A Method for Producing Protein Nanoparticles with Applications in Vaccines
A Method for Producing Protein Nanoparticles with Applications in Vaccines
David S. Jones 0 1
Christopher G. Rowe 0 1
Beth Chen 0 1
Karine Reiter 0 1
Kelly M. Rausch 0 1
David L. Narum 0 1
Yimin Wu 0 1
Patrick E. Duffy 0 1
0 Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Disease, National Institutes of Health , Rockville, Maryland, 20852 , United States of America
1 Editor: Osamu Kaneko, Institute of Tropical Medicine, Nagasaki University , JAPAN
A practical method is described for synthesizing conjugated protein nanoparticles using thioether (thiol-maleimide) cross-linking chemistry. This method fills the need for a reliable and reproducible synthesis of protein conjugate vaccines for preclinical studies, which can be adapted to produce comparable material for clinical studies. The described method appears to be generally applicable to the production of nanoparticles from a variety of soluble proteins having different structural features. Examples presented include single-component particles of the malarial antigens AMA1, CSP and Pfs25, and two component particles comprised of those antigens covalently cross-linked with the immunogenic carrier protein EPA (a detoxified form of exotoxin A from Pseudomonas aeruginosa). The average molar masses (Mw) of particles in the different preparations ranged from 487 kDa to 3,420 kDa, with hydrodynamic radii (Rh) ranging from 12.1 nm to 38.3 nm. The antigenic properties and secondary structures of the proteins within the particles appear to be largely intact, with no significant changes seen in their far UV circular dichroism spectra, or in their ability to bind conformation-dependent monoclonal antibodies. Mice vaccinated with mixed particles of Pfs25 or CSP and EPA generated significantly greater antigen-specific antibody levels compared with mice vaccinated with the respective unmodified monomeric antigens, validating the potential of antigen-EPA nanoparticles as vaccines.
Data Availability Statement: Data are all contained
within the paper.
Funding: This work was supported by the Division of
Intramural Research (DIR) of NIAID, NIH. A portion
was funded by the Malaria Vaccine Initiative (MVI).
Competing Interests: The authors have declared
that no competing interests exist.
In the course of developing conjugates of plasmodial proteins as vaccines for malaria, an
efficient and scalable method was developed for producing protein nanoparticles comprised of
antigen alone or antigen combined with an immunogenic carrier protein (carrier).
Assembly of antigens into particles to improve their immunogenicity is an often used
strategy in modern vaccine development. Nanoparticles have found applications throughout
biomedicine, and vaccines in particular have benefited from structural features and other
properties that can be incorporated into nanoparticles [
]. The most advanced malaria vaccine
to date is a virus-like particle containing a single copy of a portion of the circumsporozoite
protein (CSP) fused to a single hepatitis B surface protein molecule and mixed in a ratio of 1:4
with unfused hepatitis B surface protein molecule [
]. Self-assembled peptide nanoparticles
have been shown to improve immune responses of peptide antigens [
]. The application of
particle-based technologies toward vaccines has been reviewed [
Conjugation of antigens to protein carriers is another widely used strategy for improving
vaccine potency. Polysaccharide conjugates in particular have contributed greatly to numerous
effective childhood vaccines [
]. Poorly immunogenic peptides and proteins can also
become better immunogens when conjugated to protein carriers [
]. Conjugates of
recombinant subunit proteins found at various stages of the malaria parasite lifecycle are being actively
investigated as vaccines. Recombinant blood stage proteins AMA1 and MSP1 have been
conjugated with Exoprotein A (EPA), a detoxified form of exotoxin A from Pseudomonas aeruginosa
]. Proteins expressed in the mosquito stage (Pfs25 and Pfs28) are being investigated as
vaccines for blocking malaria transmission. Conjugates of Pfs25 with EPA, OMPC (outer
membrane protein complex) or with itself have been shown to be more immunogenic than the
unconjugated forms [
]. Conjugation of Pfs28 to EPA also improved immunogenicity
]. Various conjugated forms of CSP, expressed in the pre-erythrocytic stage of the parasite
lifecycle, have been reported [
A significant impediment to developing protein conjugate vaccines has been poor yield and
lack of reproducibility. Consequently, protein conjugate vaccines produced for early-stage
preclinical testing have been difficult to reproduce in the quantities needed for later stages. An
efficient process was needed for preparing characterizedconjugates for pre-clinical studies, which
could be adapted to scale-up studies leading to the production of clinical grade material in
conformance with current good manufacturing practices (cGMP), if warranted. Toward that end a
process was developed for producing protein conjugates by cross-linking antigen and carrier to
form conjugated protein nanoparticles of suitable size for complete biochemical and
biophysical characterization and sterile filtration. This paper describes a practical synthetic method for
producing soluble protein nanoparticles composed of one or two proteins. Examples include
recombinant malarial antigens Pfs25, CSP and AMA1 with or without inclusion of EPA as a
2. Materials and Methods
2.1 Recombinant Proteins and Monoclonal Antibodies
AMA1 from the P. falciparum FVO malaria parasite clone (molecular weight, 61,906 Da) was
expressed in Pichia pastoris [
]. EPA (molecular weight, 66,975 Da) was expressed in E. coli
]. Pfs25H from the P. falciparum NF54 isolate (molecular weight 20,438 Da) was expressed
in P. pastoris [
]. Recombinant Pfs25M from the P. falciparum NF54 isolate without a His6
fusion tag (molecular weight 18,712) was expressed in P. pastoris and characterized in a
manner similar to Pfs25H. The P. falciparum 3D7 CSP clone, CSPM3 (molecular weight 32,578),
was expressed in P. pastoris and characterized as previously described [
]. P. falciparum
specific monoclonal antibodies against AMA1, identified as 4G2, and against CSPM3, identified as
1G12, that inhibit parasite development and recognize conformation-dependent epitopes have
been previously reported [
2.2 Reagents and Buffers
N-(ε-maleimidocaproyloxy)succinimide (EMCS) (PubChem CID: 5091655),
N-(ε-maleimidocaproyloxy)sulfo-succinimide sodium salt (sulfo-EMCS) (PubChem CID: 4229287), and
Sacetylthioglycolic acid N-hydroxysuccinimidyl ester (SATA) (PubChem CID: 127532), were
purchased from Pierce Biotechnology Inc. (Rockford, IL).
Buffers used are as follows: pH 6.5 PBSE (100 mM sodium phosphate, 150 mM NaCl, 5 mM
EDTA); pH 7.2 PBSE (100 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA); deacetylation
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buffer (0.5 M NH2OH.HCl, pH 7.2 PBSE); and PBS (1.04 mM KH2PO4, 2.97 mM
Na2HPO4.7H2O, 154 mM NaCl, pH 7.4).
Protein solutions were concentrated and buffer exchanges were accomplished using centrifugal
membrane filtration devices with a 10 kDa nominal molecular weight cutoff (catalogue item,
Amicon Ultra-15 PLGC Ultracel-PL Membrane, 10 kDa) manufactured by Millipore
Corporation (Billerica, MA). Buffer exchanges were accomplished with multiple dilutions and
concentrations using the buffer of choice to achieve a 1000 fold exchange (e.g., three iterations of a
tenfold dilution step followed by tenfold concentration step). Protein concentrations were
calculated from UV absorbance at 280 nm using extinction coefficients derived from amino acid
composition (AMA1, 1.206 mL mg-1; CSPM3, 0.268 mL mg-1; EPA, 1.299 mL mg-1; Pfs25H,
0.315 mL mg-1; Pfs25M, 0.312 mL mg-1) [
]. The extinction coefficients of
maleimide-modified proteins were adjusted upwards by 515 L mol-1 per attached maleimide group. CD spectra
were acquired using a J-815 CD Spectrometer (Jasco Analytical Instruments, Easton, MD).
Reaction yields are reported as percentage of total protein recovered.
2.4 Particle Synthesis
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ml/min using PBS as the eluent. Fractions containing particles were pooled to provide 1.90 ml
of Pfs25M-EPA solution: concentration, 3.14 mg/ml; yield, 5.99 mg (64%).
2.5 Determination of linker stoichiometry
2.5.1 Determination of thiol linkers. Thiol concentrations were determined by
incubating the thiolated protein with 4,4’-dithiodipyridine (DTDP) at pH 6.5 and measuring
absorbance at 324 nm [
]. The concentration of thiol groups was calculated from the absorbance
using an extinction coefficient of 21,400 L M-1 for the 4-thiopyridone product, and the result
was validated by comparing with cysteine standards included in the assay.
2.5.2 Determination of maleimide linkers. Maleimide concentrations were determined
by measuring consumption of thiol groups when maleimide-modified proteins were mixed
with cysteine. Aliquots of the maleimide-modified protein, or buffer alone, were added to
standard cysteine solutions, and the mixtures were incubated for 1 hour at room temperature.
Thiol concentrations were measured as described above using DTDP. Maleimide
concentration was calculated as the difference between the thiol concentration of the cysteine solution
plus buffer and the cysteine solution plus maleimide-modified protein.
2.6 Determination of molar mass and hydrodynamic radius
The average molar mass (Mw) and hydrodynamic radius (Rh) of the conjugates were
determined by size exclusion chromatography coupled with multi-angle light scattering
(SEC-MALS). A Dawn Helios 18 angle light scattering detector fitted with quasi elastic light
scattering (QELS) (Wyatt Technologies, Santa Barbara, CA) was used to collect light scattering
signals, and an OptilabRx refractive index detector (Wyatt) was used to simultaneously
measure concentration. The signals from both detectors were processed using Astra software
(Wyatt) to determine Mw and Rh. The size exclusion columns used were G4000PWxl and
G5000PWxl (Tosoh Bioscience, King of Prussia, PA).
2.7 Determination of protein composition
Amino acid analyses were performed at the W.M. Keck Biotechnology Resource Lab at the
Yale School of Medicine (New Haven, CT). The molar amino acid compositions were
determined for each EPA-containing nanoparticle and for the individual unmodified proteins. The
molar ratios of the two proteins comprising the nanoparticles were calculated as previously
2.8 Vaccinations of mice
Animal protocols were carried out in compliance with National Institutes of Health guidelines
and under the auspices of Animal Care and Use committee approved protocols. For Pfs25H,
CSPM3 and particles containing Pfs25H, Pfs25M and CSPM3, female CD-1 mice were
vaccinated on days 0 and 28 by intramuscular injection of 0.05 mL of vaccine formulation
containing the doses indicated adsorbed on Alhydrogel, and the animals were bled on day 42. For
AMA1 and particles containing AMA1, female mice were vaccinated on days 0, 14 and 28 by
subcutaneous injection of 0.10 mL of vaccine formulation containing the doses indicated
adsorbed on Alhydrogel, and the animals were bled on day 42. The percentage of protein
bound to Alhydrogel in each formulation was determined to be greater than 95% (100%—free
protein). Supernatants were examined by polyacrylamide gel electrophoresis with visualization
by silver staining. In each case the amount of free protein was below the limit of detection (5%
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Animal studies were performed following the guidelines approved by the National Institutes
of Health (NIH) Animal Care and Use Committee (ACUC) and approved by the National
Institutes of Health Animal Care and Use committee, according to the Institutional Animal
Care and Use Committee (IACUC) approved protocol. The National Institutes of Allergy and
Infectious Disease (NIAID), Division of Intramural Research (DIR) Animal Care and Use
Program (ACUC), as part of the NIH Intramural Research Program (IRP), complies with all
applicable provisions of the Animal Welfare Act and other Federal statutes and regulations relating
to animals. The Program acknowledges and accepts responsibility for the care and use of
animals involved in activities covered by the NIH IRP’s PHS Assurance #A4149-01.
2.9 Measurement of antibody levels
Antigen-specific antibody levels were determined using standardized ELISA[
un-modified antigens as the plate antigens [
]. Absorbance based ELISA units (EU) were determined
relative to a reference antisera obtained from mice immunized with the un-modified antigen.
Statistical significance for differences between groups was tested using a Mann-Whitney test,
performed with Prism software (GraphPad Software, Inc., La Jolla, CA).
2.10 In vitro parasite growth inhibition assay
Groups of five female New Zealand rabbits were vaccinated intramuscularly with 0.05 mL of
formulation containing 25 μg (AMA1 content) of conjugate or monomeric AMA1 adsorbed
on 800 μg of Alhydrogel. Vaccinations occurred on days 0 and 56. The animals were bled on
day 70. IgG was purified from individual rabbits that had been vaccinated with conjugate
AMA1-AMA1 or monomeric AMA1, and the sera were tested in a standardized parasite
growth inhibition assay [
3. Results and Discussion
3.1 Preparation of protein nanoparticles
The method we describe in this communication was developed as a way to overcome
shortcomings that were identified in the preparation of malarial antigen conjugates. Conjugates with the
immunogenic carrier protein EPA, prepared using thioether chemistry, had been shown to be
significantly more immunogenic than un-modified antigens, thus establishing conjugation as an
attractive malaria vaccine strategy [
]. The antigens had been thiolated using
N-acetylhomocysteine thiolactone as the thiolating reagent  and subsequently reacted with
maleimidemodified EPA to form conjugates. We found this method of production to suffer from low yield
and variable product composition, with significant amounts of unmodified antigen remaining.
These shortcomings were attributed to low and variable degrees of thiolation.
Our approach to improving the conjugation process was to increase the degree of antigen
thiolation, which we surmised would drive the conjugation reaction toward higher molecular weight
cross-linked products and thereby minimize the amount of un-conjugated protein. We used SATA
as the thiolating reagent because it reacts very efficiently with lysine amino groups on proteins
under mild conditions [
], and the degree of thiolation is easily controlled as a function of the
concentration of SATA. Maleimide-modification of proteins was accomplished using EMCS as
previously described [
]. SATA and EMCS are both commercially available linkers, provided as
activated esters that react to form covalent amide bonds with lysine amines. After reaction with
SATA, the acetyl groups are removed from the linkers using a hydroxylamine-containing buffer to
generate free thiols. Fig 1 shows the stepwise process of attaching thiol and maleimide linkers to
proteins and subsequent cross-linking of the two modified proteins to form particles.
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Fig 1. Multiple thiol or maleimide linkers are non-specifically attached to lysine amines on the protein
molecules by treatment with excess SATA or EMCS. Particles composed of A and B are formed by mixing
thiolated protein with maleimide-modified protein.
Numerous test reactions were carried out to elucidate appropriate reaction parameters (data
not shown). This practice is highly recommended, particularly when protein quantities are
limited. The necessary concentrations of SATA or EMCS can be conveniently determined using
small-scale pilot reactions with approximately 1 mg of protein. Conditions for forming
particles can be determined using approximately 500 μg of protein. Protein concentration and
linker stoichiometry, the average number of linkers on the proteins, were found to be key
determinants of particle size (Mw and Rh). In general 3–4 linkers on each protein were found to be
sufficient for optimal reaction. The relative number of linkers on each protein component also
proved to be important. Larger particle sizes and higher yields were favored when the molar
proportions of thiol and maleimide linkers were roughly equivalent; therefore, particles
comprised of proteins with significantly different molecular weights were best prepared when the
larger protein contained proportionally more linkers than the smaller one. Reactions in which
thiolated protein comprised 40–55% of the protein mass appeared to result in optimal
crosslinking (higher Mw and yield). The mass composition of particles comprised of two different
proteins is similar to mass composition of the proteins used in the reaction.
The thiolation of Pfs25M was described in detail in the methods section. All the thiolation
reactions were performed identically except the molar excess of SATA. After reaction with
SATA, the mixtures were exchanged into fresh buffer to remove reagents and byproducts.
Deacetylation was accomplished by treating with a hydroxylamine containing buffer. The solution
was then exchanged into pH 6.5 buffer, and the number of attached thiols was determined. The
conditions and results of thiolation reactions are shown in Table 1.
a Reactions were performed as described in detail for thiolation of Pfs25M, with the exception of the
number of moles of SATA used per mole of protein.
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a Reactions were performed as described in detail for maleimide-modification of EPA, with the exception of the number of moles of EMCS used per mole
of protein and the concentration of protein.
b Sulfo-EMCS dissolved in water was used instead of EMCS.
Maleimide modifications were accomplished using EMCS as described in detail in the
methods section for the maleimide modification of EPA. All the maleimide modifications were
performed identically except the molar excess of EMCS and the protein concentrations. After
reacting the protein with EMCS, the product was exchanged in pH 6.5 buffer to remove
reagents and byproducts, and the number of attached maleimides was determined. The
conditions and results of maleimide modification reactions are shown in Table 2.
Particles were formed by mixing the thiolated and maleimide-modified proteins at a
predetermined final concentration of both proteins in pH 6.5 buffer. After one hour, excess cysteine
was added to quench possible remaining maleimide groups. A detailed description of the
formation of Pfs25M-EPA particles is presented in the Methods Section. The procedures used to
prepare the other particles were very similar, with differences in the number of attached thiol
and maleimide linkers, the molar proportions of the modified proteins, and the total protein
concentration in the reaction. Those parameters and the overall yields of protein in the purified
particle preparations are listed in Table 3.
a Reactions were performed as described in detail for Pfs25M-EPA with the exception of the concentration of protein in the reaction, number of linkers on
the proteins, and the relative masses of linker-modified proteins used. Thiolated antigens were reacted with antigen or EPA modified with maleimides.
b Total concentration of protein.
c Two pools were collected separately from one reaction (23% yield of pool with Mw of 3,420 kDa, 11% yield of pool with Mw of 909 kDa).
d Overall yield of protein, including modifications of proteins with linkers.
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Fig 2. Absorbance profile (280 nm) of fractionation of Pfs25M-EPA across a 16 mm X 60 mm column
pack with Superdex 200 matrix. The shaded area represents the fractions that were pooled.
Purification of particles was accomplished using preparative size exclusion chromatography.
The majority of UV absorbing product was collected, excluding monomeric proteins and low
Mw conjugates. Decisions on what fractions to pool were made based on polyacrylamide gel
electrophoresis of fractions on the low molecular weight end of the peak. The 280 nm
absorbance profile of the fractionation of Pfs25M-EPA particles is shown in Fig 2. Fig 3 shows
analytical SEC run on the particles before and after the fractionation process. In contrast to a
standard SEC chromatographic peak of greater than 95% purity and good symmetry for
example, it is accepted that asymmetrical peaks may be observed.
Fig 3. Absorbance profile (280 nm) of analytical HPLC of Pfs25M-EPA using a G5000PWxl column (Toso
Biosciences); A) before purification and B) after purification. The Y axis represents detector voltage.
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a Moles of antigen per mole of EPA was determined from amino acid analysis.
b Mw was calculated from multi-angle light scattering data.
c Average hydrodynamic radius was calculated from dynamic light scattering data.
d Two separate fractions were collected.
810 ± 40
487 ± 24
1,584 ± 80
1,740 ± 80
1,331 ± 65
711 ± 35
3,420 ± 150909 ± 50
3.2 Characterization of nanoparticles
Particles were characterized with respect to size and composition. The physical parameters of
average molar mass (Mw) and hydrodynamic radius (Rh) were determined from size exclusion
chromatography using static and dynamic light scattering detectors. The molar composition of
twocomponent EPA-containing particles was determined from amino acid analysis data. A summary
of the particle characterizations is reported in Table 4. The larger particle size seen for CSPM3
particles is consistent with the elongated or rod-like shape reported for CSP constructs [
Circular dichroism spectroscopy (CD) and binding to monoclonal antibodies were used to
qualitatively compare the antigens before and after incorporation into particles. Fig 4 shows far
UV optical ellipticity profiles of AMA1 vs. AMA1-AMA1 particles, CSPM3 vs.
CSPM3-CSPM3 particles and Pfs25H vs. Pfs25H-Pfs25H particles. The similarity of the spectra
before and after incorporation into particles indicates that the secondary structures of the
antigens within the particles were largely retained [
]. AMA1-AMA1 particles and CSPM3-EPA
particles were tested and shown to retain the ability to bind antigen-specific
conformationdependent monoclonal antibodies using Western transfers from polyacrylamide gels (see S1
and S2 Figs). Binding of antigen-specific conformation-dependent monoclonal antibodies to
Pfs25H-EPA was previously demonstrated [
3.3 Immunogenicity of nanoparticles
Mice were vaccinated with the particles adsorbed on alum to assess antigen-specific immunity.
Fig 5 shows ELISA antibody units (dilution to achieve 1 OD) for day 42 sera from mice
vaccinated twice intramuscularly (days 0 and 28) with Pfs25 and CSPM3 particles, and unmodified
proteins included as comparators. Antigen-specific antibody levels achieved with Pfs25 or
CSPM3 particles were as high as or higher than the levels achieved with the unmodified
proteins, significantly higher for the EPA containing particles. AMA1 particles and unmodified
AMA1 were tested under a different protocol (subcutaneous vaccination, days 0, 14 and 28).
Under these conditions AMA1, AMA1-AMA1 and AMA1-EPA generated similar levels of
antigen-specific antibody (S3 Fig).
Antisera from vaccinated mice appear to retain functionality, as evaluated using surrogate
functional assays. The growth of parasites in a standardized growth inhibition assay is similarly
inhibited by sera from mice vaccinated with AMA1 or the AMA1-AMA1 particle as shown in
Fig 6. Sera from mice vaccinated with Pfs25 and Pfs25-EPA were previously shown to inhibit
oocyte formation in mosquitoes fed with the sera using a membrane feed assay [
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Fig 4. CD spectra of un-modified proteins and single component particles: A) 4.04 μM AMA1 (solid line) and
4.04 μM (AMA1 content) AMA1-AMA1 (broken line); B) 15.34 μM CSPM3 (solid line) and 6.14 μM (CSPM3
content) CSPM3-CSPM3 (broken line); C) 9.78 μM Pfs25H (solid line) and 9.78 μM (Pfs25H content)
Pfs25H-Pfs25H (broken line). Stock solutions of proteins and particles in PBS were diluted with water to the
stated concentrations. Spectra were obtained at 20°C.
Methods were described for the preparation and characterization of soluble protein
nanoparticles from recombinant proteins. The examples that were presented included particles that were
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Fig 5. Antibody levels in mice vaccinated with un-conjugated or conjugated forms of Pfs25H, Pfs25M,
and CSPM3, expressed as ELISA units (dilution sera to obtain an OD of 1). Mice were injected IM twice,
28 days apart, with 0.5 μg antigen content (* 2.5 μg for Pfs25H) of the vaccines adsorbed on 80 μg of
Alhydrogel (** 45 μg of Alhydrogel for Pfs25M-EPA). Serum antibody levels were determined two weeks
later by ELISA on plates coated with un-modified antigen. Dots represent ELISA units reported for individual
mice. Center line represents the geometric mean, and error bars are 95% confidence levels. Statistics
(Kruskal-Wallis with Dunn’s multiple comparisons): p < 0.05 for Pfs25H-EPA vs. Pfs25H; CSPM3-EPA vs.
CSPM3- CSPM3; CSPM3-EPA vs. CSPM3.
prepared from the malaria protein antigens Pfs25, CSP and AMA1 for testing as vaccines. The
examples include particles comprised of the antigens alone or the antigens combined with the
immunogenic carrier protein EPA. Although the proteins were structurally quite different,
they each performed similarly in forming particles, indicating that the described method may
have general utility for preparing protein nanoparticles from soluble proteins.
Fig 6. Inhibition of parasite growth in vitro with purified IgG from immunized rabbits. Y axis shows %
inhibition, and X axis shows the concentration of AMA1-specific antibody used in the assay. Closed triangles
represent individual rabbits vaccinated with un-conjugated AMA1. Open squares represents rabbits
vaccinated with the AMA1-AMA1 particle.
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The particles form spontaneously by virtue of the formation of multiple thioether bonds
between thiolated and maleimide-modified protein molecules. The Mw and Rh of the particles
depend on the concentration and the number of linkers attached, and those parameters can be
used to achieve lot to lot consistency. Particles with average molar mass of 500 to 2,000 kDa
were targeted for this work because they are large enough to be readily separated from smaller
species (monomeric proteins, small conjugates and other reagents or byproducts), yet they can
be prepared at reasonable protein concentrations (less than 10 mg/ml). The particles can be
readily purified using size separation methods to remove small particles, monomeric proteins,
reagents and byproducts. Overall protein yields of greater than 50% after purification are
achievable. The described method and scale should be adaptable, with minor modifications, to
the preparation of well characterized protein nanoparticles from a wide variety of proteins.
The antigenic properties of the antigenic proteins in the particles appear to be largely
unaffected by the process as evidenced by retention of binding to conformation dependent
monoclonal antibodies that interfere with biological functions, retention of secondary structure as
determined by CD spectroscopy, and ability to generate an antigen-specific antibody responses.
Vaccination of mice with nanoparticles of Pfs25H or CSPM3 adsorbed on alum demonstrated
that immunogenicity was either elevated or unchanged compared with unmodified antigen.
Particles of Pfs25H without carrier generated higher antibody levels than unmodified Pfs25H, whereas
particles of CSPM3 without carrier did not appear more immunogenic than unmodified CSPM3.
Incorporation of EPA into the particles significantly increased antigenicity for both antigens.
A major advantage of the use of this method to prepare particles for vaccine development,
in addition to increasing immunogenicity, is that it can be readily adapted to provide particles
for clinical use, that have product profiles closely matching previous preparations used in early
phase preclinical testing. Such adaptations may include the use of different techniques or
equipment for buffer exchange, liquid handling, column purification, etc. A separate
publication describes the development of a cGMP process for scaled-up production of Pfs25H-EPA
nanoparticles for clinical use including more extensive biochemical and biophysical
]. The efficacy of the Pf25-EPA product vaccine is currently being evaluated in
clinical trials and has passed safety evaluations (clinicaltrials.gov; ID# NCT01434381) and two
additional chemically conjugated transmission blocking vaccines have entered clinical trials
(clinicaltrials.gov; ID# NCT01867463, and NCT02334462). It will be important to determine if
this strategy of chemical crosslinking to form protein nanoparticles will work for a human
vaccine, particularly against malaria.
S1 Fig. Western blot from 3–8% Tris-acetate, showing binding of conformation dependent
AMA1-specific monoclonal antibody 4G2 (see Materials and Methods for details); color
generated with alkaline phosphatase-conjugated secondary antibody and BCIP/NBT as
substrate. (A) un-conjugated AMA1; (B) AMA1-AMA1.
S2 Fig. Western blot from 3–8% Tris-acetate, showing binding of conformation dependent
anti-CSP (TSR domain-specific) monoclonal antibody 1G12 (see Materials and Methods
for details); color generated with alkaline phosphatase-conjugated secondary antibody and
BCIP/NBT as substrate. (A) pre-stained MW markers; (B) CSPM3-EPA.
S3 Fig. Antibody levels expressed as ELISA units (extrapolated reciprocal dilution of sera
to obtain an OD of 1) in mice vaccinated with AMA1-EPA, AMA1-AMA1 (self-conjugate),
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and AMA1. Mice were injected SC three times, 14 days apart, with 100 μL of formulation
containing 0.5 μg antigen content of the vaccines adsorbed on 160 μg of Alhydrogel. Serum
antibody levels were determined two weeks later by ELISA on plates coated with un-modified
antigen. Dots represent ELISA units reported for individual mice. Center line represents the
geometric mean, and error bars are 95% confidence levels.
Recombinant proteins and expression methods were developed by the LMIV Molecular
Biology Unit led by Nicholas MacDonald. Production of recombinant proteins was carried out by
the LMIV Process Development Unit. Animal studies were carried out by the LMIV Animal
Study Unit led by Lynn Lambert. Vaccines were formulated for injection by the LMIV
formulation group led by Kelly Rausch. ELISA assays were performed by the LMIV Immunoassay
Group under the supervision of Joan Aebig.
Conceived and designed the experiments: DSJ DN YW CGR BC KMR. Performed the
experiments: CGR BC KR KMR. Analyzed the data: DSJ DLN CGR BC YW KMR PED. Contributed
reagents/materials/analysis tools: DKN KR. Wrote the paper: DSJ CGR BC PED.
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