![Functional activity of spy‑VLP vaccine‑induced humoral responses. a Transmission reducing activity (TRA) of anti‑Pfs25 (day 56) sera follow‑ ing immunization of mice with the Pfs25 spy‑VLP vaccine or with the control vaccine (soluble Pfs25 + untagged AP205 VLP) as described in Fig. 2a. Y‑axis shows the number of oocysts identified in the midgut of each of 20 A. stephensi mosquitoes. Pre‑immunization mouse serum was used as additional negative control. Mann–Whitney rank sum test was used for statistical comparisons. b Binding between VAR2CSA expressing infected erythrocytes and CSA in the presence of different concentrations of serum from immunized mice. Binding in the presence of serum from non‑ immunized mice was set to 100 %. Serum pools were from groups of 5 mice immunized with the VAR2CSA spy‑VLP vaccine (black circle) or with control vaccine (empty square). The EC50 value for the spy‑VLP vaccinated mice was 8.8 fold higher [3.213–14.41] than the value from mice receiving the control vaccine](https://www.researchgate.net/profile/Susan-Thrane/publication/301480531/figure/fig1/AS:355370993242113@1461738455379/Functional-activity-of-spy-VLP-vaccine-induced-humoral-responses-a-Transmission-reducing_Q320.jpg)
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Thrane et al. J Nanobiotechnol (2016) 14:30
DOI 10.1186/s12951-016-0181-1
RESEARCH
Bacterial superglue enables easy
development ofecient virus-like particle
based vaccines
Susan Thrane1,2, Christoph M. Janitzek1,2, Sungwa Matondo3, Mafalda Resende1,2, Tobias Gustavsson1,2,
Willem Adriaan de Jongh4, Stine Clemmensen1,2,4, Will Roeffen5, Marga van de Vegte‑Bolmer5,
Geert Jan van Gemert5, Robert Sauerwein5, John T. Schiller6, Morten A. Nielsen1,2, Thor G. Theander1,2,
Ali Salanti1,2* and Adam F. Sander1,2*
Abstract
Background: Virus‑like particles (VLPs) represent a significant advance in the development of subunit vaccines, com‑
bining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal
scaffolds for display of vaccine antigens. Traditional approaches for VLP‑based antigen display require labor‑intensive
trial‑and‑error optimization, and often fail to generate dense antigen display. Here we utilize the split‑intein (SpyTag/
SpyCatcher) conjugation system to generate stable isopeptide bound antigen‑VLP complexes by simply mixing of the
antigen and VLP components.
Results: Genetic fusion of SpyTag or SpyCatcher to the N‑terminus and/or C‑terminus of the Acinetobacter phage
AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag
or two SpyTags per capsid protein. Mixing of spy‑VLPs with eleven different vaccine antigens fused to SpyCatcher or
SpyTag resulted in formation of antigen‑VLP complexes with coupling efficiencies (% occupancy of total VLP binding
sites) ranging from 22–88 %. In mice, spy‑VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly
increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing
monomeric proteins. The spy‑VLP vaccines also effectively broke B cell self‑tolerance and induced potent and durable
antibody responses upon vaccination with cancer or allergy‑associated self‑antigens (PD‑L1, CTLA‑4 and IL‑5).
Conclusions: The spy‑VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP‑
based vaccines. Our data provide proof‑of‑concept for the technology’s ability to present complex vaccine antigens
to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self‑toler‑
ance. The spy‑VLP‑system may serve as a generic tool for the cost‑effective development of effective VLP‑vaccines
against both infectious‑ and non‑communicable diseases and could facilitate rapid and unbiased screening of vac‑
cine candidate antigens.
© 2016 Thrane et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Background
Active vaccination against infectious diseases has been
one of the most effective medical interventions in human
history with a tremendous impact on global health. Due
to safety-, manufacturing- and reproducibility concerns,
global vaccine development has gradually turned its focus
away from whole-pathogen based vaccines and towards
recombinant subunit vaccines based on defined antigen
components [1]. e effectiveness of simple subunit vac-
cines is, however, considerably inferior to that of whole-
pathogen-based vaccines and the successful development
of soluble proteins as vaccine candidates has in many
cases been a disappointment. e low immunogenicity
of soluble protein antigens has been attributed to their
Open Access
Journal of Nanobiotechnology
*Correspondence: salanti@sund.ku.dk; adamsander@gmail.com
1 Centre for Medical Parasitology at the Department of Immunology
and Microbiology, University of Copenhagen, Copenhagen, Denmark
Full list of author information is available at the end of the article
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Page 2 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
small size (<10nm), susceptibility to proteolytic degrada-
tion, and a low capacity for activating the innate immune
system. Virus-like particles (VLPs) represent a specific
class of particulate subunit vaccines, which are highly
immunogenic due to sharing key characteristics with live
viruses [2]. Several VLP-vaccines have already been com-
mercialized e.g. Engerix (hepatitis B virus) and Cervarix
(human papillomavirus) by GlaxoSmithKline, Recom-
bivax HB (hepatitis B virus) and Gardasil (human papil-
lomavirus) by Merck, and Hecolin (hepatitis E virus) by
Xiamen Innovax [3]. VLPs are safe non-replicating shells
consisting solely of viral structural proteins that, when
overexpressed, self-assemble into dense multi-protein
arrays with icosahedral or rod-like structures. e size of
VLPs (20–200nm) allows for direct drainage into lymph
nodes and is optimal for uptake by antigen-presenting
cells and cross-presentation [4]. eir highly repetitive
surface structures moreover enable complement fixation
and B cell receptor clustering, altogether leading to the
activation of the innate immune system, greater B cell
activation and ultimately increased antibody production
[4–6]. Importantly, it has been established that hetorol-
ogous antigens displayed on VLPs can assume a similar
immunogenicity as the underlying particle, creating a
strong rational for using VLPs as antigen-presenting plat-
forms to increase immune responses against otherwise
poorly immunogenic antigens [2, 7]. Antigen display has
traditionally been achieved by either genetic fusion of
heterologous epitopes into the self-assembling coat pro-
tein or by conjugation to preassembled VLPs. Genetic
fusion of smaller peptides (often single epitopes) has in
several cases been successful, whereas insertion of larger
sequences generally prevents VLP-assembly [2, 8, 9]. Even
if VLP-assembly is achieved, chimeric particles are often
instable and the functional conformation of the inserted
epitope may not be retained. Consequently, the genetic
fusion approach is inevitably based on substantial trial-
and-error optimization and is largely restricted to con-
tinuous epitopes thus requiring the pre-identification of
such determinants in the target-antigen. Chemical cross-
linking chemistry has been employed to conjugate target
antigens to pre-assembled VLPs by stimulating covalent
linkage between reactive amino acid side chains in the
antigen and coat protein sequences, respectively [10, 11].
Complex antigens, however, generally present multiple
reactive sites hampering consistent directional coupling
of the antigen to the VLP required for optimal epitope
display. In addition, such chemical reactions often result
in a lower than optimal density of the VLP-displayed
antigen [10, 12]. Other strategies, involving non-covalent
antigen-VLP conjugation have also been pursued, each
with individual advantages and disadvantages [13, 14].
e most successful general approach was developed by
Cytos Biotech and involves the terminal addition of a
reactive Cysteine residue to the target-antigen followed
by addition of a hetero-bifunctional cross-linker to medi-
ate coupling between the reactive sulfhydryl group (Cys)
and the N-term of Lysine residues exposed on the sur-
face of Qbeta VLPs [10, 15]. Several promising VLP-vac-
cines have been developed by this technology, although
this method suffers from an inconsistent ability to dis-
play complex antigens with conformation-dependent
epitopes. erefore, there is a strong interest in devel-
oping new methods to obtain optimal VLP-display for
complex target-antigens. Herein we report the use of the
split-intein (SpyTag/SpyCatcher) conjugation system [16]
to facilitate conjugation of target antigens to VLPs under
physiological conditions. is conjugation system takes
advantage of the spontaneous formation of an isopeptide
bond between a Lys and an Asp present in two split units
of the Streptococcus pyogenes fibronectin-binding pro-
tein FbaB. ese split units consist of a peptide (SpyTag)
and a protein (SpyCatcher), which in solution interact to
form a highly stable amide bond. e irreversible reac-
tion occurs within minutes and so this technology offers
a simple way to conjugate antigen to VLPs. We devel-
oped a panel of genetically modified Acinetobacter phage
AP205 VLPs displaying either the SpyCatcher protein
(116 amino acids) or the SpyTag peptide (13 amino acids)
in regular arrays. We also engineered an AP205 VLP pre-
senting two SpyTags per VLP subunit (2xSpyTag-VLP).
We characterized these spy-VLPs in terms of stability
and antigen display capacity using a variety of antigens
and discuss how the different spy-VLPs can be used to
increase the versatility of the spy-VLP display system.
Using two malaria antigens, we demonstrate that the spy-
VLP system elicited high levels of high affinity IgG, which
effectively inhibited key processes in the parasite devel-
opment. Finally, we show that B cell self-tolerance can
be overcome by the spy-VLP system, which effectively
induced IgG against a range of self-antigens including
PD-L1, CTLA-4 and IL-5. us, the data demonstrate the
broad usability of the spy-VLP platform and validate its
ability to facilitate strong functional antibody responses
against complex vaccine antigens.
Results
Development, expression andcharacterization ofspy‑VLPs
A panel of SpyTag or SpyCatcher presenting VLPs was
designed based on the Acinetobacter phage AP205 coat
protein. Expression of this protein in Escherichia coli
results in the assembly of 29nm icosahedral (T=3) VLPs
consisting of 180 subunits [17]. Precisely, the 116 amino
acid SpyCatcher sequence was fused to the N-terminus
(SpyCatcher-VLP) of the AP205 coat protein (Gene ID:
956335). In addition, the 13 amino acid SpyTag peptide
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Thrane et al. J Nanobiotechnol (2016) 14:30
was fused to N-terminus (SpyTag-VLP) or to both N- and
C-terminus (2xSpyTag-VLP) of the AP205 coat protein
(Fig.1a). Recombinant E. coli expression of spy-AP205
coat proteins was confirmed by SDS-PAGE analysis of
fractions collected following density gradient ultracen-
trifugation. Reduced SDS-PAGE showed pure protein
bands of expected sizes (Additional file 1: Figure S1).
VLP-assembly of each spy-AP205 coat proteins was
evaluated by transmission electron microscopy (TEM)
(Fig. 1b) and dynamic light-scattering (DLS) analysis.
For all recombinant particles the DLS analysis revealed a
homogenous population of non-aggregated particles with
an average estimated size of 36nm [Pd=12.1] (SpyTag-
VLP), 42 nm [Pd =21.6] (2xSpyTag-VLP) and 43 nm
[Pd=9.7] (SpyCatcher-VLP). In comparison, unmodified
AP205 VLPs were determined by DLS to have an average
size of 35nm [Pd=10.7].
To test if the recombinant spy-VLPs could form a cova-
lent interaction with an antigen through their SpyTag
or SpyCatcher, individual spy-VLPs were mixed with
antigen fused to the corresponding binding-partner and
formation of antigen-VLP subunit conjugates was sub-
sequently confirmed by SDS-PAGE analysis. For all spy-
VLP types and all tested antigens the SDS-PAGE revealed
the occurrence of a protein band matching the combined
size of the antigen and VLP subunit, as exemplified in
Fig.1c. Mixing of SpyTag- or SpyCatcher-fused antigen
with unmodified AP205 VLPs did not produce this band-
ing pattern (data not shown).
Versatility ofthe spy‑VLP platform
To explore the versatility of the spy-VLP antigen display
system we cloned and expressed 11 vaccine candidate
antigens genetically fused to either a SpyTag or a Spy-
Catcher (Additional file2: TableS1). e panel of spy-
antigens, representing very diverse proteins with respect
to origin, structure and size (14.5–118 kDa) included;
(a) malaria proteins: CSP, CIDR, VAR2CSA, and Pfs25,
which are expressed at different developmental stages
of the complex life cycle of Plasmodium falciparum and
used in different vaccine strategies to reduce malaria
transmission or disease [18, 19]; (b) the Mycobacterium
tuberculosis protein, Ag85A, in development for a tuber-
culosis vaccine; (c) mouse proteins involved in cancer
(CTLA-4, PD-L1, Survivin and HER2), asthma/allergy
(IL-5) or cardiovascular disease (PCSK9). e latter self-
proteins are targets of therapies employing monoclonal
antibody. e vaccine antigens were mixed with corre-
sponding spy-VLPs and the antigen coupling efficiency (%
occupancy of total VLP binding sites) and antigen display
capacity (number of antigens per VLP) was estimated
for each reaction by SDS-PAGE densitometric analysis.
Standard molar mixing ratio was 1:1.5 (VLP:antigen) and
a
b
c
Fig. 1 The spy‑VLP antigen display platform. a Three types of spy
expressing VLPs were constructed by genetic fusion of SpyTag or
SpyCatcher to the virus‑like particle (VLP)‑forming AP205 capsid
protein. (1) “SpyTag‑VLP” had the SpyTag fused to the N‑terminus
of the AP205 capsid protein and present 180 potential SpyCatcher‑
antigen binding motifs (2) “2xSpyTag‑VLP” had SpyTag fused to both
the N‑ and C‑terminus of the AP205 capsid protein and present 360
potential SpyCatcher‑antigen binding motifs; (3) “SpyCatcher‑VLP”
had SpyCatcher fused to the N‑terminus of the AP205 capsid protein
and present 180 potential Spytag‑antigen binding motifs. b Transmis‑
sion electron microscopy (TEM) images showing the SpyTag‑VLP,
2xSpyTag‑VLP and SpyCatcher‑VLP. Purified spy‑VLP samples were
placed on carbon, adsorbed to a grid and negatively stained with
2 % phosphotungstic acid. Scale bar 50 nm. Images show uniform,
non‑aggregated particles of approximately 30 nm (SpyTag‑VLP and
2xSpyTag‑VLP) and 42 nm (SpyCatcher‑VLP). c Reduced SDS‑PAGE
gels loaded with VLP vaccines demonstrating that vaccine proteins
had formed covalent bonds to the AP205 capsid protein. Left panel
shows that mixing of SpyTag‑VLPs with SpyCatcher‑IL‑5 resulted in
three protein bands corresponding to the size of an antigen‑VLP
capsid protein conjugate (48 kDa) (top), uncoupled vaccine antigen
(33 kDa) (middle) and unconjugated SpyTag‑VLP capsid protein
(16.5 kDa) (bottom). The middle panel shows that mixing of 2xSpyTag‑
VLP with SpyCatcher‑IL‑5 resulted in four protein bands represent‑
ing; a conjugate of two vaccine antigens bound to each end of
a 2xSpyTag‑VLP capsid protein (83 kDa), a conjugate of the 2xSpyTag‑
VLP capsid protein and a single vaccine antigen (48 kDa), uncoupled
vaccine antigen (33 kDa) and unconjugated 2xSpyTag‑VLP capsid
protein (18.5 kDa). The left panel shows that mixing of SpyCatcher‑VLP
with PD‑L1‑SpyTag resulted in three protein bands representing; an
antigen‑VLP capsid protein conjugate (50 kDa), uncoupled vaccine
antigen (33 kDa) and unconjugated SpyCatcher‑VLP capsid protein
(27 kDa)
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Thrane et al. J Nanobiotechnol (2016) 14:30
reactions occurred over night at 4°C. A similar coupling
efficiency was observed using three-hour incubations at
37 °C (data not shown). Overall, antigen-coupling effi-
ciencies ranged from 22–88%, corresponding to 40–159
antigens displayed per VLP (Table1).
ere was a negative correlation between antigen size
and the number of antigens bound per VLP (Spearman
Rank Order Correlation Coeff.=−0.75, P= 0.02) (the
2xSpyTag-VLP constructs were excluded from the analy-
sis). ere was no significant difference in the antigen
coupling efficiency in SpyTag-VLP and SpyCatcher-VLP
reactions. However, the estimated coupling capacity was
higher for the reaction between SpyCatcher-IL-5 and
2xSpyTag-VLP (193 antigens per VLP) compared with
mixing similar amounts of SpyCatcher-IL-5 withSpyTag-
VLP (138 antigens per VLP) (Table1; Additional file3:
FigureS2D).
Immunogenicity ofspy‑VLP vaccines
To assess the immunogenicity of spy-VLP delivered anti-
gens, we tested two clinically relevant malaria proteins,
Pfs25 and VAR2CSA, and evaluated humoral responses
in mice after intramuscular immunizations. Pfs25 is
expressed on the P. falciparum ookinete surface within
the mosquito. Immunization with recombinant Pfs25
formulated in Montanide ISA51 induced Pfs25-specific
antibodies with capacity to block parasite infectivity to
mosquitoes in a Phase 1 human clinical trial but the vac-
cine had unacceptable side effects [20]. e Pfs25 antigen
is poorly immunogenic by itself and development of an
effective transmission-blocking Pfs25 vaccine has been
hampered by the requirement of a strong adjuvant [20–
22]. SpyCatcher was fused to the C-terminus of Pfs25 and
Pfs25-SpyCatcher was expressed and purified from E. coli
SHuffle® cells. is enabled high level expression of solu-
ble correctly folded Pfs25-SpyCatcher as verified by bind-
ing of the transmission-blocking anti-Pfs25 monoclonal
antibody, mAb 4B7 [23] (Additional file4: FigureS3).
VAR2CSA is a unique member of the P. falciparum
erythrocyte membrane protein 1 (PfEMP1) protein fam-
ily. is protein binds parasite-infected erythrocytes
to placental chrondroitin sulphate A (CSA) [24]. Anti-
VAR2CSA antibodies can prevent this binding [25, 26],
and clinical testing of a protein-based VAR2CSA vac-
cine to protect women against placental malaria has
been initiated [27].SpyTag was genetically fused to the
N-terminus of the CSA binding domain of VAR2CSA
(domains DBL1-ID2a) and expressed and purified from
E. coli SHuffle® cells. e protein expressed well and was
folded correctly as measured by its binding to decorin, as
described in [28].
Spy‑VLP vaccine inducedIgG titers
e antigen display capacities for Pfs25 and VAR2CSA
were 109 and 61 proteins per VLP, respectively (Table1;
Additional file 3: Figure S2C, K). e antigen-specific
IgG titer was measured by enzyme-linked immune-sorb-
ent assay (ELISA) 2weeks after each immunization (on
days 14, 35 and 56) as well as at day 212 (Pfs25) and 137
(VAR2CSA) (Fig.2a). e Pfs25 spy-VLP vaccine induced
higher antigen-specific IgG titers than the control vac-
cine at all the tested time-points (P<0.01 (day 14, 35 and
56); P=0.03 (day 212), Mann–Whitney Rank Sum Test)
(Fig.2a). At day 212, there was a 37-fold increase in the
geometric mean titer (GMT) of IgG in sera from spy-
VLP vaccinated mice compared to mice vaccinated with
the same amount of soluble Pfs25 plus untagged AP205
VLPs.
Table 1 Estimation ofantigen coupling eciency
a % occupancy of total VLP subunits: number of displayed antigens/total number of VLP subunits (=180)×100
Spy‑VLP Potential
binding motifs Spy‑antigen Antigen
size (kDa) Coupling
eciency (%) Display capacity
(antigens/VLP)
SpyTag 180 SpyCatcher‑CIDR 32 76 136
SpyTag 180 SpyCatcher‑IL‑5 33 77 138
SpyTag 180 SpyCatcher‑Ag85A 48 75 134
SpyTag 180 CSP‑SpyCatcher 53 48 86
SpyTag 180 SpyCatcher‑HER2 83 22 40
SpyTag 180 PCSK9‑SpyCatcher 84 23 42
2xSpyTag 360 SpyCatcher‑Survivin 30 52 (104)a187
2xSpyTag 360 SpyCatcher‑IL‑5 33 54 (107)a193
2xSpyTag 360 Pfs25‑SpyCatcher 40 30 (61)a109
SpyCatcher 180 CTLA‑4‑SpyTag 15 88 159
SpyCatcher 180 PD‑L1‑SpyTag 27 45 81
SpyCatcher 180 SpyTag‑VAR2CSA 118 34 61
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Thrane et al. J Nanobiotechnol (2016) 14:30
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Page 6 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
e GMT of VAR2CSA-specific IgG was consistently
higher in the group immunized with the VAR2CSA spy-
VLP vaccine compared to the control group vaccinated
with uncoupled VAR2CSA. However, the difference in
GMTs between the two groups was not as profound as
seen in the Pfs25 study and only reached statistical sig-
nificance at days 14 (P=0.03, Mann–Whitney Rank Sum
Test) and 137 (P=0.03, Mann–Whitney Rank Sum Test)
(Fig.2a).
Avidity ofspy‑VLP vaccine induced IgG
In order to further examine qualitative differences in
humoral responses, we investigated the avidity of IgG
antibodies induced after immunizations with spy-VLP
vaccines compared to control vaccines (uncoupled anti-
gen + untagged AP205 VLPs). e avidity index val-
ues of serum IgG were determined by measuring the
resistance of antibody-antigen complexes to 8M urea
by ELISA, as described [29]. Avidity index values can
be divided into three categories denoting: high avidity
(avidity index values higher than 50 %), intermediate
avidity (between 30 and 50%) and low avidity (>30%)
[29]. Prior to measuring avidity, pre-determined IgG
antibody levels in individual mouse serum samples were
equalized by dilution. Both the Pfs25 and VAR2CSA
spy-VLP vaccines induced antigen-specific IgG with sig-
nificantly higher avidity-index values compared to cor-
responding control vaccines P= 0.015 (Pfs25 day 35)
and P=0.032 (Pfs25day 56) and P=0.032 (VAR2CSA
day 35) and P=0.056 (VAR2CSA day 56), Mann–Whit-
ney Rank Sum Test) Fig. 2b. e mean avidity index
value of anti-Pfs25 sera obtained at day 35 was 46% for
the spy-VLP group, thus falling within the “intermediate
avidity” category, whereas the mean value of the control
group was only 22% (i.e. low avidity). e correspond-
ing values for day 56 were 57% (i.e. high avidity) and
30% (i.e. intermediate avidity) for the spy-VLP and con-
trol group, respectively. e mean avidity of antibodies
induced by the VAR2CSA spy-VLP vaccine was 63 and
62% (both high avidity) in sera obtained at day 35 and
56, respectively. In comparison, mean avidity of anti-
bodies induced by the control vaccine was 39 and 42%
(both intermediate avidity) in sera obtained at day 35
and 56, respectively.
Subclass proling ofvaccine induced IgG
e relative proportion of IgG subclasses elicited in the
spy-VLP vaccinated groups and the control groups was
also compared. Measurements were performed on sera
obtained at day 98 (Pfs25) or 88 (VAR2CSA) and cal-
culations were based on ELISA measurements using
subclass-specific (IgG1, IgG2a and IgG2b) secondary
anti-mouse IgG antibodies for quantification. Anti-
mouse total IgG secondary antibody was used for nor-
malization. IgG1 was the dominant IgG subclass in all
sera. Sera from Pfs25 vaccinated mice contained IgG1
and IgG2b and the spy-VLP vaccine induced significantly
higher IgG2b (P=0.03) and significantly lower (P=0.03)
IgG1 levels compared to the control vaccine (Fig.2c, left).
Sera from VAR2CSA vaccinated mice contained IgG
of all subclasses. e distribution of IgG subclasses was
similar in mice vaccinated with the two VAR2CSA vac-
cines. (Fig.2c, right).
Functional activity ofspy‑VLP induced humoral responses
For most vaccines only a fraction of the induced IgG is
biologically active in inhibiting the development of the
targeted microorganism and the level and overall avid-
ity of vaccine induced IgG responses does not necessarily
reflect the anti-microbial functional activity. e goal of
the Pfs-25 vaccine is to block parasite development inside
the mosquito. We therefore used the standard membrane
feeding assay (SMFA) to measure transmission-blocking
(TB) activity of the antibodies induced by the two vac-
cines [30]. e Pfs25 spy-VLP vaccine showed more than
99% transmission-reducing activity (TRA) (one oocyst
found in the 20 investigated mosquitos) compared to
pre-immune serum (82 oocysts detected) or serum from
See figure on previous page.)
Fig. 2 Antigen‑specific IgG levels in mice after immunization with soluble or spy‑VLP displayed malaria antigens. (a, upper panels) Antigen‑specific
IgG levels (OD Elisa) in serum from mice (n = 5 per group) immunized with a Pfs25 2xSpyTag‑VLP vaccine (filled circles) or with a control vaccine
consisting of soluble Pfs25 mixed with untagged AP205 VLPs (open squares). Both vaccines were formulated using aluminum hydroxide adjuvant
(Statens Serum Institut, Copenhagen, Denmark). Mice were immunized on days 0, 21 and 42 and serum was collected on the indicated days after
first immunization. Differences in median endpoint titers between vaccination groups were analyzed using Mann–Whitney Rank Sum test; day
14 (P < 0.01), day 35 (P < 0.01), day 56 (P < 0.01) and day 212 (P = 0.03). (a, lower panels) Similar results for the VAR2CSA based vaccines (VAR2CSA
SpyCactcher‑VLP and soluble VAR2CSA plus untagged AP205 VLP), which were formulated without extrinsic adjuvant. Statistical analysis; day 14
(P = 0.03), day 35 (P = 0.09), day 56 (P = 0.09) and day 137 (P = 0.03). b Antibody avidity was assessed on days 35 and 56 in serum samples from
mice vaccinated with the Pfs25 or VAR2CSA vaccines. Avidity index values were determined by measuring the resistance of antibody‑antigen com‑
plexes to 8 M urea. The avidity index was calculated as the ratio of the mean ELISA OD490 value of urea‑treated wells to PBS control wells multiplied
by 100. Mann–Whitney Rank Sum test was used for statistical comparisons. c The distribution of IgG1, IgG2a and IgG2b relative to the total vaccine‑
induced IgG response in mice (n = 5 per group) following Pfs25 or VAR2CSA immunization. Anti‑Pfs25 and anti‑VAR2CSA sera (left) were obtained
on days 98 and 88, respectively. Mann–Whitney Rank Sum test was used for statistical comparisons
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 7 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
mice immunized with the control vaccine (85 oocysts
detected) (Fig. 3a). In a second study, BALB/c mice
(n=7) were immunized with the Pfs25 spy-VLP vaccine
at days 0 and 14, and different concentrations of purified
IgG from pooled anti-Pfs25 serum samples were subse-
quently tested in the SMFA assay. At the highest IgG con-
centration (750µg/ml) serum from the Pfs25 spy-VLP
vaccinated mice completely blocked oocyst formation
(Table2).
Serum concentrations of 250 and 83.3µg/ml showed
more than 95 % transmission-reducing activity com-
pared to IgG purified from ovalbumin immunized mice
(Table2).
e aim of VAR2CSA vaccines is to induce IgG, which
inhibit the binding between parasite-infected erythro-
cytes and placental chondroitin sulfate. We therefore
compared the vaccines ability to elicit IgG inhibiting
the binding between VAR2CSA expressed on infected
erythrocytes and chondroitin sulfate in an invitro assay
(Fig. 3b). e mean EC50 calculated from the dose–
response curve of serum from VAR2CSA spy-VLP immu-
nized mice was eightfold higher, [CI 95% 3.213–14.41],
than the mean EC50 in serum from mice vaccinated with
VAR2CSA that was not bound to VLPs (Fig.3b).
Breaking self‑tolerance byspy‑VLP display
To examine the capacity of the spy-VLP system to over-
come B cell self-tolerance and induce autoantibody
responses upon vaccination with self-antigens, mouse
proteins PD-L1, CTLA-4 and interleukin-5 were recom-
binantly expressed with SpyTag or SpyCatcher and
formulated as spy-VLP vaccines. PD-L1 and CTLA-4
down regulate T cell function. Expression of these pro-
teins in tumors has been linked to poor prognosis and
Fig. 3 Functional activity of spy‑VLP vaccine‑induced humoral responses. a Transmission reducing activity (TRA) of anti‑Pfs25 (day 56) sera follow‑
ing immunization of mice with the Pfs25 spy‑VLP vaccine or with the control vaccine (soluble Pfs25 + untagged AP205 VLP) as described in Fig. 2a.
Y‑axis shows the number of oocysts identified in the midgut of each of 20 A. stephensi mosquitoes. Pre‑immunization mouse serum was used as
additional negative control. Mann–Whitney rank sum test was used for statistical comparisons. b Binding between VAR2CSA expressing infected
erythrocytes and CSA in the presence of different concentrations of serum from immunized mice. Binding in the presence of serum from non‑
immunized mice was set to 100 %. Serum pools were from groups of 5 mice immunized with the VAR2CSA spy‑VLP vaccine (black circle) or with
control vaccine (empty square). The EC50 value for the spy‑VLP vaccinated mice was 8.8 fold higher [3.213–14.41] than the value from mice receiving
the control vaccine
Table 2 Transmission-reducing activity ofPfs25 spy-VLP vaccine induced IgG
a Total oocyst count in mosquitoes fed with anti-Pfs25 IgG/total oocyst count in mosquitoes fed with control serum IgG×100
b Mann–Whitney Rank Sum Test
Control serum Serum fromPfs25 spy‑VLP immunized mice
Tested concentrations (µg/ml) of purified total serum IgG 750 750 250 83.3 27.8
Detected oocysts per 20 tested mosquitoes 133 0 1 5 97
Median (25 and 75 percentiles) 5.5 (3:10) 0 (0:0) 0 (0:0) 0 (0:0) 4.0 (2:6.75)
Transmission‑reducing activity (%)a– 100 99.2 96.2 27.1
P valueb– <0.001 <0.001 <0.001 0.704
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Page 8 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
antibody-targeting of the proteins is showing promise
in cancer treatment [31]. Antibody measurements of
anti-sera obtained at day 56 showed that the PD-L1 and
CTLA-4 spy-VLP vaccines induced significant antibody
responses against the self-antigens compared to the solu-
ble protein (P=0.01 and P<0.01, respectively) (Fig.4a,
b).
e therapeutic use in severe asthma of antibodies tar-
geting IL-5 is supported by abundant data from invitro
experiments, animal models and clinical trials [32]. e
SpyCatcher was fused at the N-terminus of mouse IL-5.
Antibody measurements of anti-IL-5 in sera obtained
at day 56 showed that the spy-VLP vaccine induced sig-
nificant antibody responses against this self-antigen
compared to the control vaccine (P = 0.02) (Fig. 4c).
e autoantibody response elicited by the spy-VLP
vaccine did not decay rapidly as the titers measured at
day 112(Fig.4d) were similar to those measured at day 56
(P=0.02) (Fig.4c).
Mixing SpyCatcher-IL-5 with 2xSpyTag-VLPs resulted
in a higher antigen coupling capacity than when mixing
the same antigen with SpyTag-VLPs (Table1; Additional
file 3: Figure S2D). ese two mixtures were adminis-
tered to mice to compare IgG levels elicited by the two
vaccines. e GMT titer of sera obtained at day 35 from
mice vaccinated with the IL-5 2xSpyTag-VLP vaccine
(GMT=580) was 13 fold higher than for the anti-IL-5
SpyTag-VLP sera (GMT=41) (P =0.06, Mann–Whit-
ney Rank Sum test), suggesting that the increased anti-
gen coupling capacity of the 2xSpyTag VLP had an effect
on the immunogenicity of the vaccine (Additional file5:
FigureS4).
Fig. 4 Breakage of self‑tolerance. IgG autoantibody responses measured by standard ELISA. a, b C57BL/6 mice (n = 10 per group) were immunized
with a PD‑L1 (a) or CTLA‑4 (b) SpyCatcher‑VLP vaccine (filled circles) or with a control vaccine (n = 3 per group) consisting of similar amounts of spy‑
antigen mixed with untagged AP205 VLPs (open squares). Both vaccines were formulated using aluminum hydroxide adjuvant (Statens Serum Insti‑
tut, Copenhagen, Denmark). Mice were immunized with a dose of 5 µg antigen on days 0, 21 and 42 and serum was collected on day 56 after first
immunization. Median endpoint titers were compared for the PD‑L1 vaccination groups (P = 0.01) and the CTLA‑4 vaccination groups (P = 0.01)
using Mann–Whitney Rank Sum test. c, d BALB/c mice (n = 4) were immunized with an IL‑5 SpyTag‑VLP vaccine or a control vaccine (soluble
IL‑5 + untagged AP205 VLP) (n = 5) which were both formulated with aluminum hydroxide (Statens Serum Institut, Copenhagen, Denmark). Mice
were immunized on days 0, 21 and 42 with antigen doses of 5, 2.5 and 2.5 µg, respectively, and serum was collected on days 56 (c) and 112 (d).
Median endpoint titers for the two vaccination groups were compared using Mann–Whitney Rank Sum test; day 56 (P = 0.2) and day 122 (P = 0.2)
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Thrane et al. J Nanobiotechnol (2016) 14:30
Discussion
Virus-like particles (VLPs) are effective at establish-
ing both prophylactic and therapeutic immunity against
their source virus or foreign antigens displayed on their
surface. Aiming at developing a versatile VLP-based anti-
gen display platform, we designed a panel of genetically
modified spy-AP205 coat proteins, which assembled into
stable, non-aggregated VLPs presenting reactive SpyTag
or SpyCatcher on their surfaces. e Acinetobacter phage
AP205 was chosen as platform as AP205 VLPs have been
shown to tolerate fusion of heterologous peptides at both
the N- and C-terminus of the structural protein [17]
and because AP205 VLPs can be produced at low cost
in E. coli. e spy-VLPs were assessed in terms of their
applicability to bind and display antigens using the Spy-
Catcher/SpyTag conjugation system [16]. is protein-
coupling tool offers several key advantages. SpyTag and
SpyCatcher polypeptides react and form an irreversible
isopeptide bond at high yield under a variety of condi-
tions (including variation in pH, temperature or buffer
composition) [16], which offer ample possibilities for
antigen-specific optimization. We have previously expe-
rienced that antigen-VLP conjugates tend to form aggre-
gates and precipitate upon mixing of antigen and VLP.
Subsequent modification of buffer conditions can pre-
vent this while still allowing efficient conjugation (data
not shown). Notably, a similar split-intein conjugation
system (Snooptag/SnoopCatcher) was recently reported,
which may be used in a similar strategy for conjugation of
antigen to pre-assembled VLPs [33].
Having developed both SpyTag and SpyCatcher fused
VLPs; either binding partner can be chosen as fusion-tag
to the antigen. In most cases the SpyTag peptide will be
the preferred choice since the minor addition of 13 amino
acid residues to the antigen is unlikely to negatively influ-
ence protein expression or polypeptide folding, which in
turn could have impact on the functional capacity of elic-
ited IgG. Interestingly, for several proteins, we observed
increased expression yields after fusing the SpyCatcher
to the antigen, creating an additional rational for employ-
ing SpyTag-VLPs for conjugation of SpyCatcher-fused
antigens. Specifically, we were able to express soluble and
correctly folded Pfs25-SpyCatcher using E. coli SHuffle®
(NEB Biolabs) strains. Notably, E. coli expression of solu-
ble Pfs25, in functional conformation, has not previously
been reported [34] and expression of naked Pfs25 using
similar E. coli SHuffle® cells resulted, in our hands, in for-
mation of inclusion bodies (data not shown).
Epitope density is an important factor for B cell acti-
vation [35]. It was thus interesting to examine whether
the spy-VLP system presented vaccine antigens at high
density. e VLPs formed by structural proteins to which
one SpyTag or SpyCatcher were fused provided 180
binding sites per VLP. We tested 11 spy-antigens in dif-
ferent combinations with these spy-VLP platforms. e
antigen density per particle varied according to antigen
and the VLP employed, but the mean display capacity
was 118 antigen molecules per spy-VLP, corresponding
to that antigens had been coupled to 65% of the available
VLP subunits. We had expected antigen-specific char-
acteristics (e.g. net surface charge) would have a greater
effect on the coupling efficiency than what was observed,
but the observed correlation between antigen size and
the density of antigen display on the VLPs, suggests that
steric hindrance plays a role. e VLPs with 180 available
antigen binding sites elicited robust IgG responses, how-
ever we also designed VLPs where each subunit protein
had two SpyTags attached. ese VLPs had 360 binding
sites available. e immunogenicity of IL-5 was higher
when administrered with the high density VLP (193 anti-
gens coupled per VLP) than withthe lower density VLP
(138 antigens coupled per VLP), but the difference only
achieved borderline statistical significance (P = 0.06).
Due to close proximity of the SpyTags on the VLPs with
360 binding sites it is likely that smaller proteins are bet-
ter able to bind effectively to these particles, whereas
larger proteins face problems of steric hindrance. us
each of the three developed spy-VLP platforms contrib-
utes to the versatility of the antigen display system.
e long-term success of a Pfs25-based transmis-
sion-blocking vaccine depends upon induction of high
and sustainable levels of functional antibody that effec-
tively block parasite development in the mosquito [36,
37]. In this regard, a single immunization with bivalent
HPV16/18 L1 VLP vaccine (Cervarix) has been shown
to induce protective antibody levels remaining stable for
more than 48months [38]. is has been attributed to
the ordered, repetitive, and dense display of epitopes on
the VLP surface. We found, that the immunogenicity of
Pfs25 on spy-VLPs was high and that the anti-Pfs25 IgG
levels was considerably higher 6months post immuniza-
tion compared to mice vaccinated with uncoupled pro-
tein, although IgG levels showed a tendency to decline
throughout the study period. Further long-term studies
are thus needed to establish if anti-Pfs25 levels will even-
tually plateau and, in that event, assay the transmission-
blocking activity of the remaining antigen-specific IgG.
Anti-VAR2CSA IgG titers were not boosted to the
same extent by the spy-VLP display as seen in the Pfs25
study. Likely, this is explained by the fact that soluble
VAR2CSA is a superior immunogen i.e. the soluble pro-
tein induced relatively high antigen specific IgG titers.
However, the comparably lower antigen display capacity
of the VAR2CSA spy-VLP vaccine (61 antigens per VLP)
may also have limited immunogenicity. Importantly, a
vaccine to protect against placental malaria should be
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Page 10 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
administered prior to conception i.e. to pre-puberty girls.
erefore, several years are likely to lapse between vac-
cination and exposure. It is thus important to note that
anti-VAR2CSA IgG titers were significantly higher in
the spy-VLP immunized group compared to the control
when examining serum samples obtained at day 137.
VLP display had a significant positive effect on the
avidity of the humoral response against both Pfs25 and
VAR2CSA. e avidity index values determined for anti-
Pfs25 IgG in sera from mice immunized with soluble
Pfs25 formulated in aluminum hydroxide were catego-
rized as “low” to “intermediate” affinity whereas IgG in
corresponding serum samples from Pfs25 spy-VLP immu-
nized mice were categorized as “high affinity”. e avidity
of anti-VAR2CSA IgG was also higher after vaccination
with VLPs compared to vaccination with uncoupled pro-
tein. is might be of importance since the avidity of
anti-VAR2CSA IgG has been associated with protection
against placental malaria [39]. is might seem somewhat
surprising as one might predict that VLP display could
lower the average avidity of induced antibodies since the
multivalent interaction of the displayed antigen with B
cell receptors would lead to activation of lower affinity B
cells. However, the ability of virus-like display to break B
cell peripheral self-tolerance and activate anergic B cells,
perhaps via signaling through IgD, may contribute to the
observed higher avidity. ese activities would permit the
generation of antibody secreting plasmablasts through
tolerized naïve B cells and self-reactive intermediates gen-
erated during somatic hypermutation and this would not
be the case with monomeric antigen. is situation could
result in a more diverse antibody repertoire that would
drive selection of higher affinity clones [40–42].
Comparison of IgG subclasses revealed a significantly
(2–2.5 fold) higher induction of anti-Pfs25 IgG2b in Pfs25
spy-VLP vaccinated mice compared to the control group
(such a difference was not observed in the VAR2CSA
study). In mice, IgG1 is associated with a T-helper cell
type 2 (2)-like response, while a 1 response is asso-
ciated with the induction of IgG2a, IgG2b, and IgG3
antibodies [43]. IgG2b (and IgG2a) exhibit the strongest
binding to Fc receptors [44] and has a higher capacity for
complement fixation than IgG1 [45]. Both Pfs25 vaccines
were formulated in aluminum hydroxide (Statens Serum
Institut, Copenhagen, Denmark), which is a known stim-
ulator of 2 responses. It is interesting that the Pfs25
spy-VLP vaccine was able to induce a significantly higher
IgG2b level, indicating a boosted 1 response. e IgG
subclasses can contribute to clearance of pathogens by
different mechanisms. Accordingly, the induction of a
broad subclass and a balanced 1/2 response might
be desirable for some vaccines. AP205 VLPs expressed
in E. coli contain host cell RNA, which can activate the
innate immune system via TLR 7 and 8. Besides, endo-
toxin levels were not measured in the VLP prepara-
tions. It is thus a possibility that bacterial RNA and/or
endotoxins have affected the profile of IgG subclasses
induced by the spy-VLP and unconjugated control vac-
cine, respectively.
For Pfs25, the spy-VLP vaccine was far more potent
than the vaccine based on soluble protein in the ability
to elicit biologically functional antibodies. Indeed when
our results are benchmarked against other Pfs25 vacci-
nation studies the tested Pfs25 spy-VLP vaccine shows
state-of-the-art efficacy [34, 46, 47]. Similar findings were
observed for VAR2CSA, the functional parasite binding-
inhibition assay showed an eight fold higher parasite
binding-inhibition capacity in sera from spy-VLP vacci-
nated mice compared to control sera. In conclusion, the
results of the Pfs25 and VAR2CSA immunization studies
demonstrate proof-of-concept for the spy-VLP system to
increase the functional antibody responses to complex
vaccine antigens.
Display of self-antigens on VLPs is an effective
approach for inducing strong antibody responses against
self-antigens providing opportunities for development of
therapeutic VLP-based vaccines [48–50]. We thus exam-
ined if the spy-VLP system was efficient in breaking self-
tolerance. ese studies, based on both cancer (PD-L1
and CTLA-4) and allergy (IL-5) associated self-antigen
targets, demonstrated a consistent ability of the spy-VLP
mediated antigen-display to facilitate induction of potent
autoantibody responses. Previously, VLP-display induced
autoantibody responses have been reported to wane over
time with a typical half-life in humans and non-human
primates of~3 months [51–53] and there has been no
evidence for boosting by endogenous self-antigen, prob-
ably since foreign T cell helper responses are required
to stimulate antibody production. It is thus interesting
that our IL-5 spy-VLP vaccine induced antigen-specific
autoantibody titers, which did not show a significant
decline over a period of 4months post immunization.
Reporting work, done in parallel and unknown to us,
Brune et al. recently published on the development of
a SpyCatcher-AP205 VLP, which was used in a similar
strategy to ours to develop VLP-vaccines based on two
malaria antigens (Pfs25 and CIDR) [54]. In that study,
proof-of-principle was established alone by showing
increased antigen-specific IgG titers in sera obtained
approximately 2weeks after immunization of mice with
the spy-VLP vaccines compared to a control vaccine. Our
study extends these results by presenting different spy-
VLP platforms, which are each used to characterize the
systems versatility for antigen display. Furthermore, spy-
VLP-induced responses in mice were analyzed over sev-
eral months and included measurement of antibody titer,
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Page 11 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
avidity and subclass distribution. Finally, we demonstrate
proof-of-concept for the spy-VLP system to increase
functional antibody responses against different pathogen
antigens as wells as to overcome B cell self-tolerance.
e requirement of simple subunit-based vaccines
to be administered with an extrinsic adjuvant, of which
only a few have been approved for human use [55], is
an impediment for vaccine development. VLP-display
of vaccine antigens might mitigate this requirement and
enable development of adjuvant-free vaccines. A generic
VLP-display platform may moreover be instrumental for
screening of vaccine candidate antigens since especially
low-immunogenic or cryptic antigens (e.g. Pfs25, HPV
L2, PfRH5) depend greatly on increased immunogenicity
for their protective potential. e ability of the spy-VLP
system to display whole antigens enables induction of
polyclonal antibody responses, which may have a supe-
rior neutralizing capacity compared to the essentially
monoclonal responses induced by epitope-based vac-
cines. Finally, the simple production of VLP vaccines that
have been made possible by the versatile spy-VLP system
raises the possibility that the spy-VLPs could be pre-
manufactured and distributed as a generic tool for use in
laboratories working on vaccine development against a
wide variety of target antigens.
Methods
Design, expression andpurication ofspy‑AP205 virus‑like
particles
e Acinetobacter phage AP205 coat protein (Gene
ID: 956335) was used to design three “spy-VLPs”, pre-
senting either SpyTag or SpyCatcher on their surface.
SpyTag-VLP was constructed by adding the SpyTag
peptide sequence (AHIVMVDAYKPTK) to N-termi-
nus of the AP205 coat protein. 2xSpyTag-VLP had the
SpyTag added to both the N- and C-terminus of the
AP205 coat protein. SpyCatcher-VLP had the Spy-
Catcher protein added to the N-terminus of AP205 coat
protein. e constructs were all designed to contain a
flexible linker between the binding tag and the AP205
coat protein; GSGTAGGGSGS (SpyTag-VLP), GGSGS
(SpyCatcher-VLP), GSGTAGGGSGS (N-terminus of
2xSpyTag-VLP) and GTASGGSGGSG (C-terminus of
2xSpyTag-VLP). Gene sequences were further modified
to contain NcoI and NotI restriction sites at the N- and
C-termini, respectively, and were codon-optimized for
recombinant expression in E. coli before being synthe-
sized by (GeneArt® Life Technologies, Germany). All
spy-VLP expression sequences were cloned into a pET-
15b vector and transformed into One Shot® BL21 Star™
(DE3) (ermo Scientific) cells. Transformed colonies
were screened by small-scale (10 mL) protein expres-
sion followed by SDS-PAGE analysis to identify the
transformed clones with the highest level of protein
expression. Expression was done in 3L shake flasks con-
taining 400 mL 2xYT media (100 µg/mL ampicillin).
Bacterial cultures were incubated for approximately 3h
(OD600= 0.6) at 37°C before they were induced with
1mM IPTG and then allowed to incubated for additional
16 h at 20 °C. Cultures were harvested by centrifuga-
tion (10,000g) and pellets were resuspended in 1xPBS
(pH=7.2) and lysed by sonication at 80% power with 5
pulsations for 2×5min on ice. Cleared bacterial lysates
were then purified by ultracentrifugation (UC) through
an Optiprep™ (Sigma) step (23, 29 and 35%) gradient,
modified from [56]. In brief, 1.2ml bacterial lysate was
loaded on top of the gradient in Polyallomer Centrifuge
open top tubes (11×60mm) (Bechmann Coulter) and
spun at 307.900 RCF (SW60Ti rotor, Beckmann Coul-
ter) for 3.30h at 16°C. UC fractions were subsequently
analyses by SDS-PAGE and VLP-containing fractions
were pooled and dialyzed in PBS (0.02% PS80, pH 7.2)
using a 300.000 MWCO membrane (Spectrum). Dialyzed
VLP samples were finally spun down at 16,000g for 2min
at 4°C to remove aggregates. Protein concentration was
determined by bicinchoninic acid assay (Sigma), follow-
ing manufacturer’s instructions.
Electron microscopy
Using the droplet method, an aliquot of VLPs was diluted
to 0.2 mg/mL in PBS. Diluted VLPs were adsorbed to
carbon and negatively stained with 2% phosphotungstic
acid (pH=7.0) for 1min. A grid was placed on the car-
bon floating on top of the 2% phosphotungstic acid stain
droplet. Excess stain was removed with filter paper. e
sample was examined with a CM 100 BioTWIN electron
microscope (Phillips) at an accelerating voltage of 80kV.
Photographic records were obtained on an Olympus
Veleta camera.
Particle size measurement bydynamic light scattering
(DLS)
VLPs were diluted to 0.2 mg/mL in PBS and spun at
15,000g for 10min at 4°C to remove aggregates. 70µl
of each VLP sample was loaded into disposable low vol-
ume cuvettes and mounted into the DLS chamber. Size
distribution was obtained by DLS measurements at 25°C
using WYATT Technology, DynaPro NanoStar, equipped
with a 658nm laser. Each sample was measured twice
with 20 runs. e mean size of the most predominant
particles in the population was calculated together with
the % polydispersity (% Pd).
Design, expression andpurication ofspy‑antigen
All vaccine constructs were designed with a VLP bind-
ing-tag (SpyCatcher or SpyTag) and a hexa histidine
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Page 12 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
purification tag at opposite termini of the antigen. A flex-
ible serine/glycine linker was inserted between the spy
binding-tag and the antigen. Gene sequences codon-opti-
mized for expression in E. coli were modified to contain
NcoI and NotI restriction sites at the N- and C-termi-
nus, respectively. Gene sequences codon-optimized for
expression in Spodoptera frugiperda insect cells were
designed with BamHI and NotI restriction sites at the
N- and C-terminus, respectively. All sequences were syn-
thesized by (GeneArt® Life Technologies, Germany). A
complete list of antigens used in this study is shown in
(Additional file2: TableS1). For expression in E. coli One
Shot® BL21 Star™ (DE3) (ermo Scientific) or SHuf-
fle® T7 Express (C3029H New England BioLabs) cells,
gene sequences were cloned in into a pET-15b vector
and expressed, as described previously (see Expression
and purification of spy-AP205 virus-like particles). For
expression in baculovirus-transfected insect cells gene
fragments were cloned into the BamHI/NotI sites of the
pAcGP67A vector (BD Biosciences). To generate recom-
binant virus particles, linearized BakPak viral DNA (BD
Biosciences) was co-transfected with pAcGP67A/Avi-L1
into Sf9 insect cells using Lipofectamine 2000 10 Rea-
gent (Invitrogen, 11668–019) and incubated at 28°C for
3–5days. Recombinant baculovirus was harvested from
the supernatant and used to generate a high-titer virus
stock, which was used for infection of High-Five insect
cells. Infected High-Five cells were incubated for 48 h
at 28°C with shaking. Expression in S2 insect cells were
done as previously described [57].
Conjugation ofvaccine antigens toVLPs
To attach vaccine antigen to VLPs, spy-VLPs and spy-
antigen were incubated overnight at 4°C at a 1:1.5 M
ratio. Antigen and VLPs were mixed in a standard phos-
phate-buffered saline buffer supplemented with 0.2 %
Polysorbate 80, pH 7.2. To prevent aggregation/precipi-
tation of the antigen-VLP conjugate it was sometimes
necessary to optimize buffer conditions by adjusting the
pH and/or ionic strength of the buffer (PD-L1 VLP vac-
cine was mixed in PBS, 0.8 M NaCl, pH 6.2). Coupling
efficiencies reported in Table1 were estimated by den-
sitometric analysis of SDS-PAGE gels (Additional file3:
FigureS2) using ImageQuant TL software. e percent-
age of VLP subunits, which had been conjugated to an
antigen via interaction between SpyTag and SpyCatcher
(% coupling efficiency), was estimated by dividing the
intensity value of the VLP subunit protein band before
reaction with antigen with the corresponding intensity
value after reaction with antigen, multiplied with 100.
is percentage was subsequently multiplied by 180 or
360 to calculate the antigen-display capacity (number of
antigens/VLP).
Immunization ofmice
Mice were immunized with the described VLP vaccines
or control vaccines prepared in a similar way as the VLP
vaccines, but where the Spy-tagged vaccine antigens
were incubated with untagged AP205 VLPs. us, the
only difference between the VLP vaccines and the con-
trol vaccines was that vaccine antigens were coupled to
the VLPs in the VLP vaccines, whereas the control vac-
cines contained soluble vaccine antigen and uncoupled
VLPs. Stock solutions of spy-VLPs contained 1 mg/ml
spy-AP205 protein and were estimated by SDS-PAGE
to be >95 % pure. All vaccines, except the VAR2CSA
based vaccines, were formulated with 2 % Alhydrogel
(Statens Serum Institut, Copenhagen, Denmark) to a
final concentration of 2mg/ml aluminum hydroxide. e
Alhydrogel was added 1h prior to intramuscular immu-
nization (50 µL/mice). e VAR2CSA vaccines were
diluted with PBS and administrated without adjuvant.
e indicated vaccine dose refers solely to the amount
of antigen. Vaccines were administered on days 0, 21 and
42. Blood samples were obtained 2weeks after the first,
second and third immunizations. Additional blood sam-
ples were collected according to the vaccine antigen as
follows: Pfs25 vaccines on days 98 and 212; VAR2CSA
vaccines on days 88 and 137; IL-5 vaccines on day 112.
BALB/c mice were used for the VAR2CSA and IL-5
immunization studies as well as for generating anti-
Pfs25 sera used in the SMFA study testing different IgG
concentrations (Table2). C57/BL6 mice were used in the
CTLA-4 and PD-L1 study as well as in the other Pfs25
immunization study (Figs.2, 3).
Antibody response measured bystandard ELISA
Serum IgG levels were measured by standard enzyme-
linked immunosorbent assay (ELISA). 96-well plates
(Nunc MaxiSorp) were coated over night at 4 °C with
vaccine protein, without SpyTag or SpyCatcher com-
ponent, (1µg/ml in PBS). Plates were blocked with 1%
BSA buffer for 1 h at room temperature (RT). Mouse
serum diluted 1:100 in blocking buffer were added in
three-fold dilutions to triplicate wells and incubated for
1h at RT. Plates were washed three times in PBS with
0.05% TWEEN 20 in between different steps. Horserad-
ish peroxidase (HRP)-conjugated polyclonal goat anti-
mouse IgG (A16072, Life Technologies, Denmark) was
diluted 1:3000 in blocking buffer and incubated for 1h.
Finally, color reactions were developed for 7min by add-
ing o-phenylenediamine substrate. e HRP enzymatic
reaction was terminated by addition of 2.5 M H2SO4
and the optical density was measured at 490 nm using
an ELISA plate reader (VersaMax Molecular Devices).
Serum IgG endpoint titers were estimated using a cutoff
of OD490=0.2.
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Thrane et al. J Nanobiotechnol (2016) 14:30
Serum IgG subclass proling byELISA
IgG subclass profiling of anti-Pfs25 and anti-VAR2CSA
mouse sera followed a standard ELISA protocol, as
described above. However, serum IgG levels were first
normalized based on pre-determined ELISA OD490 val-
ues by dilution. Subsequent to incubation of IgG-nor-
malized serum samples, secondary HRP-conjugated
antibodies were used for detection of total mouse IgG
(A16072, Life Technologies, Denmark) along with
mouse IgG subclasses; IgG1 (A10551, ermo Fisher),
IgG2a (M32307, ermo Fisher) and IgG2b (M32507,
ermo Fischer). For each serum sample, the OD490
value obtained by detection with subclass-specific anti-
mouse IgG secondary antibodies was divided by the
OD490 obtained by detection with anti-mouse total IgG
secondary antibody. ese relative measures were finally
used for comparison of IgG subclass profiles in anti-Pfs25
and anti-VAR2CSA sera from spy-VLP immunized and
mice vaccinated by the uncoupled soluble protein control
vaccines.
Measuring avidity ofserum IgG
Recombinant Pfs25 or VAR2CSA (1µg/ml in PBS) was
coated on Nunc MaxiSorp plates overnight at 4°C. Plates
were incubated with blocking buffer (1% BSA) for 1h at
room temperature (RT). Plates were washed three times
in between different steps. Before adding the anti-sera for
incubation with the capture antigen, IgG levels in individ-
ual serum samples were equalized by dilution. is was
done based on pre-determined OD490 ELISA values. IgG-
normalized serum samples were added (50µl per well) to
the ELISA plate in triplicates, and incubated for 1h at RT.
e ELISA wells were subsequently washed three times
in PBS with 0.05% TWEEN 20 and 50µl of freshly made
8M Urea was then added to the wells for 5min (reference
plates were incubated with PBS). ELISA plates were then
washed three times with PBS (0.05% TWEEN 20). HRP-
conjugated polyclonal goat anti-mouse IgG (A16072, Life
Technologies, Denmark) was diluted 1:3000 in blocking
buffer and incubated for 1h. Finally, color reactions were
developed for 7min by adding o-phenylenediamine sub-
strate. e HRP enzymatic reaction was stopped by add-
ing 2.5M H2SO4 and the optical density was measured at
490nm using an ELISA plate reader (VersaMax Molecu-
lar Devices). IgG avidity index values were calculated as
the ratio of the mean OD value of urea-treated wells to
PBS control wells multiplied by 100.
Purication ofIgG fromanti‑Pfs25 mouse serum
Total IgG was purified using Protein G columns (Pierce,
USA) from anti-Pfs25 mouse serum samples. Briefly,
protein G columns were equilibrated with binding
buffer (Immunopure IgG, Pierce, USA) after which a 1:1
mixture of sera and binding buffer was allowed to flow
through under gravity, the columns were then washed
and eluted with elution buffer (Immunopure IgG, Pierce,
USA). e eluted fraction was collected in 1 M Tris–
HCl (pH 9.0, Teknova, USA) and transferred for buffer
exchange to Amicon centrifugal filters (Millipore, USA)
using PBS. e eluent was concentrated in PBS (Invitro-
gen, UK) and filtered using a 0.22 μm Millipore Ultrafree
sterile centrifugal unit.
Standard Membrane‑Feeding Assay (SMFA)
Two independent SMFA analyses were performed. e
first SMFA analysis was done to compare the transmis-
sion blocking (TB) activity of anti-Pfs25 sera obtained by
immunization of mice with the Pfs25 spy-VLP vaccine
or with the corresponding control vaccine. is SMFA
experiment was conducted as previously described [30,
58]. Briefly, 30μl of mouse serum obtained at day 56 was
mixed with 90μl of naïve human serum and 150μl of
invitro gametocyte cultures of the P. falciparum (NF54)
laboratory line. e mixture was fed to Anopheles ste-
phensi mosquitoes through a membrane feeding appara-
tus. Pre-immune sera served as controls. Fully engorged
mosquitoes were separated and held at 26 °C. Seven
days later, midguts of 20 mosquitoes were examined for
oocysts. e observed transmission reducing activity of
serum was determined as the percentage reduction in the
median oocyst number in test samples compared to that
in controls. e experiment was considered valid when
at least 85% of the mosquitoes feeding on control sera
were infected. e other SMFA study, testing different
concentrations of IgG purified from anti-Pfs25 spy-VLP
mouse sera, was done as described [59]. Briefly, mature
P. falciparum strain NF54 Stage V gametocytes (adjusted
to parasiteamia of 0.15% ± 0.05%) were mixed with dif-
ferent concentrations of purified IgG from Pfs25 spy-VLP
vaccinated mice. is mixture was then fed to 4–6day
old starved female A. stephensi (SDA 500) mosquitoes via
a parafilm® membrane. e mosquitoes were maintained
at 26 °C and 80% relative humidity. After 7days mosqui-
toes (n=20 per group) were dissected and the number
of oocysts counted per mosquito midgut was recorded.
Percent reduction in infection intensity was calculated
relative to the respective control IgG (anti-ovalbumin
mouse serum) tested in the same assay.
Inhibition ofbinding assays
Plasmodium falciparum (FCR3 genotype) parasites were
maintained in culture as described [60]. Parasites were
panned on BeWo cells to select for a chondroitin sulphate
A (CSA)-binding phenotype, as described [61]. Parasite
DNA was labeled with Tritium by overnight incorpora-
tion of titrated hypoxanthine. A 96-well plate (Falcon)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 16
Thrane et al. J Nanobiotechnol (2016) 14:30
was coated with 2 μg/ml of Decorin (Sigma-Aldrich)
overnight and blocked with 2% bovine serum albumin
(Sigma) as described [60]. Tritium labeled late-stage
infected erythrocytes (IEs) were MACS purified and
added to the 96-well plate in a concentration of 200,000
cells per well. Titrations of serum were added in a total
volume of 100μl in triplicate wells. After incubation for
90min at 37 °C, unbound IEs were washed away by a
pipetting robot (Beckman-Coulter). e remaining IEs
were harvested onto a filter plate (Perkin-Elmer). After
addition of scintillation fluid (Perkin-Elmer) the counts
per minute (CPM) recording the number of non-inhib-
ited IE was determined by liquid scintillation counting
on a Topcount NXT (Perkin-Elmer). Data were adjusted
to percentage of binding by dividing test result with the
mean value of wells with IE incubated without serum.
Statistical analysis
All statistical analysis was done using non-parametric,
two tailed, Mann–Whitney Rank Sum Test. Statistical
significance was defined as P<0.05.
Ethical statements
e animal studies were approved by the Danish Ani-
mal Experiments Inspectorate. Approval number:
2013-15-2934-00902/BES.
Additional les
Additional le1: Figure S1. Recombinant expression of spy‑VLP in E.
coli. Reduced SDS‑PAGE showing unmodified AP205 structural protein
and structural AP205 proteins modified by Spy elements. Protein bands
match the theoretical sizes of SpyCatcher‑AP205 (27 kDa), 2xSpyTag‑
AP205 (18.5 kDa), SpyTag‑AP205 (16.5 kDa) and unmodified AP205
(14.5 kDa).
Additional le2: Table S1. Tested spy‑vaccine antigens.
Additional le3: Figure S2. Estimated coupling efficiencies of spy‑
VLP/antigen reactions. SDS‑PAGE gels demonstrating that SpyTag or
SpyCatcher fused antigens had reacted with corresponding spy‑VLPs. (A)
Survivin, (B) CIDR, (C) IL‑5, (D) Pfs25, (E) Ag85A, (F) CSP, (G) HER2, (H) PCSK9,
(I) CTLA‑4, (J) PD‑L1 and (K) VAR2CSA. Reactions were incubated for at
least 12 h at 4OC.
Additional le4: Figure S3. SDS‑PAGE and Western blot of Pfs25‑Spy‑
Catcher. The figure shows a SDS‑PAGE of recombinant Pfs25‑SpyCatcher
run under reducing and non‑reducing conditions (left) and a correspond‑
ing western blot (right) where the Pfs25‑Spycatcher was detected using
mAb 4B7 antibody followed by hrp‑conjugated goat anti‑mouse second‑
ary antibody.
Additional le5: Figure S4. Comparison of the immunogenicity of IL‑5
SpyTag‑VLP and IL‑5 2xSpyTag‑VLP vaccines. BALB/c mice were immu‑
nized with 5 µg antigen (SpyCatcher‑IL‑5) mixed with either SpyTag‑VLP
with 180 binding motifs (n = 4, open squares) or 2xSpyTag‑VLP (n = 5,
filled circles) with 360 potential binding motifs. Anti‑IL‑5 IgG titers were
determined by standard ELISA using sera obtained two weeks the 3rd
immunization. Anti‑IL‑5 IgG titers were higher in mice immunized with
the 2xSpyTag‑VLP vaccine (filled circles) compared to the SpyTag‑VLP
vaccine (open squares). The difference approached statistical significance
(P = 0.06, Mann–Whitney Rank Sum test).
Authors’ contributions
ST and CMJ performed all experiments, except WR, MVDVB, GJVG and RS per‑
formed the SMFA, SM, WADJ, SC and MR generated vaccine antigens and MAN
performed the parasite‑binding‑inhibition assay. ST, TG, JTS, TGT, AS and AFS
designed the experiments. AFS and ST wrote the paper. All authors analyzed
the data. All authors read and approved the final manuscript.
Author details
1 Centre for Medical Parasitology at the Department of Immunology
and Microbiology, University of Copenhagen, Copenhagen, Denmark.
2 Department of Infectious Diseases, Copenhagen University Hospital,
Copenhagen, Denmark. 3 Kilimanjaro Clinical Research Institute, KCMC, Moshi,
Tanzania. 4 ExpreS2ion Biotechnologies, SCION‑DTU Science Park, Hørsholm,
Denmark. 5 Department of Medical Microbiology, Radboud University Medical
Center, Nijmegen, The Netherlands. 6 Laboratory of Cellular Oncology, National
Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Acknowledgements
The authors would like to thank Jens Hedelund Madsen, Anne Corfitz, Elham
Marjan Mohammad Alijazaeri and Nahla Chehabi for technical assistance. We
would like to thank Susheel Kumar Sinhg for the kind donation of mAb 4B7
and The Biophysics Facility—Protein Structure and Function Program from
Center for Protein Research, Copenhagen, for assisting us with DLS measure‑
ments. We would like to thank Dr. Carole Long and Dr. Kazutoyo Miura, NIH,
for assisting with SMFA analysis, and the PATH Malaria Vaccine Initiative for
their funding support to the NIH for this effort. This study was supported by
DANIDA, IdMalVac from the Danish Research Councils (Grant 13127), PlacMal‑
Vac under the European Union Seventh Framework Programme, FP7‑HEALTH‑
2012‑INNOVATION under grant agreement no. 304815 and through a grant
from Bill and Melinda Gates Foundation Grant ID APP178218.
Competing interests
ST, CMJ, TG, MAN, TGT, AS and AFS are inventors on a patent application cover‑
ing the described technology.
Received: 25 February 2016 Accepted: 1 April 2016
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