-EPA conjugates failed to
enhance MSP142 immunogenicity, the conjugation procedure
including the use of design of experiments may be used as a
platform for development of other protein-protein conjugates
through chemical modification of both antigen and carrier, or use
of innate or genetically engineered free sulfhydryl groups for
protein-protein coupling. Further investigation is required to
understand the benefit of this protein-protein conjugation strategy
for the development of investigational malaria vaccines or other
We appreciate the support provided by the LMIV core vaccine
Conceived and designed the experiments: FQ GEDM LBM CAL LHM
DLN. Performed the experiments: FQ KR YZ RLS JAA KMR LL.
Analyzed the data: FQ KR VN DZ DLN. Contributed reagents/
materials/analysis tools: VN DZ. Wrote the paper: FQ DLN.
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0.880.770.69 0.70 0.580.51
0.73 0.710.50 0.710.62 0.57
Immunogenicity of Recombinant MSP142Conjugates
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Immunogenicity of Recombinant MSP142Conjugates
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