REVIEW ARTICLE

Plasmodium meets AAV—the (un)likely marriage of

parasitology and virology, and how to make the match
Franziska Hentzschel1,2,3, Anne-Kathrin Herrmann2,3, Ann-Kristin Mueller1 and Dirk Grimm2,3,4
1 Department of Parasitology, Center for Infectious Diseases, Heidelberg University Hospital, Germany
2 Department of Virology, Center for Infectious Diseases, Heidelberg University Hospital, Germany
3 Cluster of Excellence CellNetworks, Heidelberg, Germany
4 German Center for Infection Research (DZIF), Heidelberg, Germany

Correspondence
D. Grimm, Heidelberg University Hospital,
BioQuant BQ0030, Im Neuenheimer Feld
267, D-69120 Heidelberg, Germany
Fax: +49 6221 5451481
Tel: +49 6221 5451339
E-mail: dirk.grimm@bioquant.uni-heidelberg.
de
The first two authors contributed equally
(Received 3 March 2016, revised 24 March
2016, accepted 21 April 2016, available
online 9 May 2016)
The increasing use of screening technologies in malaria research has substan-
tially expanded our knowledge on cellular factors hijacked by the Plasmod-
ium parasite in the infected host, including those that participate in the
clinically silent liver stage. This rapid gain in our understanding of the hep-
atic interaction partners now requires a means to validate and further disen-
tangle parasite–host networks in physiologically relevant liver model systems.
Here, we outline seminal work that contributed to our present knowledge on
the intrahepatic Plasmodium host factors, followed by a discussion of surro-
gate models of mammalian livers or hepatocytes. We finally describe how
Adeno-associated viruses could be engineered and used as hepatotropic tools
to dissect Plasmodium–host interactions, and to deliberately control these
networks for antimalaria vaccination or therapy.

doi:10.1002/1873-3468.12187 Keywords: 3D culture; AAV; adeno-associated virus; liver; malaria;

Plasmodium
Edited by Wilhelm Just

Malaria is a devastating infectious disease that kills
438 000 people a year worldwide [1], mainly children
—one every 30 s. An effective vaccine is widely
regarded as an essential tool for sustainable malaria
control. To date, attenuated whole parasites and wild-
type parasites under drug cover have been shown to
induce complete sterilizing immunity [2–6], but numer-
ous practical issues make it unlikely that this vaccine
type could be licensed for common use in humans.
This may differ for the latest subunit of malaria vac-
cine candidates, which are best represented by Mos-
quirix/RTS,S and which have recently obtained market
authorization by the European Medicines Agency [7].
While a pivotal step in the field and a significant scien-
tific achievement, the enthusiasm is unfortunately
dampened by clinical data implying that Mosquirix
only yields modest protection against malaria infection
[8–10]. In fact, it was concluded that a bed net remains
more effective than this vaccine.
In general, a seminal reason for the persisting lack
of better vaccines is the gaps in our understanding of
the interactions of the malaria parasite, Plasmodium,
with its human host in vivo. This is especially the case
for the first site in the infected body, namely, the liver
—the bridgehead of malaria infection where Plasmod-
ium develops further after the bite of an infected

Abbreviations
AAV, Adeno-associated viral; CSP, circumsporozoite protein; ER, endoplasmic reticulum; FGF, fibroblast growth factor; HBV, Hepatitis B
virus; HCV, Hepatitis C virus; HGF, hepatocyte growth factor; HO-1, heme oxygenase 1; HSPGs, heparan sulfate proteogylcans; IFN, inter-
feron; iPSCs, induced pluripotent stem cells; ISGs, interferon-stimulated genes; L-FABP, liver-fatty acid-binding protein; LSEC, liver sinusoidal
endothelial cells; MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma differentiation-associated gene 5; miRNA, microRNA;
MPCC, micropatterned cocultivation; MSC, mesenchymal stem cells; ORFs, open reading frames; PV, parasitophorous vacuole; ss, single-
stranded; TRAP, thrombospondin-related adhesion protein; UIS3, up-regulated in sporozoites 3.

FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies 2027
Plasmodium meets AAV F. Hentzschel et al.

Anopheles mosquito. In principle, a single infectious
sporozoite reaching the liver and completing its intra-
hepatic development suffices to give rise to up to
10 000 merozoites capable of invading erythrocytes
and causing malaria pathology [11,12]. Before produc-
tively invading a final host hepatocyte, sporozoites
transmigrate through multiple hepatocytes, whereby
the parasites breach the cell plasma membrane and
glide through the cytosol before exiting the cell [13]
(Fig. 1). Eventually sporozoites initiate a transforma-
tion process in their final host cell and develop into
liver trophozoites. Multiple rounds of DNA replication
and nuclear divisions without subsequent mitosis (i.e.,
schizogony) then lead to the formation of a multinu-
clear syncytium, the so-called schizont. Subsequent
repeated invaginations of the schizont plasma mem-
brane result in the generation of individual first-gen-
eration merozoites. Finally, the infected host cell
detaches from its surroundings, and merozoites pack-
aged in vesicles called merosomes bud off the detached
cell and are released into the blood stream, to initiate
the intraerythrocytic stage [14].
Curiously, as noted, parasite propagation within hep-
atocytes is not accompanied by disease symptoms;
instead, malaria pathology starts with the transition of
parasites from the liver into the blood where they
repeatedly infect red blood cells. Hence, only interven-
tions that target the clinically silent pre-erythrocytic
stages, that is, before the parasite enters the blood, can
mediate complete sterilizing protection and thereby pre-
vent malaria disease. In turn, this creates an urgent need
to fully dissect the molecular repertoire of intrahepatic
Plasmodium stages and their interplay with the host
cells. It is, thus, even more puzzling how poorly these
factors and mechanisms have been characterized to
date, as compared to the pathogenic erythrocytic Plas-
modium stages and also considering that the intrahepatic
stages were discovered more than 60 years ago.
In this review, we first summarize recent pivotal
advances in identifying host factors that promote or
restrict parasite liver-stage development, and point out
remaining gaps in our knowledge of host contributions
to hepatic malaria infection. Subsequently, we high-
light newly emerging key technologies and strategies
that should ultimately permit dissection of parasite–
host interactions within hepatocytes in much greater
detail. Our specific focus is on (a) novel surrogate
models of whole livers that could replace animal

Fig. 1. Intrahepatic stages in the Plasmodium life cycle. (1) Traversal through Kupffer cell(s); (2) traversal through hepatocyte(s); (3)
attachment to final hepatocyte; (4) invasion via moving junctions; (5) dedifferentiation; (6) schizogony; (7) invagination of parasitophorous
vacuolar membrane; (8) egress. Indicated are major interacting host factors and, where occurring, their dysregulation (arrows up or down).
CSP, circumsporozoite protein; FABP, fatty acid binding protein; HSPG, heparan sulfate proteoglycans; IFN, interferon; ISG, interferon-
stimulated genes; LSEC, liver sinusoidal endothelial cell; PVM, parasitophorous vacuolar membrane; TRAP, thrombospondin-related
adhesion protein; UIS3, up-regulated in sporozoites 3.

2028 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
experimentation in the future, as well as on (b) recom-
binant Adeno-associated viruses as a powerful, versa-
tile, and ideally suited tool to study or perturb
Plasmodium–host interactions ex or in vivo.
Wanted ex or in vivo—host factors
involved in intrahepatic Plasmodium
development
Sporozoite infection follows a very specific path in the
mammalian host, largely dictated by interactions with
distinct cellular factors. It is initiated by the extravascu-
lar inoculation of infectious single-celled sporozoites
into the skin by anopheline mosquito bites [15,16]. Once
deposited into the skin, they reach and invade the vascu-
lature to be passively transported by the blood flow until
they eventually arrive at the liver [17]. There, the infec-
tious malarial sporozoites arrest at the liver endothelium
due to a specific interaction between both, their major
surface proteins, circumsporozoite protein (CSP) and
thrombospondin-related adhesion protein (TRAP), with
highly sulfated heparan sulfate proteogylcans (HSPGs)
presented by stellate cells and hepatocytes [18–20].
Within the infected hepatocyte, the highly differentiat-
ing and replicative parasite resides in a membranous
compartment. In addition to its own plasma membrane,
this so-called parasitophorous vacuole (PV) physically
separates the parasite from the host cell cytoplasm
[11,21] (see also Fig. 1).
To date, only few studies have investigated which
host-specific factors next to HSPGs are exploited by
the parasite, and thereby contribute to the establish-
ment and maintenance of its intrahepatic develop-
ment. The first host factors involved in Plasmodium
sporozoite invasion were identified either as a direct
protein interaction partner of a Plasmodium-specific
protein, or because they were already known to be
important for infection with other hepatotropic patho-
gens such as Hepatitis C virus (HCV). In this manner,
the tetraspanin CD81, a surface molecule on hepato-
cytes and one of the main receptors for HCV [22,23],
was identified as a crucial host factor for hepatocyte
invasion of both, P. falciparum and P. yoelii sporo-
zoites [23–25]. Similarly, Cha and coworkers deter-
mined a critical role of host CD68 as a putative
receptor for sporozoite invasion and traversal of
Kupffer cells [26]. A final relevant example is the host
protein L-FABP (liver-fatty acid-binding protein) that
was found to specifically interact with the PV mem-
brane-resident protein, UIS3 (up-regulated in sporo-
zoites 3, [27]) within the infected hepatocyte, and
which is presumably required for fatty acid uptake
into the parasite [28].
FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
Plasmodium meets AAV
A limitation of these approaches, which focus on
single factors, is that the assessment of individual pro-
teins is labor-intensive and requires a priori knowledge
of promising candidates. Therefore, a number of
groups conducted high-throughput screens with the
aim to evaluate the contribution of a wider variety of
potential host factors to Plasmodium liver-stage devel-
opment. Typically, these screens were based on siRNA
knock-down of a set of genes in human hepatoma cell
lines prior to infection with the rodent parasite P. ber-
ghei. In one such comprehensive approach, 727 genes
belonging to the human kinome were assessed individ-
ually and stringently, resulting in the identification of
five kinases that are required for P. berghei liver stages
[29]. One of the hits was PKCf, a member of the PKC
family. More specifically, it belongs to the atypical
PKCs that are involved in various cellular processes
such as cell growth and survival as well as regulation
of NFΚB [30]. Further in vitro experiments revealed
that PKCf is required for sporozoite invasion into
hepatocytes, but not for liver-stage development. Inter-
estingly, mice that had received a siRNA-targeting
PKCf had reduced liver burdens and exhibited a delay
in the onset of blood-stage parasitemia [29].
In a similar approach, all lipoprotein-related host
factors—a total of 53 genes—were screened for their
contribution to Plasmodium infection, leading to the
identification of the class B, type I scavenger receptor
(SR-BI) as an important candidate [31]. The authors
of this work could show that SR-BI is required for
both, invasion and intracellular development of P. ber-
ghei and P. falciparum, respectively. A lack of SR-BI
impedes liver-stage development in vitro and in vivo,
albeit studies with blocking antibodies revealed that no
direct interaction is needed between SR-BI and the
infectious sporozoite [31–33]. It is intriguing to specu-
late that the cholesterol, which is provided by SR-BI
and incorporated into the plasma membrane, is impor-
tant for parasite invasion; perhaps it may also serve as
a lipid source during growth. Notably, HCV entry
depends on SR-BI as well, and cholesterol and antago-
nists against this receptor are currently under clinical
investigation [34–37].
In the aforementioned studies, the assays were
restricted to a preselected group of genes due to the
limitations of RNAi-based screening platforms. Also,
only loss-of-function effects on parasite development
could be assessed, but not the natural response of the
host cell to infection. In fact, transcriptional changes
upon productive hepatocyte infection are not restricted
to the Plasmodium parasite, but the host cell, too,
responds with numerous alterations in gene expression.
To begin to unravel these, Albuquerque and colleagues
2029
Plasmodium meets AAV
performed an unbiased assessment of the global host
response to Plasmodium infection, using high-through-
put microarray technology to measure genome-wide
transcriptional changes in murine hepatoma cells upon
infection with P. berghei [38]. Strikingly, they found
1064 host genes to be expressed differentially at one or
more of the selected time points (6, 12, 18, or 24 h
post infection), and noted that transcriptional changes
followed a coordinated and timed pattern. Based on
their data, it seems that once infected, the host cell ini-
tially mounts a stress response to the presence of the
parasite. This is followed by subsequent engagement of
diverse metabolic pathways, presumably to cope with
the parasite’s rapid proliferation and energy needs. At
all times, genes involved in cell death and apoptosis
are regulated differentially in order to maintain viabil-
ity of the host cell [39–41]. Furthermore, the develop-
ing intrahepatic parasite is highly metabolically active
and up-regulates various pathways, such as redox,
mitochondrial citric acid cycle, apicoplastic FAS II
pathway, and glycolysis, among several others [42].
Notably, some of the hits from the work mentioned
above have already been further validated in follow-up
studies in vivo. For example, In
acio and colleagues
showed that parasite infection induces an ER (endo-
plasmic reticulum) stress response in hepatocytes, acti-
vating proteins of the unfolded protein response
pathway [43]. Exogenous pharmaceutical activation of
the ER stress response in vivo prior to infection with
P. berghei sporozoites yielded a higher number of liver
stages, but no size change (i.e., maturation status), at
24 h after infection. Another example is heme oxyge-
nase 1 (HO-1) which was identified as a main host fac-
tor required for rodent Plasmodium infection [44].
Interestingly, while the initial screening that identified
HO-1 to be up-regulated upon infection was per-
formed in hepatoma cell lines, immunohistochemical
stainings of liver sections revealed that HO-1 is
induced even more robustly in Kupffer cells (liver-resi-
dent macrophages). Congruently, mechanistic studies
revealed that HO-1 plays an anti-inflammatory role
protecting liver stages from the immune response of
the host, and siRNA-mediated knock-down of this fac-
tor in vivo nearly abolished liver-stage infection. This
example nicely illustrates that while in vitro screenings
in single cell types can provide clues on which factors
might be involved, it is key to dissect the parasite–host
interactions in a more physiologically relevant system
that encompasses all cell types, to fully understand the
host’s contribution(s).
Accordingly, it should be rewarding to try and iden-
tify host factors related to Plasmodium infection
directly in vivo in the mouse liver. Indeed, a recent
2030 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
measurement of transcriptome changes in the murine
liver upon Plasmodium infection has revealed a strong
induction of 89 interferon-stimulated genes (ISGs)
linked to the type I interferon (IFN) signaling pathway
[45,46]. It was demonstrated that the host hepatocyte
senses intrahepatic parasite infection by pattern recog-
nition of cytoplasmic Plasmodium RNA via the
MDA5 (melanoma differentiation-associated gene 5)/
MAVS (mitochondrial antiviral signaling protein)
pathway. This in turn leads to the transcription of
IFN-a/b that finally triggers a type I IFN response in
hepatocytes and leukocytes, in an autocrine and para-
crine manner. Knock-out of this type I IFN response
increased parasite liver infection, while an induction of
type I IFN prior to Plasmodium infection led to a sig-
nificant reduction in liver parasite load. While it
remains unclear how parasite RNA reaches the hepa-
tocyte cytoplasm, the study by Liehl et al. demon-
strates that, in contrast to previous assumptions, the
host innate immune response indeed recognizes and
acts against Plasmodium liver-stage infection [45].
Importantly, not all host responses to Plasmodium
infection can be measured as changes in mRNA levels.
Instead, the host cell might also respond with alter-
ations on the translational or post-translational level,
for example, by adjusting the phosphorylation status
of members of different signaling cascades. This vital
type of host responses would therefore be missed when
only quantifying transcript levels. Consequently, one
study by Kaushansky et al. addressed perturbations on
the protein level after Plasmodium infection using
reverse-phase protein microarray technology [47].
Although restricted to a subset of proteins by the anti-
body selection, the authors were able to detect a gener-
ally antiapoptotic, proliferative, and antiautophagic
environment in the host cell. Specifically, the tumor
suppressor, p53, was largely decreased in infected hep-
atocytes, while the phosphorylated form of Bcl-2,
involved in antiapoptotic signaling, was elevated. A
deliberate increase in p53 levels, either by overexpres-
sion or by application of the drug, nutlin-3, that inhi-
bits p53 degradation, reduced liver-stage burden
in vitro and in vivo. Of note, drug-mediated increase in
p53 is currently being evaluated in several clinical trials
as a potential treatment for a variety of cancers [48].
This exemplifies how the study of malaria host factors
as therapeutic targets might benefit from synergisms
with other research areas sharing the same focus.
Next to proteins, an additional class of important
host factors are microRNA (miRNA), roughly 22-
nucleotide-long RNA that are master regulators of at
least 60% of all mammalian genes and as such play
central roles in pathological processes, including cancer
F. Hentzschel et al.
and pathogen infections [49]. Recently, we dissected
the effects of Plasmodium sporozoite infection on host
miRNA expression under physiologically relevant con-
ditions in livers of adult mice [50]. Combined microar-
ray and PCR screens using total liver RNA obtained
after infection with either wild-type or attenuated Plas-
modium strains revealed not only an up-regulation of
IFN-associated genes (confirming and extending previ-
ous work, see above and [2,3]) but also a dysregulation
of 11 of 698 studied miRNA. In particular, by focus-
ing on known immunoregulatory miRNA, we found
miR-155 to be highly up-regulated upon Plasmodium
liver-stage infection. Moreover, we could identify
Kupffer cells as the main source of this particular
miRNA in the adult mouse liver. Intriguingly, miR-
155 is one of the best characterized proinflammatory
miRNA and plays a fundamental role in mammalian
host defense mechanisms against pathogen challenge
[51]. In the context of Plasmodium, we thus concluded
that miR-155 boosts the immune response against liver
stages. Additional evidence was that exogenous overex-
pression of this miRNA using hepatotropic Adeno-
associated viral (AAV) gene delivery vectors improved
the vaccination of mice with genetically attenuated
parasites [3,4]. In turn, this reduced the number of
immunizations needed to achieve sterile protection
against rechallenge with wild-type parasites from three
to one [50].
Particularly noteworthy about this study is that it
illustrates two major persisting challenges in the field
of malaria liver-stage research that need to be over-
come and that are in the focus of the remainder of this
article. (1) The first is related to the facts that (i) the
infection rate of liver stages is typically very low,
implying that subtle changes around the local infection
site are easily overlooked among the vast majority of
uninfected cells, and that (ii) there are differences
between rodent and human Plasmodium strains that
have to be accounted for. For example, P. berghei
infection seems to be supported by the hepatocyte
receptor c-Met that is activated by the hepatocyte
growth factor, while this is not the case for P. falci-
parum infections [52,53]. In addition, (iii) although
rodent models are physiologically highly relevant, their
routine use is hampered by ethical, financial, and logis-
tical concerns that are inherently associated with ani-
mal experimentation. Consequently, there is a strong
desire to implement and harness novel surrogate
ex vivo models of an intact liver or of specific hepatic
cell types that circumvent the use of live animals, and
that can be customized for a given Plasmodium strain
and/or experimental question. (2) The second challenge
in the field is to concurrently devise means to
FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
Plasmodium meets AAV
deliberately, specifically, and robustly control the
expression of putative host factors in live animals and/
or in these new surrogate systems, including gene over-
expression or inhibition, in order to fully dissect their
role in the hepatic Plasmodium stages. Above, we have
already briefly mentioned AAV vectors as one possible
solution for this second challenge. We further make a
case for these particular tools below, by highlighting
most recent progress in their successful use in livers in
in vivo or in ex vivo organotypic models, including
their engineering toward functionality in human hepa-
tocytes. First, however, we discuss selected examples
of promising liver replacement systems for the study of
intrahepatic Plasmodium stages (Fig. 2).
Back to the future—From whole livers
to organotypic models and back to
chimeric organs
At first glance, it may appear straight-forward to use
established hepatic cell lines as a liver surrogate, con-
sidering the benefits of indefinite growth and limited
costs that are inherent to transformed cell lines. Yet,
there are numerous reasons that argue against this par-
ticular strategy, most notably the fact that the histo-
typic and phenotypic characteristics of cell lines as
well as their gene/miRNA expression profiles differ
substantially from their cellular counterparts within an
intact liver. Adding to the seminal differences is that
cells in culture typically grow in two dimensions (2D),
whereas in the liver, hepatocytes and all the other cell
types are embedded in a three-dimensional (3D) envi-
ronment providing special architecture and hemody-
namic properties. Using primary liver cells instead of
cell lines increases the physiological relevance but does
not solve the problem of an unnatural and inadequate
microenvironment, comprising the lack of an extracel-
lular matrix. Moreover, primary cells are difficult to
obtain and culture in a differentiated state, particularly
for longer periods. The latter, however, may be
required to fully mimic processes that can take days or
weeks in the intact liver, such as the establishment of
an anti-Plasmodium immune response.
For all these reasons, major efforts have been under-
taken and are currently intensifying to create more
sophisticated, multicellular, and organotypic ex vivo
liver models that are as close as possible to the natural
environment, and that are ideally also compatible with
medium- to high-throughput experimental strategies.
Examples for such systems that have been developed
and tested before include 3D sandwich cell cultures,
microfluidic perfusion arrays, bioreactors, biochips, or
hanging droplets, to name a few. These systems find
2031
Plasmodium meets AAV F. Hentzschel et al.

Fig. 2. Surrogate liver models to study Plasmodium liver stages. (A) Types of ex vivo cell culture systems and cell sources. See text for
details. (B) In vivo systems. Top: Creation of humanized mice by partial depletion of murine (mu) hepatocytes, followed by transplantation
of, and liver repopulation with, human (hu) hepatocytes. Bottom: Isolated livers can be decellularized using combinations of physical and
chemical methods, followed by recellularization of the remaining liver scaffold with human hepatocytes (and other cell types).

broad utility in biomedical research, from hepatotoxic-
ity testing of pharmaceutical compounds, to investiga-
tions into liver cell physiology and pathology. For
more details on these various systems, we refer the
reader to excellent previous review articles (e.g., [54–
56]). In the following, we will specifically highlight two
ex vivo cell culture models that have recently been
shown to support Plasmodium liver-stage infections
and that, in our opinion, hold great promise for future
malaria research for reasons explained below.
The first of these two systems, reported by Ng and
colleagues in 2015 [57], capitalizes on the possibility to
differentiate induced pluripotent stem cells (iPSCs,
[58,59]) into so-called iPSC-derived hepatocyte-like
cells or, for short, iHLCs (for review see [60])
(Fig. 2A). This was achieved using a 20-day differenti-
ation protocol involving iPSC culturing in a mono-
layer on matrigel and exposure to various agents
including fibroblast growth factor (FGF), hepatocyte
growth factor (HGF), and oncostatin M. Remarkably,
as demonstrated via immunostaining for specific mark-
ers of parasite liver stages (HSP70 and MSP-1), the
iHLCs were susceptible to infection with sporozoites
from four different Plasmodium species—berghei, yoelii
(both rodent), falciparum, and vivax (both human).
This was in line with the typical hepatocyte morphol-
ogy of the iHLCs as well as their expression of proto-
typical hepatocyte markers such as albumin and
alpha-1-antitrypsin. Moreover, they expressed SR-B1
and CD81, which are both host entry factors for Plas-
modium (as well as for HCV). Further interesting was
that the cells acquired Plasmodium susceptibility
already at the hepatoblast stage on day 15 in the dif-
ferentiation protocol, albeit liver-stage development
seemed to be delayed or perturbed as compared to the
more mature cells on day 20. Finally noteworthy is
that additional treatment of the cells with the small
molecule FPH1 advanced the maturation process and
eventually rendered the cells sensitive to the anti-
malaria drug primaquine, exemplifying the potential of
this system for future phenotypic drug screens.
As compared to established hepatocyte cell lines, the
use of iHLCs provides a variety of advantages, most
important of which is that they more accurately reca-
pitulate parasite–host interactions. At least from a
technical standpoint, the iHLC system is also superior
to primary human cells since the iPSC-derived cells are
renewable in culture. Another unique trait and benefit

2032 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
is that iPSCs and thus iHLCs can be derived from
somatic cells from any human donor, offering the pos-
sibility to include a broad spectrum of the population
in future assays with this system. This is essential as it
will permit to assess the effects of different genotypes
related to highly polymorphic genetic variants and
diverse ethnic groups. In turn, this equips these cells
with a much higher predictive value in drug screens or
biological assays than primary cells derived from a sin-
gle donor. On top of the natural heterogeneity, it
should be possible to deliberately modulate the gen-
ome of iPSC/iHLCs, by exploiting easily accessible
and customizable CRISPR gene-editing technology.
Notably, the two essential CRISPR components (guide
RNA and Cas9 nuclease) can be delivered by recombi-
nant AAV vectors (see also below), which makes the
combination of these powerful technologies—CRISPR,
iHLCs, and AAV—very promising. Support for this
enthusiasm comes from data provided by us and
others (A-K. Herrmann & D. Grimm, unpublished
results and [61]) showing that iPSC/iHLCs can be
transduced very efficiently with AAV vectors, depend-
ing on the capsid variant used for vector generation.
In this context, a recent study by Egan et al. [62] is
worth highlighting as well, in which the authors used
a forward shRNA screen to identify host factors for
P. falciparum blood-stage development within the ery-
throcyte. As these cells are denucleated and therefore
impervious to genetic modification, the shRNA were
instead delivered to hematopoietic progenitor cells,
which were subsequently matured into erythroblasts
and then infected with the parasite. The fact that this
permitted the identification of CD55 as an essential
factor for parasite invasion into the erythrocyte further
exemplifies the power of stem cell-based strategies and
thus complements the work by Ng and colleagues [57].
Taken together, the key features of the iHLC system
—natural genetic diversity, possibility of deliberate
mutation, and susceptibility to Plasmodium sporozoite
infection during iPSC-to-iHLC differentiation—suggest
a large potential for the study and dissection of para-
site–host interactions. To even further enhance these
prospects, it would be beneficial to standardize the dif-
ferentiation protocol to facilitate its rapid implementa-
tion in other laboratories. Additionally, efforts should
be undertaken to improve the degree of cell matura-
tion, to ensure that the iHLCs truly mimic the host
factor repertoire of primary hepatocytes and thus even
more accurately recapitulate the situation in an intact
liver.
Importantly, even with all these improvements in
place, a limitation of the current iHLC system remains
its concentration on a single cell type, that is,
FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
Plasmodium meets AAV
hepatocytes. However, the liver comprises a variety of
other cell types that are most likely also involved in
Plasmodium infection and establishment of liver stages,
especially Kupffer cells, stellate cells, and liver sinu-
soidal endothelial cells (LSEC). In fact, there is evi-
dence that prior to hepatocyte infection, the parasite
traverses Kupffer cells and LSECs, and that this may
be essential for subsequent liver-stage formation
[63,64].
In this respect, a second novel system should be
highlighted that has recently been reported by March
and colleagues [65,66]. This study is noteworthy as it
describes a robust and multicellular ex vivo platform
that recapitulates the complete liver stage of Plasmod-
ium sporozoite infection, including progression to the
schizont and merozoite stages. In more detail, the hall-
mark of this platform is micropatterned cocultivation
(MPCC, [67]) of cryopreserved primary human hepato-
cytes together with supportive stromal cells (murine
embryonic fibroblasts) (Fig. 2A). Notably, the hepato-
cytes grown in this system polarize, exhibit typical
drug and energy metabolism, do not proliferate, and
maintain a functional phenotype for up to 6 weeks. In
addition, high reproducibility is guaranteed through
the use of cryopreserved human cells, which minimizes
donor-to-donor interexperimental variability, and like-
wise through the use of cryopreserved, purified, vialed,
and aseptic sporozoites (P. falciparum or vivax). Using
a battery of assays, March and coworkers were able to
validate the viability of these sporozoites and to prove
their ability to mature into hepatic schizonts. Remark-
ably, the latter were also shown to release merozoites
that were capable of infecting human red blood cells,
which were added to the MPCC culture.
In comparison to regular cultures of primary human
liver cells, an advantage of this system and a promis-
ing feature for its future use in malaria research is that
it combines high reproducibility, robustness, and relia-
bility, with the possibility to maintain and study func-
tional cellular phenotypes over extended periods of
time. Equally remarkable is the ability of this platform
to support the full Plasmodium liver-stage infection,
from sporozoite invasion to schizont/merozoite release.
In this regard, it may even surpass the capabilities of
the iHLC system highlighted above (albeit a direct
comparison has not yet been performed). However, a
potential drawback of the MPCC approach may be
the lower amenability to stable genetic engineering for
it is based on nonproliferating primary hepatocytes,
rather than on expandable stem cells. In fact, the
authors had to prescreen various batches of cryo-pre-
served cells in order to eventually identify two which
mediated high-level sporozoite infection; similar
2033
Plasmodium meets AAV
variations in efficiency were also noted for three differ-
ent frozen parasite batches. Together, this suggests
that it may be more challenging with this particular
system to cover the natural genotypic variation in the
human population, and hence the predictive value may
be lower than that of the iHLC approach. Moreover,
to fully mimic an intact liver, the MPCC system will
need to be expanded by other physiologically relevant
cell types, especially Kupffer cells, which are likely
required for Plasmodium liver infection [26,63].
In conclusion, there are numerous promising ave-
nues in the field of ex vivo liver surrogate models for
malaria research, two of which were highlighted above
—use of iPSC as an unlimited cell source and multicel-
lular coculture systems. An intriguing and rewarding
next step should be to try and juxtapose the best fea-
tures of the two strategies, that is, to establish robust
protocols for iPSC differentiation into other relevant
hepatic cell types, such as Kupffer cells, and to then
combine all the different cell types in a single
micropatterned culture vessel. Ideally, this will occur
in a 3D format comprising an adequate extracellular
matrix and fully mimicking the architecture and hemo-
dynamics in an intact liver. In this respect, we also
wish to note a recent series of encouraging reports on
newly developed bioartificial 3D scaffolds (Fig. 2B
bottom). This includes a study by Schanz et al. [68]
who extracted small bowel segments from pigs, chemi-
cally removed the residual porcine cells, seeded the
vascular structures with human endothelial cells, and
eventually populated this vascularized scaffold with
primary human hepatocytes. While not tested yet for
susceptibility to Plasmodium infection, the fact that the
engrafted hepatocytes were morphologically intact as
well as functionally and metabolically highly active is
certainly auspicious. Even more promising may be,
however, to engineer decellularized liver scaffolds
instead of bowel tissue as a biomimetic 3D matrix pro-
viding optimal gas and nutrient diffusion. In one
example, Jiang et al. [69] used a combination of physi-
cal and chemical methods to decellularize whole
extracted livers of mice, which were subsequently
repopularized with mesenchymal stem cells (MSC) and
attached to a perfusion culture system. Notably, the
MSC efficiently and rapidly differentiated into HLC
showing typical hepatocyte morphology, gene expres-
sion, and functionality that was maintained even after
transplantion of the entire recellularized scaffold into
recipient mice. Similarly, the Kurreck group has more
recently reported successful decellularization of iso-
lated rat liver 3D scaffolds and subsequent repopula-
tion with HepG2 cells, a human hepatoma cell line
[70]. Particularly noteworthy is that the authors were
2034 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
moreover able to demonstrate potent transduction of
the engrafted cells with AAV vectors. This again exem-
plifies the great potential of these vectors for func-
tional studies in ex vivo liver models, as already noted
above in the context of the iHLC strategy and as fur-
ther discussed in the next chapter. Akin to the other
organotypic systems, it will now be important to
include additional cell types that represent the physio-
logical situation better, especially Kupffer cells and
LSECs. To this end, it may again be beneficial to com-
bine the best of multiple strategies, for example, to
replace the HepG2 cells in the rat liver scaffold with
MSC or iPSC, and to then differentiate them into vari-
ous hepatic cell populations.
Last but not least, we wish to point out another
related strategy, which is in vivo repopulation of livers
with human hepatocytes in a living mouse (Fig. 2B
top). Therefore, a subpopulation of the murine hepato-
cytes is destroyed deliberately (e.g., by overexpression
of a suicide gene), triggering liver regeneration and
creating a short window during which human hepato-
cytes can be transplanted and engrafted. The resulting
‘humanized’ mice support the life cycle of the human
Plasmodium strains P. falciparum and P. vivax [71–74],
thus complementing wild-type mice that are routinely
used to propagate the rodent parasite strains. How-
ever, such human-chimeric mice are costly and labori-
ous to generate. Moreover, one should consider that
without further transplantation of, for instance,
human immune cells or hematopoietic stem cells, all
the other cell types in these mice remain of murine ori-
gin. Importantly, as described in more detail below, it
is possible to engineer AAV vectors that readily trans-
duce the human hepatocytes within these chimeric liv-
ers, again illustrating the potential of this particular
vector for malaria research in various ex or in vivo
liver models.
Fighting fire with fire—engineering of
hepatotropic viruses to dissect and
control Plasmodium liver stages
Adeno-associated viruses are a large group of viruses
that are endemic in many species including humans,
and that have been studied intensely for the past five
decades. One reason for their attractiveness is their
unique biology, since AAVs strictly depend on coin-
fection with a second unrelated, so-called helper
virus, typically Adenovirus or Herpes simplex virus.
This is because AAV has only two open reading
frames (ORFs; Fig. 3A left) whose differential
expression results in eight proteins which play essen-
tial roles in viral gene expression, replication, and
F. Hentzschel et al. Plasmodium meets AAV

Fig. 3. Adaptation of the AAV vector system for use in malaria research. (A) Left: Wild-type genome consisting of two open reading frames
rep and cap, which are flanked by inverted terminal repeats (ITR, typically derived from AAV2). Right: Three possible recombinant vector
configurations, that is, (i) a conventional single-stranded vector containing a transgene expression cassette of up to 4.5 kb, (ii) a self-
complementary genome for expression of shRNA (usually together with a second cassette, for example, a gfp reporter), and (iii) a similar
variant of self-complementary AAVs for gRNA expression (including a Cas9-specific gRNA scaffold, shown in orange). Note that the self-
complementary genotype in (ii) and (iii) is created through specific mutation of one of the ITRs (indicated with the asterisk, see main text for
details). (B) Four options to retarget AAV vectors to specific liver cell types of potential interest for malaria research. Specificity can be
achieved through either (i) modification of the target cell surface, resulting in de novo binding of a selected AAV capsid; (ii) vice versa,
genetic modulation of the AAV capsid surface, creating novel binding sites for particular cellular receptors; (iii) selection of cell type-specific
promoters (arrows); and/or (iv) inclusion of binding sites for miRNA (red triangle) that are abundant in off-target cells where gene expression
is unwanted, but missing in the actual on-target cell(s). HC, hepatocyte; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell.

capsid formation, but which fail to mediate the
entire AAV life cycle. To compensate for this defi-
ciency, AAV hijacks multiple proteins and RNA (in
the case of Adenovirus) from its helper virus, to fos-
ter its own transcription, translation, and particle
egress [75]. Yet, even more appealing than this
intriguing biology is an exclusive combination of fea-
tures that makes AAV highly attractive and impor-
tant as a basis for recombinant vectors for human
gene therapy. First and foremost, AAV is believed to
be apathogenic in humans, and hence AAV vectors
are considered as the safest of all recombinant
viruses currently under (pre-) clinical investigation. In
fact, there has not been a single case of serious
adverse events in any human patient treated with
AAV vectors in over 100 clinical trials thus far
[76,77]. Secondly, the viral genome is very easy to
engineer due its small size (roughly 4.7 kilobases)
and due to the availability of AAV vector plasmids
that can be readily customized using standard
cloning techniques [78]. Thirdly, there are over 100
naturally occurring isolates of AAV which differ in
cell specificity and infection efficiency, providing a
vast toolkit for vector engineering [79]. To even fur-
ther expand on this natural diversity, we and others
implemented a variety of methods for molecular evo-
lution of ‘designer’ AAV capsids that are tailored for
a given application, for example, transduction of a
specific cell type in the presence of neutralizing anti-
AAV antibodies [80–82].
In the following sections, we discuss why we believe
that AAV vectors are ideally suited as a tool to study
Plasmodium liver stages in ex or in vivo model systems,
based on accumulating data from the recent literature
and from our own research.
Versatility of the genome
In principle, there are at least three possibilities to
identify, validate, or further dissect Plasmodium host

FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies 2035
Plasmodium meets AAV
factors and associated networks in liver cells: (a) over-
expression, (b) RNAi-mediated inhibition (knock-
down) on the mRNA level, and (c) nuclease-mediated
suppression (knock-out) on the DNA level (Fig. 3A
right). Importantly, all three are fully compatible with
AAV vectors, as extensively demonstrated by us and
others in the past. Their implementation is facilitated
by the aforementioned fact that there are widely avail-
able, user-friendly, high-copy number AAV vector
plasmids. They contain the only viral element which is
required for vector generation in cis, namely, the
inverted terminal repeats or ITRs. These are short
(about 145 bp) hairpin sequences that flank the viral
genes or the transgene expression cassette, respectively,
and mediate its replication and encapsidation. Since
the two AAV ORFs can be replaced entirely with for-
eign DNA, and since the capsid can package slightly
oversized DNA (up to 120% of the length of the wild-
type genome), an AAV vector construct can accommo-
date roughly 5.2 kb of DNA. Considering a typical
size of promoter and polyadenylation elements in a
range of 1 kb, this leaves about 4 to 4.5 kb for the
cDNA of choice, which is probably sufficient for the
majority of mammalian cDNA of interest (Fig. 3A[i]).
Notably, in case the cDNA is sufficiently short (less
than 2.4 kb including promoter and polyadenylation
signal), there is also the option to embed it in a ‘self-
complementary’ (sc)AAV vector genome. This is a
variant of standard AAV vectors in which one of the
ITRs is mutated by deleting the so-called ‘terminal res-
olution site’, that is, a short sequence that is normally
nicked during replication of the AAV vector genome
from single-stranded (ss) to double-stranded DNA.
[83]. Consequently, scAAV genomes package as a
molecule consisting of two inverted copies of the trans-
gene; hence the reduction in the capacity for the for-
eign DNA cassette to about 50% of the wild-type
genome (Fig. 3A[ii/iii]; the mutated ITR is labeled
with an asterisk). Importantly, scAAV vectors express
more rapidly and more robustly than conventional
AAV vectors, because the latter package as ssDNA
which has to be converted into a double-stranded
molecule prior to transcription. It may thus take a
ssAAV vector up to 2 weeks to reach the peak of
transgene expression in cultured cells or in the liver, as
compared to a few days for a scAAV counterpart
[83,84]. Therefore, one should carefully weigh the pros
and cons of the two possible vector configurations
when designing an experiment involving overexpres-
sion of a (putative) Plasmodium host factor from AAV
vectors—larger capacity (ss vectors) versus more potent
and faster transgene expression, at the cost of size (sc
vectors). Clearly, if the latter has no limitation to
2036 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
begin with, we strongly recommend the use of scAAV
vector genomes.
Unlike the situation with cDNA that vary in size,
scAAV vectors are always the preferred option for
strategies requiring knock-down or knock-out of Plas-
modium host factors. This is because the expression
cassettes to trigger RNAi with shRNA, or CRISPR
with g(uide)RNA, respectively, are typically in a range
of 100–700 bp, depending on the promoter used for
small RNA expression (e.g., H1: 100 bp, or U6:
600 bp) [85]. This is far below the packaging limit of
scAAV vectors and thus even permits the inclusion of
a second expression cassette, for instance encoding a
fluorescence marker to track the targeted cells
(Fig. 3A[ii/iii]). In fact, we have recently created a
toolbox of scAAV vector templates that allow for
coexpression of any shRNA or gRNA together with
various fluorescence reporters, and that can be easily
customized for any desired Plasmodium host factor
([86], and F. Schmidt, K. B€ orner & D. Grimm, unpub-
lished results). This vector collection, which is freely
available upon request, should largely foster and accel-
erate the future dissection of parasite–host interactions
ex or in vivo.
Notably, CRISPR-based knock-out of Plasmodium
host factors will also entail expression of the Cas9
nuclease, whose currently known orthologs are in a
size range of 3.1–4.2 kb [87,88]. Since all of these
cDNA exceed the capacity of scAAV vectors, coex-
pression of Cas9 from an AAV vector requires the use
of ss genomes which, as noted before, express more
slowly and less efficiently than sc vectors (encoding the
gRNA). Still, this is no major limitation as recently
demonstrated by a variety of reports describing very
efficient AAV-mediated Cas9 expression and gRNA-
induced gene editing in cultured cells and livers of
adult mice [86,89–91]. In addition, the increasing avail-
ability of stable Cas9-expressing cell lines or of Cas9-
transgenic mice [90] will soon entirely alleviate the
need for vector-mediated Cas9 expression, at least in
an exploratory context.
Efficiency in liver cells
Adeno-associated viral vectors are predestined for use
in the liver or in isolated liver cells due to the fact that
this organ is one of the main natural targets for the
wild-type virus, in particular for the most commonly
used AAV serotypes including the AAV2 prototype.
This explains why vectors derived from these serotypes
can readily transduce 100% of the hepatocytes in an
intact mouse liver, as consistently found with, for
example, AAV8 or AAV9 vectors by us and many
F. Hentzschel et al.
others [84,92,93]. Of note, this complete liver transduc-
tion is observed after peripheral vector application via
tail vein injection, which is a simple and rapid proce-
dure that can be quickly adapted in any laboratory.
One should also not get discouraged by the AAV par-
ticle numbers that have to be administered to achieve
100% transduction in vivo, and that are typically in a
range of 1 9 1011 per mouse or higher. In fact, thanks
to the highly advanced AAV vector production tech-
nology, it is feasible to generate stocks with titers of
greater than 1 9 1013 particles per mL within 2 weeks,
using standard cell culture techniques that are already
available in the majority of laboratories [78].
Next to the mouse, AAV vectors are also very effi-
cient at transducing livers of larger animal species,
such as dogs and nonhuman primates. While not all
of these animals are already used in malaria research,
it is important to realize that, in principle, the same
vector constructs exploited in mice to identify or vali-
date parasite host factors can also be translated into
these larger animals. This is particularly beneficial for
candidates that hold biomedical potential as malaria
therapeutics or vaccines, and hence require extensive
preclinical investigation in different small and large
animal species. Likewise, important in this context is
to again highlight the extensive clinical experience with
AAV vectors in humans, which has been gathered in
over 100 patient trials thus far and which is utmost
encouraging. Most notable with respect to malaria
research on Plasmodium liver stages are the latest clini-
cal trials with scAAV8 vectors in patients suffering
from hemophilia B, a bleeding disorder caused by
defects in the human blood clotting factor IX. Follow-
ing a single peripheral AAV vector administration, the
majority of treated patients expressed stable factor IX
levels above the critical threshold needed to provide
therapeutic benefit (50 ng per mL, that is, 1% of nor-
mal) [94,95]. None of the patients, neither in this trial
nor in any other AAV-based clinical study experienced
serious adverse events related to the vector, again illus-
trating the safety of the recombinant AAV system. It
is finally also essential in this context to understand
that a recent report claiming an association of AAV
infection with hepatocellular carcinoma in humans
attributed this effect to wild-type AAV2, but not to
AAV vectors [96]. Moreover, this study has been
highly criticized within the AAV community for
potentially overstating the results [76,77], and based
on the aforementioned clinical evidence that AAV
vectors are exceptionally safe in human patients.
Besides, further evidence for the safety and promise of
AAV vectors in humans is that the first gene therapy
product approved in the Western world in 2012,
FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
Plasmodium meets AAV
Glybera, is derived from a recombinant AAV serotype
1 vector [97].
In addition to their great potential in vivo, AAV
vectors are likewise highly efficient in liver cell lines or
primary cells that are cultured ex vivo. This comprises
numerous hepatoma cell lines, such as Huh7 or
HepG2, all of which can be transduced very robustly
with, for example, AAV2 or AAV6 vectors. Impor-
tantly, these cell lines retain their high susceptibility to
AAV transduction even when grown in a 3D environ-
ment rather than a 2D monolayer. This has recently
been exemplified in the aforementioned study by Wag-
ner and colleagues [70] who engrafted HepG2 cells
onto decellularized rat liver scaffolds and then demon-
strated wide-spread and efficient transduction with an
AAV6 vector. Comparable findings were made with
primary mouse or human hepatocytes that can be
transduced with various AAV serotypes when cultured
in both, 2D or 3D structures such as spheroids [80,98]
(K. Börner & D. Grimm, unpublished results).
In view of these attractive features, it is not surpris-
ing that one can find an exhaustive list of published
examples for biomedical AAV vector applications in
cultured liver cells or whole livers in vivo, including in
humans as mentioned above. Particularly noteworthy
with respect to the topic of the current article are
studies showing the successful development and use of
AAV vectors expressing shRNA or shmiRNA (differ-
ent types of RNAi triggers) directed against liver
pathogens. For instance, we and several other groups
have previously engineered scAAV vectors from hepa-
totropic serotypes, such as AAV7, 8, or 9, to express
shRNA against Hepatitis B virus (HBV) and then
proved their efficacy in cultured liver cell lines as well
as in livers of HBV-transgenic mice [84,99,100]. Best
results include the robust and persistent suppression
of HBV gene expression in adult mice for over 1 year,
achieved with a scAAV8 vector that was optimized
for hepatocyte-specific shRNA expression from a
RNA polymerase II promoter [99]. An important les-
son learned in this and other in vivo studies using
AAV/RNAi vectors is that vector-mediated shRNA
expression needs to be tightly controlled, in order to
avoid adverse oversaturation of the endogenous cellu-
lar miRNA pathway and ensuing cytotoxicity. This
risk was first discovered by us in a 2006 study in
which we had inadvertently triggered liver damage
and animal fatalities during our evaluation of a bat-
tery of anti-HBV AAV/shRNA vectors in livers of
HBV-transgenic mice [84]. As these effects are pre-
dominantly dose-dependent, we and others subse-
quently designed optimized AAV vectors that use
weaker and/or hepatocyte-specific promoters (see
2037
Plasmodium meets AAV F. Hentzschel et al.

above) for shRNA expression and thus alleviate the Adaptability of the capsid
oversaturation concern. In addition, concurrent over-
As described in the previous two paragraphs, there are
expression of Exportin-5 and Argonaute-2, two major
a variety of naturally occurring AAV serotypes that
rate-limiting factors in the mammalian RNAi path-
robustly transduce cultured liver cell lines (e.g., AAV2
way, can further improve AAV/RNAi vector safety
or AAV6) or livers of newborn or adult mice (e.g.,
and efficiency in cells and mouse livers [84,101,102].
AAV8 or AAV9). Important to point out, the hepatic
Yet another critical parameter is the on- versus off-
cell type that is predominantly targeted by all these
target specificity of the encoded shRNA, which has to
capsid variants are hepatocytes (or hepatocyte-derived
be considered for both shRNA strands, that is, the
cell lines, respectively). In contrast, other, non-
antisense strand directed against the actual target as
parenchymal liver cell types that are relevant and
well as the passenger strand. In fact, we have found
interesting in the malaria liver-stage context, that is,
recently that both shRNA strands are usually active,
Kupffer cells, stellate cells, and LSECs, are largely
and that deliberate suppression of passenger strand
refractory to transduction with wild-type AAV capsids.
activity using a ‘tough decoy’ inhibitor improves
In our previous work, we did observe AAV8 activity
potency and efficiency of AAV/shRNA vectors target-
in Kupffer cells in mouse livers following systemic vec-
ing HCV or HBV [103] (T. Michler, S. Grosse,
tor administration, but the efficiency was clearly below
M. Heikenwalder, U. Protzer & D. Grimm, unpub-
that in hepatocytes in the same animals [50]. We have
lished results). Altogether, these findings yield a vari-
recently made similar findings for another wild-type
ety of options to fine-tune the activity and in
serotype, AAV6, and stellate cells in mouse liver
particular the safety of shRNA-expressing AAV vec-
(D. Grimm & H. Willenbring, unpublished results).
tors for use in animal or human liver in vivo, which
Fortunately, there are at least two major possibili-
provides a very solid basis for their future develop-
ties to improve AAV targeting to nonhepatocyte cell
ment in the context of malaria research. To this end,
types in the liver (Fig 3B). One is to alter the host
it is highly beneficial that AAV/RNAi vectors can
environment, or, more specifically, to modify the sur-
also be evaluated and optimized in ex vivo 3D organ-
face composition of the cells of interest, in order to
otypic liver models, as recently exemplified by up to
increase the binding avidity of AAV serotypes (Fig 3B
90% knock-down of human cyclophilin B gene
[i]). However, a drawback of this strategy, especially
expression in a recellularized rat liver scaffold [70].
when used in combination with in vivo Plasmodium
Next to RNAi, similarly encouraging data are also
infection, is that it globally alters the configuration of
already available for the combination of AAV vectors
cells in the liver and is thus prone to creating artifacts.
and the CRISPR gene editing system in liver cell lines
Nonetheless, a notable example was reported by the
or mouse livers in vivo, although CRISPR technology
Asokan group who treated mice with intravenously
is much younger than RNAi. Most notable results
administered recombinant sialidase, knowing that this
thus far include a report of over 40% in vivo modifica-
would lead to cell surface exposure of N-terminal
tion of the Pcsk9 gene, resulting in significant reduc-
galactose, a receptor for AAV9. Indeed, this resu-
tions in serum cholesterol [89], as well as a recent
lted in a marked change in the overall tropism of
study achieving functional correction of a mutation in
AAV9, including a sequestration in Kupffer cells and
the ornithine transcarbamylase gene in livers of new-
LSECs [105].
born mice [104]. In both cases, appropriate gRNA
Because of the practical restrictions of this
were delivered with an AAV8 vector together with
approach, it is more adequate and more promising for
Cas9 from Staphylococcus aureus, the smallest of all
malaria research to pursue a second strategy to retar-
currently known Cas9 variants (3.1 kb); the latter was
get AAV vectors to nonparenchymal liver cells,
either encoded in a second [104] or on the same [89]
namely, genetic modification of the viral capsid
vector. Moreover, as noted above, our own groups
(Fig 3B[ii]). For details on the underlying complex
have reported a user-friendly AAV vector toolbox for
technology, we refer the reader to a set of excellent
cloning and expression of custom gRNA, alone or
recent reviews [81,106–108]. Here, suffice it to highlight
from an ‘all-in-one’ vector also comprising the Cas9
what we consider as the most versatile strategy for
cDNA [86]. Collectively, these first in vivo data and
AAV capsid diversification, that is, ‘shuffling’ of the
the availability of customizable gRNA templates
capsid genes from multiple different AAV serotypes
should pave the way for the looming application of
through enzymatic fragmentation and subsequent
the CRISPR system as a novel, powerful, and versatile
reassembly based on partial homologies. In 2008, we
tool to further dissect Plasmodium liver stages and
have introduced this technology into the AAV field
associated host factors.

2038 FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
F. Hentzschel et al.
and used it to molecularly evolve the capsid AAV-DJ,
a unique chimera between wild-type serotypes AAV2,
8, and 9 that combines high activity in both, cultured
cells including hepatoma lines and livers (hepatocytes)
of adult mice [80]. In addition, more recently [109],
Lisowski and colleagues reported the capsid LK03
which had been enriched from a shuffled AAV capsid
library by iterative screening in livers of human-chi-
meric mice. Interestingly, this capsid appears to have a
higher efficiency in human hepatocytes than AAV8,
which was tested in parallel as the gold standard
(based on its high efficiency in mouse hepatocytes).
Most likely, the elevated potency of LK03 is related to
the fact over 95% of its sequence are identical to wild-
type AAV3, which others also found to work well in
human hepatocytes [110,111].
Because these data clearly illustrate the tremendous
potential of DNA shuffling technology, we consider it
highly rewarding to now apply the same capsid evolu-
tion and selection strategy to other cell types in the
liver as well. Ideally, this will eventually result in an
AAV vector toolbox permitting to specifically target
any given cell population, either in an intact liver or in
complex cell culture systems. This will in turn allow to
overexpress or inhibit any desired putative Plasmodium
host factor (provided its cDNA size is compatible with
AAV, see above) and, ultimately, to thus fully unravel
the intricate parasite–host interactions in all relevant
liver cell types. Toward this aim, it should be useful to
further juxtapose retargeting strategies based on AAV
capsid modification with synergistic other approaches,
such as the use of cell type-specific promoters (Fig 3B
[iii]), or cellular detargeting through incorporation of
selected miRNA-binding sites into the vector (Fig 3B
[iv]). For instance, one candidate miRNA that could
be exploited for this purpose is miR-122, as it is exclu-
sively present in hepatocytes and can thus be har-
nessed to detarget AAV vectors from this cell type
[86,112,113].
Experience in malaria context
In light of the numerous benefits and features of AAV
vectors outlined above, it should be evident why they
are increasingly used in malaria research. Already, one
can distinguish at least four distinct applications in the
literature, and we can readily anticipate that many
more will be tested soon. Firstly, several reports
described the use of AAV1 vectors to express different
malarial antigens—MSP4 (merozoite surface protein 4,
P. falciparum) or MSP4/5 (P. yoelii)—in the muscle of
mice. These antigens were either expressed alone [114],
or, in efforts to augment the immune response, as
FEBS Letters 590 (2016) 2027–2045 ª 2016 Federation of European Biochemical Societies
Plasmodium meets AAV
fusions with, for example, C3d3 [115] or CTLA4-Ig
[116]. Curiously, all these efforts resulted in antigen-
specific immunity but failed to elicit protection against
Plasmodium infection, implying that further improve-
ments to this particular approach are required. Sec-
ondly, Huang and colleagues exemplified the
usefulness of AAV9 vectors to foster reconstitution of
immunodeficient mice with a human immune system
[117]. Therefore, the vectors were engineered to deliver
a cocktail of HLA class II genes, genes encoding vari-
ous human cytokines and human B-cell activation fac-
tor. Vector administration followed by engraftment of
hematopoietic stem cells resulted in humanized mice
carrying human CD4+ T- and B-cells. Notably, these
animals could be immunized with recombinant P. fal-
ciparum CSP and were subsequently protected from
in vivo challenge with sporozoites. Also, their sera ini-
hibited parasite invasion into HepG2 hepatoma cells
in vitro, further validating the power of this mouse
model to investigate human immune mechanisms of
antibody-based malaria vaccines. Thirdly, Deal et al.
studied a vectored immunoprophylaxis (VIP) strategy
by exploiting intramuscularly injected AAV8 vectors
to express antibodies against P. falciparum CSP [118].
Remarkably, up to 70% of treated mice were pro-
tected long-term from either intravenous or mosquito
bite challenge with sporozoites, in an antibody dose-
dependent manner. This clearly shows the great poten-
tial of AAV-based VIP approaches to confer immunity
to infection, albeit a number of parameters still need
to be elucidated and optimized prior to clinical transla-
tion in humans, such as the effective antibody levels
and the best sporozoite and/or pre-erythrocytic antigen
targets.
Lastly, our own two groups have previously har-
nessed AAV8 vectors to dysregulate miR-155 in
mouse livers prior to vaccination with genetically
attenuated P. berghei parasites (GAP, [3,4]), following
our discovery that this immunoregulatory host
miRNA is highly elevated upon Plasmodium liver-
stage infection [50]. Our concomitant result that GAP
infusion stimulates expression of TNFa (tumor necro-
sis factor alpha) and IFNc shed light on the underly-
ing molecular mechanism, considering that these two
cytokines are known upstream regulators of miR-155
expression. Most notable was, however, our finding
that AAV8 vector-mediated miR-155 overexpression
in hepatocytes and Kupffer cells boosts the protective
capacity of the GAP vaccine. This exemplifies how
AAV vector technology can be used not only to bet-
ter dissect host–parasite interactions in the liver, but
concurrently also to develop novel biomedically rele-
vant tools and strategies.
2039
Plasmodium meets AAV
Concluding remarks
Over the past decade, there has been tremendous and
impressive progress in all research areas that were in
the center of this article, culminating in an improved
understanding of Plasmodium–host interactions, an
arsenal of novel surrogate models of mammalian liv-
ers, and an ever expanding repertoire of technologies
for AAV vector engineering. In view of the rapid pace
at which these advances were made, we can readily
anticipate that the continued optimization and combi-
nation of these synergistic avenues holds great poten-
tial to propel both basic and applied malaria research
fast forward, and to hopefully result in the eventual
eradication of this devastating disease.
Acknowledgements
All four authors appreciate the support of their
research by the DFG (German Research Foundation)
through the Collaborative Research Center SFB1129.
F.H., A.-K.H & D.G. are grateful for support from
the DFG Cluster of Excellence CellNetworks
(EXC81), and A.-K.M. is thankful for support from
the DFG SPP1580 program. Finally, we appreciate
critical reading of the manuscript by several members
of our two (A.-K.M. & D.G.) laboratories. The figures
in this article use clipart from Servier Medical Art.
Author contributions
A-KM and DG conceived this review. All four authors
jointly wrote and edited the manuscript.
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