ACCEPTED MANUSCRIPT
A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites Ben C L van Schaijk, Ivo H J Ploemen, Takeshi Annoura, Martijn W Vos, Foquet Lander, Geert-Jan van Gemert, Severine Chevalley-Maurel, Marga van de Vegte-Bolmer, Mohammed Sajid, Jean-Francois Franetich, Audrey Lorthiois, Geert Leroux-Roels, Philip Meuleman, Cornelius C Hermsen, Dominique Mazier, Stephen L Hoffman, Chris J Janse, Khan M Shahid, Robert W Sauerwein
DOI: http://dx.doi.org/10.7554/eLife.03582 Cite as: eLife 2014;10.7554/eLife.03582 Received: 4 June 2014 Accepted: 19 November 2014 Published: 19 November 2014
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A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites One sentence summary:
Preclinical evaluation of a malaria GAP vaccine candidate
Ben C.L. van Schaijk1†, Ivo H.J. Ploemen1†‡, Takeshi Annoura2,‡‡, Martijn W. Vos1, Lander Foquet3 Geert-Jan van Gemert1, Severine Chevalley-Maurel2, Marga van de Vegte-Bolmer1, Mohammed Sajid2, Jean‐Francois Franetich4,5, Audrey Lorthiois4,5, Geert Leroux-Roels3, Philip Meuleman3, Cornelus C. Hermsen1, Dominique Mazier4,5,6, Stephen L. Hoffman7, Chris J. Janse2, Shahid M. Khan2, Robert W. Sauerwein1* 1
Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
2
The Leiden Malaria Research Group, Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
3
Center for Vaccinology, Ghent University and University Hospital, Ghent, Belgium.
4
Centre d'Immunologie et des Maladies Infectieuses, CIMI-Paris ; INSERM, U1135, Paris, F-75013 France.
5
Centre d'Immunologie et des Maladies Infectieuses, CIMI-Paris; Université Pierre et Marie Curie-Paris 6,
UMRS CR7, Paris, France. 6 7
AP-HP, Groupe hospitalier Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, F-75013 France.
Sanaria Inc. 9800 Medical Center Drive, Suite A209 Rockville, MD, USA.
1
Email addresses: BvS :
[email protected] IP:
[email protected] TA:
[email protected] MV:
[email protected] LF :
[email protected] GvG:
[email protected] SCC:
[email protected] MvdV:
[email protected] MS:
[email protected] JF:
[email protected] AL:
[email protected] GLR:
[email protected] PM:
[email protected] CH:
[email protected] DM:
[email protected] SH:
[email protected] CJ:
[email protected] SK:
[email protected] RS:
[email protected]
†
‡
Authors contributed equally Present address: Institute for Translational Vaccinology, Bilthoven, The Netherlands.
‡‡
Present address: Department of Tropical Medicine, The Jikei University School of Medicine, Tokyo, Japan.
* Corresponding author: Tel: +31 24 3614306; Fax: +31 24 341666 Email address:
[email protected] (Robert Sauerwein) Geert Grooteplein 28, 6525 GA, Nijmegen, The Netherlands
2
Abstract 1
A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of
2
malaria but is currently unavailable. High-level protection in humans can be achieved by experimental
3
immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug
4
coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach.
5
Here we present data on safety and protective efficacy using sporozoites with deletions of two genes i.e. the
6
newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage
7
development. In the rodent malaria model, Pb∆b9∆slarpGAP was completely attenuated showing no
8
breakthrough infections while efficiently inducing high level protection. The human Pf∆b9∆slarpGAP generated
9
without drug-resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted
10
with human hepatocytes in vivo but completely aborted development after infection. These findings support the
11
clinical development of a Pf∆b9∆slarpSPZ vaccine.
12 13 14
Keywords: malaria, vaccine, genetically attenuated parasite (GAP), sporozoite, liver, PfSPZ.
15
3
16
Introduction
17
A vaccine that induces high-level (>90%) sterile protection by inducing immunity that attacks the non-
18
pathologic, asymptomatic pre-erythrocytic stages of Plasmodium falciparum (Pf) will prevent infection, disease,
19
and transmission and could be a powerful instrument to eliminate Pf malaria from geographically defined areas
20
(1, 2). In rodent models, sterile protection can be induced by immunization with live Plasmodium sporozoites
21
attenuated by either irradiation, genetic modification (GAP) or by concomitant anti-parasitic drug treatment (for
22
reviews see(3-6)). In humans, induction of complete sustained protective immunity against a challenge infection
23
has been achieved by previous exposure to the bites of mosquitoes infected with i) live radiation-attenuated
24
Plasmodium sporozoites that invade but then completely arrest in the liver (7, 8) and ii) live sporozoites in
25
volunteers taking chloroquine chemoprophylaxis (CPS) with full parasite liver stage development; once released
26
into the circulation asexual blood stages are killed by chloroquine (9, 10). More recently it has been
27
demonstrated for the first time that sterile immunity can be achieved by intravenous immunization with
28
radiation-attenuated aseptic, purified, cryopreserved Pf sporozoites (SPZ) called PfSPZ Vaccine (11).
29
From a product manufacturing perspective, GAPs have the clear advantage of representing a
30
homogeneous parasite population with a defined genetic identity. The genetic attenuation is an irreversible,
31
intrinsic characteristic of the parasite that does not require additional manufacturing steps like irradiation.
32
Furthermore, in the manufacturing process of GAP-infected mosquitoes, operators are never exposed to Pf
33
parasites that can cause disease. However, clinical development of GAPs has suffered from safety problems
34
related to breakthrough infections during immunization leading to pathological blood stage infections
35
responsible for clinical symptoms and complications. Strains of mice showed differential susceptibility to
36
breakthrough infections after injection of sporozoites of rodent malaria GAPs, demonstrating the need for
37
extensive preclinical rodent screening (12). The P. falciparum GAP Pf∆p52∆p36, is the only GAP so far that has
38
been assessed in humans but the trial in which the Pf sporozoites were administered by mosquito bite had to be
39
terminated, because of breakthrough infections in one volunteer during immunization (13). Our in vitro
40
experiments with Pf∆p52∆p36 confirm that this double gene deletion GAP (i.e. two genes removed from the
41
genome) is not fully attenuated similar to the equivalent rodent GAP, Pb∆p52∆p36 in the P. berghei/ C57BL/6
42
model(12) . Therefore, identification of additional genes critical and uniquely selective for liver stage
43
development has become a major challenge for GAP vaccine development (4, 12, 14). Furthermore, single gene
44
deletion GAPs will most likely not be adequate (14).
4
45
This prompted us to generate and test a GAP with deletions of two independent genes critical for liver
46
stage development. We recently identified a novel P. berghei (Pb) gene deletion mutant, Pb∆b9 lacking the
47
expression of the B9 protein (Pf ortholog: PFC_0750w; PF3D7_0317100)(15). This protein is a newly identified
48
member of the Plasmodium 6-Cys family of proteins. Initial safety evaluation in rodents demonstrated that
49
Pb∆b9 mutants have a stronger attenuation phenotype than mutants lacking the 6-Cys proteins P52 and P36 (15-
50
18). As second target gene for liver stage attenuation, we selected the slarp and sap1 orthologs reported in Pb
51
and P. yoelii (Py) respectively (Pf ortholog: PF11_0480; PF3D7_1147000; hereafter termed slarp). These slarp
52
mutants show an excellent safety profile by full arrest in the liver in mice (19, 20). The SLARP protein is
53
expressed in sporozoites and in early liver stages and is involved in the regulation of transcription (20, 21).
54
Here we report the generation and evaluation of a rodent GAP lacking the genes encoding for B9 and
55
SLARP (Pb∆b9∆slarp) and the generation and evaluation of the equivalent human Pf GAP lacking the Pf
56
ortholog genes. Pf∆b9∆slarp was generated using constructs that allowed for the removal of the drug selectable
57
marker from the genome by FRT/FLPe recombinase methodology (22). The safety and efficacy of Pb∆b9∆slarp
58
and the lack of development of Pf∆b9∆slarp in human hepatocytes, in vitro and in vivo in chimeric mice provide
59
strong support for clinical development of a Pf∆b9∆slarp PfSPZ vaccine.
5
60
Results
61
Arrest of liver stage development and induced protection after P. berghei ∆b9∆slarp GAP
62
Previously, we generated a Pb mutant with disruption of the b9 locus (Pb∆b9) by standard genetic
63
modification using a double cross-over integration event, followed by removal of the drug-selectable marker
64
cassette by negative selection (23). Characterization of the Pb∆b9 phenotype showed that liver stage
65
development was fully abrogated in BALB/c mice and severely compromised in the more stringent C57BL/6
66
murine model for P. berghei(15). Immunization of a single dose of 10K (i.e. 10.000 sporozoites) or5KPb∆b9,
67
protected BALB/c mice against a 10K WT-sporozoite challenge, while 80% of mice were still protected after a
68
single 1K immunizing dose (Table 1). InC57BL/6mice,immunizationwith50K/20K/20KofPb∆b9 resulted in
69
complete protection lasting up to 180 days, reducing to 45% protection when challenged at 1 year post-
70
immunization. However, sporozoite administration occasionally resulted in blood stage infections after
71
administration of high doses thereby compromising the safety profile(15).
72
Previously it has been shown by others that Pb∆slarp parasites are completely arrested in liver-stage
73
development with a complete lack of breakthrough blood-stage infections(19, 20). Therefore, we generated a
74
new single gene deletion mutant Pb∆slarp in a parasite line that constitutively expressed a fusion of the reporter
75
proteins GFP and luciferase using a slarp-targeting DNA-construct for deletion by double cross-over
76
homologous integration (Fig. 1- supplement 1). The Pb∆slarp mutant showed blood stage growth and mosquito
77
infections with functional sporozoites similar to wild-type (Supplementary File 1). However, intravenous
78
injection of up to 500k Pb∆slarp sporozoites never led to full development of parasites in the liver as assayed by
79
in vivo imaging (Fig. 1- supplement 1) or analysis of blood stage infection (Table 2). PbΔslarp sporozoites
80
arrested very soon after invasion of cultured Huh7 hepatocytes corroborating the excellent safety findings by
81
Silvie et al (20).
82
Therefore, in order to create a completely attenuated and safe rodent GAP, we additionally disrupted the
83
slarp gene in the Pb∆b9 genome by double cross-over integration (Fig. 1). Asexual growth and sporogonic
84
development/function equaled wild-type (Supplementary File 1). However, Pb∆b9Δslarp sporozoites arrested
85
soon after invasion of cultured Huh7 hepatocytes (Fig. 1) and intravenous injection of 150-200K Pb∆b9∆slarp
86
sporozoites never resulted in breakthrough blood-stage infections in mice (Table 2). Finally, protective efficacy
87
induced by Pb∆b9∆slarp was studied in both BALB/c and C57BL/6 mice. A single immunization dose of 10K,
88
5K or even 1K of Pb∆b9∆slarp sporozoites in BALB/c mice induced full protection against a 10K wild-type
89
sporozoite challenge (Table 1). C57BL/6 mice were 100% protected after 3x10K immunization with
6
90
Pb∆b9∆slarp sporozoites, and the protective efficacy reduced to 60% after a 3x1K immunization dose. A
91
challenge at day 180 post-immunization of a 50/20/20 K dose still resulted in complete protection. The combined
92
data showed that Pb∆b9∆slarp completely arrest during liver-stage development and induce a highly efficient
93
protective immunity in two different strains of mice.
94 95
Generation of a P. falciparum ∆b9∆slarp GAP
96
Considering the desired phenotype as observed in P. berghei, we generated a Pf mutant lacking
97
expression of both B9 (PF3D7_0317100) and SLARP (PF3D7_1147000; sporozoite asparagine-rich protein).
98
These genes are conserved between rodent and human species, both at the level of syntenic location in their
99
respective genomes on chromosomes 3 and 11 respectively, and at the sequence level. Pfb9 shows 37% amino
100
acid sequence identity and 54% sequence similarity with Pbb9(15); Pfslarp shows 28% amino acid sequence
101
identity and 46% sequence similarity with Pbslarp.
102
First, we generated two independent Pf mutants lacking slarp by standard double cross-over integration
103
of a DNA construct and analyzed their phenotype throughout the parasite life cycle (Fig. 2- supplement 1 and
104
2). Blood-stage development of two independently derived Pf∆slarp (i.e. Pf∆slarp-a and –b) parasites was
105
comparable to WT parasites. Pf∆slarp mutants produced WT numbers of gametocytes, oocysts and sporozoites
106
(Fig. 2). Intracellular PfΔslarp-a and -b
107
significantly different in number and morphologically identical to WT parasites at 3 and 24 hours post-infection
108
(hpi) (Fig. 3). However, their number was more than 10-fold reduced at 48 hpi and not detectable from day 3
109
onwards to day 7 post-infection. Parasites lacking b9 in P. falciparum arrested before day 2 post-infection of
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primary human hepatocytes with the exception of one observed liver schizont at a later timepoint(15). PfΔslarp-
111
a and -b parasites still showed positive HSP70 staining and morphologically normal parasites at 48 hpi in
112
primary human hepatocytes indicating later time point of arrestcomparedtoPf∆b9 parasites.
parasite development in primary human hepatocytes was not
113
Next, we generated double gene-deletion Pf∆b9∆slarp mutants using the FRT/FLPe recombinase
114
methodology (22). This methodology employs FLPe recombinase to remove a FRT-site flanked drug-resistance
115
marker cassette introduced into the Pf genome when the target gene has been removed by double cross-over
116
homologous recombination asshownforPfΔslarp-b parasites in Fig. 2- supplement 1 and 2. After cloning, this
117
‘marker-free’line was subsequently transfected with the Pfb9 gene-targeting construct pHHT-FRT-GFP-b9(15)
118
to delete the b9 locusfromthePfΔslarp-b genome (Fig. 2- supplement 1 and 2). Subsequently two ‘marker-
119
free’clones, Pf∆b9∆slarp-F7 and Pf∆b9∆slarp-G9 were obtained containing the correct genotype i.e. removal of
7
120
the slarp and b9 gene loci as well as both respective drug-selection cassettes (Fig. 2- supplement 2). In addition,
121
we confirmed loss of expression of both slarp and b9 by RT-PCR analysis by demonstrating the absence of
122
transcripts in mRNA collected from Pf∆b9∆slarp-F7 and Pf∆b9∆slarp-G9 salivary gland sporozoites (Fig. 2-
123
supplement 2). We then examined the phenotype of Pf∆b9∆slarp-F7 and Pf∆b9∆slarp-G9 mutants during blood
124
stage and mosquito development. Asexual blood stage growth of Pf∆b9∆slarp parasites was normal as both
125
clones reached an asexual parasitemia between 0.5 and 5% during cloning within 21 days and Pf∆b9∆slarp
126
clones produced WT-like numbers of gametocytes, oocysts and sporozoites (Fig. 2).
127 128
Developmental arrest of Pf∆b9∆slarp GAPs in human hepatocytes
129
We next analyzed the development of Pf∆b9∆slarp in human hepatocytes using cultured primary
130
hepatocytes and uPA+/+-SCID mice engrafted with human hepatocytes (human liver-uPA-SCID mice)(24).
131
Pf∆b9∆slarp sporozoites showed normal gliding motility, hepatic cell traversal (Fig. 2) as well as invasion of
132
primary human hepatocytes, but parasites were completely absent in two independent experiments at day 2 up to
133
day 7 post-infection following inoculation of primary human hepatocytes with 40.000 Pf∆b9∆slarp F7 or G9
134
sporozoites (Fig. 3). Detailed analyses of 80 individual wells at day 4 post-infection did not result in
135
identification of a single developing parasite. The combined day 2 and day 4 data ofPf∆b9∆slarp indicated that
136
the timing of arrest is similar to Pf∆b9 (15) and there had been complete arrest of liver-stage development,
137
similar to Pf∆slarp parasites.
138
In addition, human liver-uPA-SCID mice were intravenously inoculated with 1x106 WT or
139
PfΔb9Δslarp sporozoites. Two heterozygous uPA+/--SCID mice, not engrafted with human hepatocytes, served
140
as controls and were also challenged with P. falciparum sporozoites. Livers were collected either at 24 hpi or 5
141
days post-infection for detection of P. falciparum 18S DNA by quantitative real-time PCR (25). Both mice
142
infected with WT Pf and 1 of the 2 mice infected with PfΔb9Δslarp were positive for Pf 18S DNA at 24 hours
143
post-infection, demonstrating successful sporozoite infection in human hepatocytes (Fig. 3). A lower signal was
144
observed in PfΔb9Δslarp-infected mice at day 1 after infection compared to WT parasites, likely reflecting the
145
early time point of arrest of this GAP. All mice infected with Pf WT (3/3) showed a strong increase in parasite
146
18S DNA at day 5 post-infection, representing successful liver stage development. In contrast, none of the
147
human liver-uPA-SCID mice infected with PfΔb9Δslarp sporozoites showed 18S DNA higher than
148
heterozygous uPA+/--SCID mice, not engrafted with human hepatocytes that had been infected with PfΔb9Δslarp
149
sporozoites (Fig. 3). Although these studies were performed with a limited number of mice, these findings
8
150
indicate that PfΔb9Δslarp parasites can invade but do not develop in livers of humanized mice. Our combined
151
results demonstrate abrogation of development of PfΔb9Δslarp inside human hepatocytes.
9
152
Discussion
153
The Pf GAP PfΔb9Δslarp containing two gene deletions, is proposed as a whole-parasite malaria vaccine
154
candidate. Rationale and arguments are based on in vitro and in vivo experiments and supported by safety and
155
protection data with rodent Pb GAP with deletions of the orthologous genes. The rodent GAP PbΔb9Δslarp
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completely arrested early in liver stage development in two different mouse strains after injection of very high
157
numbers of sporozoites. In addition, immunizations with PbΔb9Δslarp efficiently induced sterile and long-
158
lasting protective immunity in both BALB/c and C57BL/6 mice. Similarly, the Pf GAP PfΔslarpΔb9,
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completely aborted development in human hepatocytes one day after invasion, while sporozoites were fully
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motile and invasive with infectivity comparable to Pf WT sporozoites. Importantly, asexual parasite growth and
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production of salivary gland sporozoites in the mosquito were unaffected ensuring normal GAP production.
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Pb∆b9∆slarp is to our knowledge the first completely attenuated rodent mutant in which multiple genes have
163
been deleted that are critical for two independent biological processes during liver stage development, i.e.
164
regulation of parasite genes/transcripts that play a role in early liver stage development stages (20, 21) and the
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establishment of the PV within the infected hepatocyte(15).
166
A number of Pb and Py GAPs have previously been reported to arrest at different time points during
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development in the liver (4, 6). These include GAPs based on genes essential for i) the formation and
168
maintenance of a parasitophorous vacuole (PV) (b9, p52, p36, uis3 and uis4; (15, 16, 26) and ii) type II fatty acid
169
synthesis (i.e. fabb/f, fabz, pdh e1α; (12, 27)), and iii) the regulation of gene expression in the liver stages
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(sap1/slarp (19-21)). A critical safety requirement for GAPs in order to qualify as vaccine candidate is total
171
absence of blood infections during immunization and therefore the complete abrogation of liver-stage
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development. Unfortunately many of the above mentioned target genes including p52, p36 and those involved in
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type II fatty acid synthesis show a leaky phenotype, resulting in blood stage infections after administration of
174
high numbers of sporozoites. Incomplete liver stage arrest obviously disqualifies GAPs for further clinical
175
development for safety reasons.
176
In P. falciparum, GAPs have been generated that lack both the p52 and p36 genes (17, 18). In the Pb
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rodent model this GAP was not completely attenuated (12). Similarly, this Pf GAP while severely attenuated by
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the lack of both genes, a low percentage of parasites of this GAP are able to develop into mature liver-stage (12).
179
These observations indicate a partially redundant function for these proteins; indeed a breakthrough blood
180
infection was observed in one out of 6 volunteers after exposure to the bite of mosquitoes infected with
181
sporozoites of a PfΔp52Δp36 GAP (13) .
10
182
Since functional redundancy of related genes has been reported more often in Plasmodium (28-31), we
183
pursued the generation of GAPs from which multiple genes were removed from the genome each governing a
184
critical yet independent cellular process. The selection of those target genes excluded type II fatty acid synthesis
185
(FAS II) because P. falciparum mutants lacking FAS II enzymes fail to generate sporozoites inside the oocyst,
186
indicating that the FAS II pathway is essential for sporogony (32). The gene encoding liver stage antigen 1
187
(LSA-1) may be an attractive candidate but no orthologues are present in rodent or non-human primate
188
Plasmodium species precluding sufficient preclinical testing (33). The reverse is true for two published rodent
189
GAPs with deletions of the genes uis3 or uis4 of which unequivocal orthologues are absent in the P. falciparum
190
genome. Alternatively, genes encoding proteins with a role in late stage parasite liver development could be an
191
attractive target, since induction of protection by late arresting GAPs may be superior to early arresting GAPs (6,
192
34) However, late arresting GAPs are likely more risky and prone to breakthrough infection as shown for GAPs
193
lacking the genes palm or lisp (4).
194
Therefore, we decided to focus on early liver stage arrest and selected the newly identified b9 as a prime
195
candidate. PbΔb9 elicits long-lived protective immune responses in mice and only few breakthrough blood
196
infections occur in mice albeit less than were observed with PbΔp52Δp36 GAP sporozoites (12). The genes p52,
197
p36 and b9, all belong to the recently expanded 6-Cys family of Plasmodium proteins and may share a similar
198
function in formation or maintenance of the PV membrane at the interface of parasite and host cell. Indeed, a
199
triple gene-deletion mutant lacking p52, p36 and b9 is no more attenuated than a mutant lacking b9, suggesting
200
that these genes do not drive independent biological pathways (14-16). To date the early arresting slarp mutant is
201
the only rodent GAP with a Pf orthologue without a record of breakthrough blood infections in mice. Indeed, our
202
data confirm that rodent sporozoites lacking slarp are fully capable of hepatocyte invasion and formation of a PV
203
but completely abort development soon after invasion as previously reported(19-21). Here, we report for the first
204
time that P. falciparum mutants lacking slarp, i.e.PfΔslarp completely arrest at day 3 post-infection of primary
205
human hepatocytes while morphologically normal liver stage parasites are still observed at 48hpi. PfΔb9
206
parasites arrest at a point in time before day 2 after hepatocyte invasion, with the exception of a single liver
207
schizont observed at a later time point(15). The multiple attenuated Pb∆b9∆slarp indeed passed our stringent
208
preclinical safety screen and no breakthrough blood infections were observed in all conditions tested. In
209
addition, we showedthatimmunizationwithPb∆b9∆slarp sporozoites induced strong and sustained protective
210
immunity in BALB/c and C57BL/6 mice with similar efficacy as reported for mutant sporozoites lacking P52 (or
211
P52 and P36) or γ-radiated sporozoites (16, 35-37).
11
212
Live vaccine strains (attenuated by natural selection or genetic engineering) may be potentially released
213
into the environment. Therefore, safety issues concerning the medical as well as environmental aspects must be
214
considered including the absence of heterologous DNA sequences (in particular drug resistance genes) from the
215
genome of GAPs (38, 39). Thus, a Pf∆b9∆slarp GAP was generated free of a drug-resistance marker using
216
FRT/FLPe-recombinase methodology. This approach permits the removal of drug-resistance markers that were
217
introduced to generate the mutant and results in an altered genome that retains only two 34 nucleotide FRT
218
sequences. The removal of the drug resistance marker has the additional advantage that these parasites are easily
219
amenable to further genetic modification (22).
220
The Pf∆b9∆slarp GAP aborted early development in cultured primary human hepatocytes with a
221
phenotypeandtimingsimilartoPf∆b9 and studies performed in a limited number of chimeric mice engrafted
222
with human hepatocytes confirm this arrest phenotype. From the combined Pb and Pf data one can conclude that
223
Δb9 attenuation phenotype induces highly effective protection, although it may at a low frequency produce a
224
breakthrough blood infection. Therefore the additional deletion of slarp in these mutants provides these parasites
225
with complete attenuation that is essential in order to proceed with human trials.
226
An important prerequisite for further downstream clinical development and manufacturing(11) is to
227
show that production of Pf∆b9∆slarp sporozoites is unabated and similar to WT parasites. We have shown that
228
thePf∆b9∆slarp GAP produces WT numbers of sporozoites that are fully capable of infecting hepatocytes. In
229
addition we have produced aseptic, purified, cryopreserved PfΔb9Δslarp sporozoites (data not shown).
230
Preliminary data from a 6 day attenuation assay in HC-04 cells showed that like irradiated PfSPZ (5, 40), none of
231
the PfΔb9Δslarp sporozoites developed to mature liver stage parasites expressing PfMSP-1 (data not shown), as
232
do aseptic, purified, cryopreserved WT sporozoites(41).
233
In conclusion, we have generated a multiply attenuated Pf∆b9∆slarp GAP, free of any drug-resistance
234
gene, and demonstrated that Pf∆b9∆slarp sporozoites invade hepatocytes comparably to WT sporozoites and are
235
completely attenuated. These findings provide a solid foundation for clinical development and testing of a
236
PfSPZ∆b9∆slarp vaccine.
12
237
Material and methods
238 239
P. berghei reference parasite lines
240
The following reference lines of the ANKA strain of P. berghei were used: line cl15cy1(42, 43) and line
241
676m1cl1 (PbGFP-Luccon; see RMgm-29 in www.pberghei.eu). PbGFP-Luccon expresses a fusion protein of GFP
242
and Luciferase from the eef1a promoter (42, 44).
243 244
P. falciparum parasites and culture
245
For transfections, the parasite used was directly from a characterized good manufacturing process
246
(GMP) produced working cell bank (WCB) of the P. falciparum NF54 wild type strain (45), produced by
247
Sanaria Inc, identical to that described previously (5, 40, 41). Blood stages of wt, PfΔslarp-a, PfΔslarp-b,
248
PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 were cultured in a semi-automated culture system using standard in vitro
249
culture conditions for P. falciparum and induction of gametocyte production in these cultures was performed as
250
previously described (46-48). Fresh human red blood cells and serum were obtained from Dutch National blood
251
bank (Sanquin Nijmegen, NL; permission granted from donors for the use of blood products for malaria
252
research). Cloning of transgenic parasites was performed by the method of limiting dilution in 96-well plates as
253
described (49). Parasites of the positive wells were transferred to the semi-automated culture system and cultured
254
for further phenotype and genotype analyses (see below).
255 256
Experimental animals
257
For P. berghei infections, female C57BL/6J and BALB/c (12 weeks old; Janvier France) and Swiss OF1
258
(8 weeks old Charles River) were used. All animal experiments with rodent parasites performed at the LUMC
259
(Netherlands) were approved by the Animal Experiments Committee of the Leiden University Medical Center
260
(DEC 07171; DEC 10099) and at the RUNMC (Netherlands) by the Radboud University Experimental Animal
261
Ethical Committee (RUDEC 2008-123, RUDEC 2008-148, RUDEC 2010-250, RUDEC 2011-022, RUDEC
262
2011-208). The Dutch Experiments on Animal Act is established under European guidelines (EU directive
263
86/609/CEE) regarding the Protection of Animals used for Experimental and Other Scientific Purposes.
264
Human liver-uPA-SCID mice (chimeric mice) were produced as described before (24). The study
265
protocol for infecting these mice with P. falciparum sporozoites was approved by the animal ethics committee of
266
the Faculty of Medicine and Health Sciences of the Ghent University.
267 13
268
Generation and genotyping of P. berghei mutants
269
To disrupt the P. berghei slarp gene (PBANKA_090210) a construct was generated using the adapted
270
‘Anchor-tagging’ PCR-based method as described (12) (Fig. 1 supplement 1). The two targeting fragments
271
(1195 bp and 823 bp) of slarp were amplified using genomic DNA (parasite line cl15cy1) as template with the
272
primerpairs5960/5961(5’targetsequence)and5962/5963(3’targetsequence).SeeSupplementary File 2a for
273
the sequence of the primers. Using this PCR-based targeting construct (pL1740) the mutant Pb∆slarp-a
274
(1839cl3) was generated in the PbGFP-Luccon reference line using standard methods of transfection and positive
275
selection with pyrimethamine (Fig. 1 supplement 1). The generation of the drug-selectable marker-free mutant
276
Pb∆b9∆sm (1309cl1m0cl2; RMgmDB no. 934) has been described by(15). This mutant, which contains a
277
disrupted b9 gene and is drug-selectable marker free, was used for deleting the slarp gene (PBANKA_090210).
278
To delete the slarp gene the gene-deletion construct pL1740 was used as described above. Using this construct
279
the mutant PbΔb9Δslarp (line 1844cl1) was generated in the Pb∆b9∆sm line using standard methods of
280
transfection and positive selection with pyrimethamine (Fig. 1).
281
Correct integration of the constructs into the genome of mutant parasites was analysed by diagnostic
282
PCR-analysis and Southern analysis of PFG-separated chromosomes as shown in Fig. 1 and Fig. 1 supplement
283
1. PFG-separated chromosomes were hybridized with a probe recognizing hdhfr or the 3’-UTR dhfr/ts of P.
284
berghei (43).
285 286
Generation and genotyping of P. falciparum mutants
287
The slarp gene (PF3D7_1147000) in P. falciparum WT parasites, (NF54wcb) was deleted using a
288
modified construct based on plasmid pHHT-FRT-(GFP)-Pf52 (22) (Fig. 2- supplement 1). Targeting regions
289
were generated by PCR using primers BVS179 and BVS180forthe5’targetregionandprimersBVS182and
290
BVS184forthe3’targetregion(seeSupplementary File 2b forprimersequences).The5’and3’targetregions
291
were cloned into pHHT-FRT-(GFP)-Pf52 digested with BsiWI, BssHII and NcoI, XmaI, respectively, resulting in
292
the plasmid pHHT-FRT-GFP-slarp. The b9 gene (PF3D7_0317100) of PfΔslarp-b P. falciparum parasites was
293
deleted using a modified construct based on plasmid pHHT-FRT-(GFP)-Pf52 (22) (Fig. 2- supplement 1).
294
Targeting regions were generated by PCR using primers BVS84 and BVS85forthe5’targetregionandprimers
295
BVS88 and BVS89 for the 3’ target region. The 5’and 3’ target regions were cloned into pHHT-FRT-(GFP)-
296
Pf52 digested with NcoI, XmaI and MluI, BssHII resulting in the plasmid pHHT-FRT-GFP- b9. All DNA
297
fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54
14
298
strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen) sub-cloning. Transfection of WT
299
(NF54wcb) parasites with the plasmid pHHT-FRT-GFP-slarp and selection of mutant parasites was performed
300
as described (22) resulting in the selection of the parasite line PfΔslarp -a. The second PfΔslarp parasite line,
301
originating from an independent transfection, was subsequently transfected with pMV-FLPe to remove the drug-
302
selectable marker cassette using FLPe as described (22) and cloned resulting in the parasite clone PfΔslarp -b.
303
Subsequent transfection of PfΔslarp-b parasites with the plasmid pHHT-FRT-GFP-b9 and selection were
304
performed as described above resulting in the parasite line PfΔb9Δslarp. The parasite line PfΔb9Δslarp was
305
subsequently transfected with pMV-FLPe to remove the drug- selectable marker cassette using FLPe and cloned
306
as described above resulting in the cloned parasite lines PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 that are free of
307
drug-resistance markers.
308
Genotype analysis of PfΔslarp and PfΔb9Δslarp parasites was performed by Expand Long range
309
dNTPack (Roche) diagnostic, long-range, PCR (LR-PCR) and Southern blot analysis (Fig. 2- supplement 2).
310
Genomic DNA of blood stages of WT or mutant parasites was isolated and analyzed by LR-PCR using primer
311
pair p1, p2 (slarp) and p3, p4 (b9) (see Supplementary File 2b for primer sequences) for correct integration of
312
the constructs in the respective slarp and b9 loci by double cross-over homologous recombination. The LR-PCR
313
program has an annealing step of 48°C for 30 seconds and an elongation step of 62°C for 10-15 minutes. All
314
other PCR settings were according to manufacturer’s instructions. PCR products were directly analyzed by
315
standard agarose gel electrophoresis or first digested with restriction enzymes for further confirmation of the
316
genotype and removal of resistance markers was confirmed by sequencing. For Southern blot analysis, genomic
317
DNA was digested with TaqI or RcaI restriction enzymes for analysis of integration into the slarp and b9 loci
318
respectively. Southern blot was generated by capillary transfer as described (50) and DNA was hybridized to
319
radioactive probes specific for the targeting regions used for the generation of the mutants and generated by PCR
320
(see above).
321
The presence or absence of slarp and b9 transcripts in WT and mutant sporozoites was analyzed by
322
reverse transcriptase-PCR (Fig. 2- supplement 2). Total RNA was isolated using the RNeasy mini Kit (Qiagen)
323
from 106 salivary gland sporozoites collected by dissection of mosquitoes 16 days after feeding with WT,
324
PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7 and PfΔb9Δslarp -G9 parasites. Remaining DNA was degraded using
325
DNAseI (Invitrogen). cDNA was synthesized using the First Strand cDNA synthesis Kit for RT-PCR AMV
326
(Roche). As a negative control for the presence of genomic DNA, reactions were performed without reverse
327
transcriptase (RT-). PCR amplification was performed for regions of slarp using primers BVS290, BVS292 and
15
328
for regions of b9 using primers BVS286 and BVS288. Positive control was performed by PCR of 18S rRNA
329
using primers 18Sf and 18Sr.
330 331
Phenotype analyses of blood stages of P. berghei and P. falciparum mutants
332
Asexual multiplication rate and gametocyte production of P. berghei blood stages were determined as
333
described (12). The P. berghei mutants were maintained in Swiss mice. The multiplication rate of blood stages
334
and gametocyte production were determined during the cloning procedure (43) and were not different from
335
parasites of the reference ANKA lines. P. falciparum blood stage development and gametocyte production was
336
analyzed as described(22).
337 338
Analysis of P. berghei and P. falciparum sporozoite production and in vitro motility, hepatocyte traversal and
339
infectivity of sporozoites
340
Feeding of A. stephensi mosquitoes with P. berghei and P. falciparum, determination of oocyst
341
production and sporozoite collection as well as P. berghei gliding motility were performed as described (12). P.
342
falciparum gliding motility of sporozoites was determined as described(17, 51). P. falciparum cell traversal and
343
invasion of hepatocytes was determined in Huh7 cells and primary human hepatocytes respectively as
344
described(17). Infectivity of P. berghei sporozoites and development was determined in cultures of Huh7 cells as
345
described(15). For analysis of liver stage development by immunofluorescence, parasites were stained with the
346
following primary antibodies: anti-PbEXP1 (PBANKA_092670; raised in chicken(52)) and anti-PbHSP70
347
(PBANKA_081890; raised in mouse (53)). Infectivity of P. falciparum sporozoites and development was
348
analysed in primary human hepatocytes as described(17). Briefly for analysis of development by
349
immunofluorescence,
350
(PF3D7_0930300 (54)); anti-CSP (PF3D7_0304600; 3SP2) using double labeling. Anti-mouse secondary
351
antibodies, conjugated to Alexa-488 or Alexa-594 (Invitrogen) were used for visualization. Primary human
352
hepatocytes were isolated from healthy parts of human liver fragments which were collected during unrelated
353
surgery in agreement with French national ethical regulations(55) and after oral informed consent from adult
354
patients undergoing partial hepatectomy as part of their medical treatment (Service de Chirurgie Digestive,
355
Hépato-Bilio-Pancréatique et Transplantation Hépatique, Hôpital Pitié-Salpêtrière, Paris, France). The collection
356
and use of this material for the purposes of the study presented here were undertaken in accordance with French
357
national ethical guidelines under Article L. 1121-1of the‘‘Code de la Santé Publique’’.Given that the tissue
parasites
were
stained
with
the
following
primary
antibodies:
anti-HSP70
16
358
samplesareclassedassurgicalwaste,thattheywereusedanonymously(thepatient’sidentityisinaccessible to
359
the researchers), and that they were not in any way genetically manipulated, article L. 1211-2 stipulates that their
360
use for research purposes is allowed provided that the patient does not express any opposition to the surgeon
361
prior to surgery and after being informed of the nature of the research in which they might be potentially
362
employed. Within this framework, the collection and use of this material was furthermore approved by the
363
Institutional Review Board (Comité de Protection des Personnes) of the Centre Hospitalo-Universitaire Pitié-
364
Salpêtrière, Assistance Publique-Hôpitaux de Paris, France.
365 366
Analysis of P. berghei sporozoite infectivity in mice and in vivo imaging of liver stage development
367
C57BL/6 or BALB/c mice were inoculated with sporozoites by intravenous injection of different
368
sporozoite numbers, ranging from 1x104–5x105. Blood stage infections were monitored by analysis of Giemsa-
369
stained thin smears of tail blood collected on day 4–14 after inoculation of sporozoites. The prepatent period
370
(measured in days post sporozoite infection) is defined as the day when a blood stage infection with a
371
parasitemia of 0.5–2% is observed. Liver stage development in live mice was monitored by real time in vivo
372
imaging of liver stages as described (56). Liver stages were visualized by measuring luciferase activity of
373
parasites (expressing luciferase under the eef1a promoter) in whole bodies of mice (57).
374 375
Immunizations of mice with P. berghei sporozoites
376
Prior to immunization, P. berghei sporozoites were collected at day 21-27 after mosquito infection by
377
hand-dissection. Salivary glands were collected in DMEM (Dulbecco's Modified Eagle Medium from GIBCO)
378
and homogenized in a homemade glass grinder. The number of sporozoites was determined by counting in
379
triplicate in a Bürker-Türk counting chamber using phase-contrast microscopy. BALB/c and C57BL/6 mice were
380
immunized by intravenous injection using different numbers of mutant sporozoites. BALB/c mice received one
381
immunization and C57BL/6 mice received three immunizations with two 7 day intervals. Immunized mice were
382
monitored for blood infections by analysis of Giemsa stained films of tail blood at day 4‐16 after immunization.
383
Immunized mice were challenged at different time points after immunization by intravenous injection of 1x104
384
sporozoites from the P. berghei ANKA reference line cl15cy1. In each experiment, age matched naive mice
385
were included to verify infectivity of the sporozoites used for challenge. After challenge, mice were monitored
386
for blood infections by analysis of Giemsa stained films of tail blood at day 4‐21.
387
17
388
Development of Pf Δb9Δslarp GAP in chimeric mice engrafted with human hepatocytes
389
Human liver-uPA-SCID mice were produced as described before (24). Briefly, within two weeks after
390
birth homozygous uPA+/+-SCID mice (25) were transplanted with approximately 106 cryopreserved primary
391
human hepatocytes obtained from a single donor (BD Biosciences, Erembodegem, Belgium). To evaluate
392
successful engraftment, human albumin was quantified in mouse plasma with an in-house ELISA (Bethyl
393
Laboratories Inc., Montgomery, TX). The study protocol was approved by the animal ethics committee of the
394
Faculty of Medicine and Health Sciences of the Ghent University. Human liver-uPA-SCID mice (n=10) and
395
non-chimeric heterozygous uPA+/--SCID mice (control, n=2) were intravenously injected with 106 fresh isolated
396
PfΔb9Δslarp -G9 or as a control WT sporozoites. One and 5 days post-infection livers were removed and each
397
liver was cut into 12 standardized sections and stored in RNALater (Sigma) at 4 °C until analysis as
398
described(25). From each part DNA was extracted to assess the parasite load by Pf18S qPCR and to assess the
399
number of human and mouse hepatocytes by Multiplex qPCR PTGER2 analysis(25).
400
18
401
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37.
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22
606
Acknowledgments
607
We would like to thank the following people from RUMC (Nijmegen) for technical support: Claudia Lagarde,
608
Alex Ignacio, Daniëlle Janssen, Rianne Siebelink-Stoter, Wouter Graumans, Jolanda Klaassen, Laura Pelser-
609
Posthumus, Astrid Pouwelsen and Jacqueline Kuhnen; and the following people from LUMC (Leiden) for
610
technical support: Jai Ramesar, Jing-wen Lin and Hans Kroeze. We acknowledge the Sanaria Manufacturing
611
Team for the GMP produced working cell bank of PfNF54.
612
613
Author Contributions
614
Conceived and designed the experiments: BVS IP TA LF CJ SK RS. Performed the experiments: BVS IP TA
615
MV LF GJVG SCM MVDVB MS JFF AL. Analyzed the data: BVS IP TA MV CJ DM SK CCH. Contributed
616
Reagents/materials/analysis tools: SH GLR PM. Wrote the paper: BVS IP CJ SK RS. All authors commented
617
and corrected the manuscript.
618 619
Competing Interests
620
The authors have declared that no competing interests exist. However, SLH, is CEO of Sanaria Inc., a
621
biotechnology company focused on whole sporozoite malaria vaccines.
622
Funding: This study was performed within the framework of Top Institute Pharma (Netherlands) project: T4-
623
102. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
624
the manuscript.
625 626
Data and Materials availability
627
The materials described in this study must be acquired through a material transfer agreement.
628
23
629
Table 1: Protection of mice after immunization with P. berghei Pb∆b9 or Pb∆b9∆slarp sporozoites Mouse Strain
Pb Mutant
Day of Challenge a
Immunization regimes No. protected/ No challenged 10k b 5k
BALB/c Pb∆b9 Pb∆b9∆slarp
10 10
C57Bl6
1k
10/10c 20/20
18/20 10/10
8/10 20/20
50/20/20k d
10/10/10k
1/1/1k
Pb∆b9
10 90 180 365
4/4 5/5 9/9 e 5/11
nd
nd
Pb∆b9∆slarp
10 180
nd 6/6
10/10 nd
6/10 nd
630 631
a
Number of days post last immunization;104 wild type sporozoites were injected by IV route.
632
b
Immunization dose: number of sporozoites x1000.
633
c
Protected/total # of immunized mice (%); protection was 0/15 in naive control BALB/c and 0/10 in C57BL/6
634
mice.
635
d
Immunization dose with 7 day intervals between immunizations.
636
e
Immunization dose 50/10/20k with 7 day intervals between immunizations.
637
nd= not done.
638 639
Table 2: Breakthrough blood-stage infections after intravenous injection of Pb∆slarp and
640
Pb∆b9∆slarp sporozoites. Infectiona Spz x 103
Breakthrough blood infection/total # mice
Pre-patent periodc (days)
WTb
10
5/5
4-5
Pb∆slarp Pb∆slarp Pb∆b9∆slarp
50 25 25
0/5 0/10 0/10
WTb
10
5/5
Pb∆slarp Pb∆slarp Pb∆b9∆slarp Pb∆b9∆slarp
500 200 200 150
0/5 0/10 0/10 0/5
Mouse Strain
Mutant
BALB/c
C57BL/6
4-5
641 642
a
Inoculation dose of sporozoites administered IV
643
b
P. berghei ANKA strain: line cl15cy1
644
c
Day with parasitemia of 0.5–2%
24
645 646
LEGENDS TO FIGURES
647 648
Fig. 1: Generation and genotype analyses of P. berghei mutant PbΔb9Δslarp
649
A. GenerationofmutantPbΔb9Δslarp.ForPbΔb9Δslarp the DNA-construct pL1740 was generated containing
650
the positive/negative selectable marker cassette hdhfr/yfcy. This construct was subsequently used to generate the
651
mutantPbΔb9Δslarp inthePbΔb9Δsm mutant. See Supplementary File 2a for the sequence of the primers.
652
B. Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant
653
PbΔb9Δslarp confirming correct disruption of the slarp and the b9 locus. See Supplementary File 2a for the
654
sequenceoftheprimersusedfortheselectablemarkergene(SM);5’-integrationevent(5’);3’-integration event
655
(3’)andtheslarp and the b9 ORF. For Southern analysis, PFG-separated chromosomes were hybridized using a
656
3’UTRpbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the
657
endogenous locus of dhfr/ts onchromosome7anda3’UTRpbdhfr probe that recognizes the construct integrated
658
into P. berghei b9 locus on chromosome 8. In addition, the chromosomes were hybridized with the hdhfr probe
659
recognizing the integrated construct into the slarp locus on chromosome 9.
660
C. Development of liver-stages in cultured hepatocytes as visualized by staining with antibodies recognizing the
661
parasitophorous vacuole membrane (anti-EXP1; green) and the parasite cytoplasm (anti-HSP70; red). Nuclei are
662
stained with Hoechst-33342. Hpi: hours post infection. Scale bar represents 10µm.
663 664
Fig. 1- supplement 1: Generation and genotype analyses of P. berghei mutant PbΔslarp-a
665
A. GenerationofmutantPbΔslarp-a.ForPbΔslarp-a the DNA-construct pL1740 was generated containing the
666
positive/negative selectable marker cassette hdhfr/yfcy. This construct was subsequently used to generate the
667
mutantPbΔslarp-a (1839cl3) in the PbGFP-Luccon reference line. See Supplementary File 2a for the sequence
668
of the primers.
669
B. Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separatedchromosomesofmutantΔslarp-a
670
confirming correct disruption of the slarp-locus. See Supplementary File 2afor the sequence of the primers
671
usedfortheselectablemarkergene(SM);5’-integrationevent(5’);3’-integrationevent(3’)andtheslarp ORF.
672
MutantPbΔslarp-a has been generated in the reference P. berghei ANKA line PbGFP-Luccon which has a gfp-
673
luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern
674
analysis, PFG-separated chromosomes were hybridized using a 3’UTR pbdhfr probe that recognizes the
675
construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on
676
chromosome 7 and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were
677
hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9.
678
C. Real time in vivo imaging of ∆slarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35 and
679
45 hours post infection. C57BL/6 mice were IV injected with either 5x104 Pb-GFPLuccon sporozoites (n=5)
680
resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 x10 5
681
Pb∆slarp-a sporozoites (n=5) (lower panel: representative image of Pb∆slarp-luc infected mice).
682 683
Fig. 2. Phenotypes of P. falciparum Pf∆slarp and Pf∆b9∆slarp parasites
25
684
A. Gametocyte, oocyst and sporozoite production. Gametocyte numbers (stage II and IV-V) per 1000
685
erythrocytes at day 8 and day 14 after the start of gametocyte cultures. Exflagellation (Exfl) of male gametocytes
686
in stimulated samples from day 14 cultures (++ score = >10 exflagellation centers per microscope field at 400x
687
magnification). Median number of oocysts at day 7, IQR is the inter quartile range and sporozoite (day 21)
688
production (x1000) in A. stephensi mosquitoes.
689
B. Gliding motility of P. falciparum WT (cytochalasin D treated and untreated), PfΔslarp-b, PfΔb9Δslarp-F7
690
and PfΔb9Δslarp-G9 parasites. Gliding motility was quantified by determining the mean percentage ± standard
691
deviation of parasites that exhibited gliding motility by producing characteristic CSP trails (≥ 1 circles) or
692
parasites that did not produce CSP trails (0 circles).
693
C. Cell traversal ability of P. falciparum NF54,PfΔslarp-b andPfΔb9Δslarp-F7 sporozoites as determined by
694
FACS counting of Dextran positive Huh7 cells. Shown is the mean percentage ± standard deviation of FITC
695
positive cells. Dextran control (control): hepatocytes cultured in the presence of Dextran but without the addition
696
of sporozoites.
697 698
Fig. 2- supplement 1: Consecutive gene deletion of slarp and b9 in P. falciparum
699
Schematic representation of the genomic loci of A. slarp (PF11_0480; PF3D7_1147000) on chromosome 11
700
(Chr. 11) and B. b9 (PFC_0750w; PF3D7_0317100) on chromosome 3 (Chr. 3) of wild-type (wt; NF54wcb),
701
PfΔslarp and PfΔb9Δslarp gene deletion mutants before (PfΔslarp a and PfΔb9Δslarp) and after the FLPe
702
mediated removal of the hdhfr::gfp resistancemarker(PfΔslarp b and PfΔb9Δslarp clones F7/G9) respectively.
703
The constructs for the targeted deletion of slarp (pHHT-FRT-
704
GFP slarp) and b9 (pHHT-FRT-GFP-B9) contain two FRT sequences (red triangles) that are recognized by
705
FLPe. P1, P2 and P3, P4 primer pairs for LR-PCR analysis of slarp and b9 loci respectively; T (TaqI) and R
706
(RcaI): restriction sites used for Southern blot analysis and sizes of restriction fragments are indicated; cam:
707
calmodulin; hrp: histidine rich protein; hsp: heatshock protein; fcu: cytosine deaminase/uracil phosphoribosyl-
708
transferase; hdhfr::gfp: human dihydrofolate reductase fusion with green fluorescent protein; pbdt: P.berghei
709
dhfr terminator.
710 711
Fig. 2- supplement 2: Genotype analysis of the generated Pf∆slarp and Pf∆b9∆slarp parasites
712
A. Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms
713
the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal
714
of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for
715
b9 (see A and B respectively; for primer sequences see primer table in Supplementary File 2b) and PCR
716
products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation
717
(i.e. slarp LR-PCR product sizes: WT, 12kb, is undigested; Δslarp-a, 5.4kb is digested into 1.3kb and 4.0kb
718
fragments,Δslarp-b, 2.4kb is digested into 1.3kb and 1.1kb fragments. b9 LR-PCR product sizes: WT, 5.5kb, is
26
719
digested into 756bp, 793bp and 4.0kb fragments; Δb9-b, 2.6kb is digested into 756bp, 793bp and 1.1kb
720
fragments).
721
B. Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7 and
722
PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the
723
5’slarp targetingregion(P:5’ slarp-T; see A) on the left side of the slarp Southernorprobedwiththe3’slarp
724
targeting region (P: 3’ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9 integration
725
DNA was digested with restriction enzymes (E: RcaI) and probed withthe5’b9 targetingregion(P:5’ b9-T; see
726
A) on the right panel. The expected fragment sizes are indicated in panel A.
727
C. RT-PCR analysis showing absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b,
728
PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA
729
was performed either in the presence or absence of reverse transcriptase (RT+ or RT-, respectively) and
730
generated the expected 506bp and 580bp fragments for slarp and b9 respectively, the positive control was
731
performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary File 2b)
732
and generated the expected 130bp fragment.
733 734
Fig. 3. Development of P. falciparum Pf∆slarp and Pf∆b9∆slarp parasites in human primary hepatocytes
735
A. In vitro invasion of P. falciparum wt, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9
736
sporozoites in primary human hepatocytes. Invasion is represented as the mean ratio ± standard deviation of
737
extra- and intracellular sporozoites by double staining at 3 and 24 hours post infection, determined after 3 wash
738
steps to remove sporozoites in suspension.
739
B. Immunofluorescence assay of PfΔslarp-b parasites in human primary hepatocytes at 3 and 24 hours post
740
infection. Parasites are visualized by staining with anti-PfCSP antibodies (green; Alexa-488) and parasite and
741
hepatocyte nuclei are stained with DAPI (blue). Images were photographed on an Olympus FV1000 confocal
742
microscope. Scale bar represents 5μm.
743
C. Development of P. falciparum wt,PfΔslarp-a PfΔslarp-b(toppanel),PfΔb9Δslarp-F7 and PfΔb9Δslarp-G9
744
(bottom panel) liver-stages in primary human hepatocytes following inoculation with 40.000 sporozoites. From
745
day 2 to 7 the mean number ± standard deviation of parasites per 96-well was determined by counting parasites
746
stained with anti-P. falciparum HSP70 antibodies. The bottom panel represents experiments performed in
747
primary human hepatocytes from 2 different donors. No parasites present (NP).
748
D. DevelopmentofliverstagesofPfΔb9Δslarp GAP in chimeric mice engrafted with human hepatocytes. Mice
749
were infected with 106 wtorPfΔb9Δslarp-G9 sporozoites by intravenous inoculation. At 24 hours or at 5 days
750
after sporozoite infection, livers were collected from the mice and the presence of parasites determined by qPCR
751
of the parasite-specific 18S DNA. uPA HuHEP; chimeric homozygous uPA+/+-SCID mice engrafted with human
27
752
hepatocytes. As controls, uPA mice; heterozygous uPA+/--SCID mice not engrafted with human hepatocytes
753
were used.
28
Figure 1
A 5’ target
SM (hdhfr/yfcu) 875 bp 6128 6127 slarp
5’ target
DNA construct pL1740
3’ target
slarp locus (PBANKA_090210) in ȴb9ȴsm genome (1309cl1m0cl2)
3’ target
1631 bp 1075 bp 1287 bp 6347 4771 6125 6349 6346 6126 5’ target
SM (hdhfr/yfcu)
3’ target
1225 bp 4438 4437 b9
B
Wďѐb9ѐslarp
Disrupted locus (1844cl1)
Wild-type locus (PBANKA_080810)
Wďѐb9ѐslarp
WT
SM 5’ 3’ O B9 SM 5’ 3’ O B9 kb 1.5 1
Chr 9 8
7 3’UTR hdhfr dhfr
ɲ yWϭ WT (24 hpi)
Wďѐb9ѐslarp (24 hpi)
ɲ,^WϳϬ
,ŽĞĐŚƐƚ
Merge
Figure 2
A Phenotypes of P. falciparum WĨѐslarp and WĨѐď9ѐslarp parasites. Parasite
WT WĨȴslarp-a WĨȴslarp-b WĨȴb9ȴslarp-F7 WĨȴb9ȴslarp-G9
Gametocyte Gametocyte stage IV-V (range) stage II (range) 6,6 (1-13)
51 (29-61)
8,8 (3-15) 9,1 (2-27) 8,2 (1-140 8,6 (4-12)
58 (41-65) 49 (27-70) 39 (22-47) 49 (27-65)
džĨů.
% Infected Oocyst Mean no. of mosquitoes production sporozoites (Infected/dissected) (IQR) (std)
++ ++ ++ ++ ++
27 (12-42)
95% (104/110)
48 (23-95)
23 (8-59) 36 (17-59) 34 (20-54) 33 (13-64)
93% (37-40) 96% (106/110) 96% (77/80) 94% (75/80)
50 (22-97) 77 (22-174) 81 (33-106) 62 (22-105)
C
B Gliding motility
,ĞƉĂƚŽĐLJƚĞƚƌĂǀĞƌƐĂů
Figure 3
A
wt WĨȴslarp-a WĨȴslarp-b WĨȴb9ȴslarp-F7 WĨȴb9ȴslarp-G9
B
ɲ^W
C
DAPI
ĞǀĞůŽƉŵĞŶƚŽĨWĨȴslarp parasites wt Pfȴslarp-a Pfȴslarp-b
EŽ͘ůŝǀĞƌƐƚĂŐĞƉĂƌĂƐŝƚĞƐ
WT (3 hpi)
WĨѐslarp-b (3 hpi)
WT (24 hpi)
Day 2
NP
NP
NP
NP
NP
3
4
5
6
7
ĞǀĞůŽƉŵĞŶƚŽĨWĨȴb9ȴslarp parasites wt Pfȴslarp-b Pfȴb9ȴslarp-F7 Pfȴb9ȴslarp -G9
WĨѐslarp-b (24 hpi) NP
Day 2
D
NP
NP
NP
NP
3
4
5
6
Total Pf/106 ,Ƶ,ĞƉ
wt WĨȴb9ȴslarp-G9
Day
1
1
5 uPA ,Ƶ, W
5
5 uPA
NP
7
