IAI Accepted Manuscript Posted Online 13 July 2015 Infect. Immun. doi:10.1128/IAI.00262-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Immunization with the MAEBL M2 domain protects against
2
lethal Plasmodium yoelii infection
3 4
Running title: MAEBL M2 domain as a malaria vaccine candidate.
5 6
Juliana A Leite1; Daniel Y Bargieri2; Bruna O Carvalho1,3; Letusa Albrecht1,4;
7
Stefanie CP Lopes1,5; Ana Carolina AV Kayano1; Alessandro S Farias1;
8
Wan Ni Chia6; Carla Claser6; Benoit Malleret6; Bruce Russell6,#;
9
Catarina Castiñeiras1; Leonilda MB Santos1; Marcelo Brocchi1;
10
Gerhard Wunderlich2; Irene S Soares7; Mauricio M Rodrigues8;
11
Laurent Rénia6; Fabio TM Costa1*.
12 13 14
1
15
UNICAMP. Campinas, SP. Brazil.
16
2
17
Brazil.
18
3
19
RJ, Brazil.
20
4
21
Brazil.
22
5
23
Manaus, AM, Brazil.
24
6
25
Research (A*STAR), Singapore, Singapore.
Department of Genetics, Evolution and Bioagents. University of Campinas –
Department of Parasitology, University of São Paulo – USP. São Paulo, SP,
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz – FIOCRUZ. Rio de Janeiro,
Instituto Carlos Chagas, Fundação Oswaldo Cruz – FIOCRUZ. Curitiba, PR,
Instituto Leônidas e Maria Deane, Fundação Oswaldo Cruz – FIOCRUZ.
Singapore Immunology Network, Agency for Science, Technology and
1
26
7
27
University of São Paulo - USP, São Paulo, SP, Brazil.
28
8
29
São Paulo, São Paulo, SP, Brazil.
Department of Clinical and Toxicological Analysis, Pharmaceutical Sciences,
Center for Cellular and Molecular Therapy - CTCMol, Federal University of
30 31
Running head: MAEBL, M2 domain, malaria vaccine, Plasmodium yoelii.
32 33
#
34
Singapore – NUS, Singapore, Singapore.
Present address: Department of Microbiology, National University of
35 36
*Address correspondence to: Fabio T M. Costa, [email protected]
37 38
Abstract
39
Malaria remains a world threatening disease largely due to the lack of a
40
long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane
41
molecule with a chimeric cysteine-rich ectodomain homologous to regions of
42
DBL-EBP and AMA1 antigens. Although MAEBL does not appear to be
43
essential for the survival of blood-stage forms, the ectodomains M1 and M2,
44
homologous to AMA1, seem to be involved in parasite attachment to
45
erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of
46
mosquito salivary glands and is expressed in liver stages. Here, the
47
Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector.
48
C57BL/6J mice were immunized with doses of the P. yoelii recombinant
49
protein (rPyM2-MAEBL). High levels of antibodies, with balanced IgG1/IgG2c
50
subclasses, were achieved. rPyM2-MAEBL antisera were capable of
2
51
recognizing the native antigen. Anti-MAEBL antibodies recognized different
52
MAEBL fragments expressed in CHO cells showing stronger IgM and IgG
53
response against the M2 domain and repeat region, respectively. After
54
challenge with P. yoelii YM (lethal strain)-infected erythrocytes, up to 90% of
55
immunized animals survived, and a reduction of parasitemia was observed.
56
Moreover, splenocytes harvested from immunized animals proliferated in a
57
dose-dependent manner in the presence of rPyM2-MAEBL. Protection was
58
highly dependent on CD4+, but not CD8+ T cells, towards Th1. rPyM2-
59
MAEBL antisera were also able to significantly inhibit parasite development as
60
observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these
61
findings support the use of MAEBL as a vaccine candidate and open
62
perspectives to understand the mechanisms involved in protection.
63 64
Introduction
65
Malaria remains one of the most devastating infectious diseases in
66
inter-tropical countries, affecting mainly children under the age of five and
67
pregnant women. Approximately 600,000 deaths occur every year (1). People
68
repeatedly exposed to malarial infections in endemic areas develop immunity
69
against clinical disease and subsequently against parasitemia (2-5).
70
Antibodies have been shown to be responsible for naturally acquired immunity,
71
since passive transfer of immune IgG from adults can protect against
72
Plasmodium falciparum blood-stage infection (2, 6-8), suggesting that a
73
malaria vaccine based on asexual antigens is feasible. Unfortunately, none of
74
the vaccines currently tested achieved a convincing rate of protected
3
75
individuals (9-11) and the observed protection was often short–lived or highly
76
strain-specific (12-16).
77
The stakes for blood stage vaccines are even higher when malaria
78
eradication is the goal because the vaccines must not only reduce disease but
79
also reduce the parasitic burden to a degree that reduces the transmission
80
(17). Despite considerable efforts, none of the blood stage vaccine candidates
81
have exhibited satisfactory clinical and sterile protection in field tests (18, 19).
82
Many of the current vaccine candidates were encountered based on
83
the finding that partly immune individuals possess high titers of antibodies
84
against the tested antigens. Recently, the finding that antibodies against
85
PfRH5 are highly effective in blocking merozoite reinvasion but are rarely
86
detected in significant quantities in semi-immune carriers was reported (20).
87
This finding suggests that other merozoite-exposed antigens against which no
88
significant response is developed in natural infections may also be effective
89
as vaccines.
90
MAEBL is a 200-kDa type 1 membrane protein that belongs to the
91
erythrocyte binding protein (ebl) family (21-24). It has a carboxy cysteine-rich
92
region homologous to region IV of the DBL-EBP (Duffy binding like –
93
Erythrocyte binding protein). Additionally, the MAEBL amino cysteine-rich
94
domains (M1 and M2 ligand domains) exhibit partial similarity with the apical
95
membrane antigen 1 (AMA1) (23, 25). The M1 and M2 domains from P. yoelii
96
have been shown to be functionally equivalent to the DBL ligand domain, as
97
they bind to mouse erythrocytes (25).
98
MAEBL is essential for the development of the parasite during
99
sporozoite infection of mosquito salivary glands (26, 27) and is also
4
100
expressed in the salivary gland sporozoite and during the late liver stage (28).
101
Weak expression of MAEBL can also be detected in blood merozoite forms,
102
although maebl deletion has no impact on blood parasite development (26).
103
Coincidently, only few antibodies are found in naturally infected individuals
104
from areas with low transmission rates (29). MAEBL is a highly conserved
105
gene between evolutionarily distinct species of Plasmodium (25). Among the
106
clones of P. falciparum and field isolates, there is little amino acid sequence
107
variation on M1 and M2 domains (21). Because MAEBL is a well conserved
108
gene and expressed at different parasite stages, it is considered an
109
interesting potential vaccine candidate (30).
110
The current knowledge of the mechanisms and interactions during
111
Plasmodium invasion of the erythrocytes are still limited, which impairs the
112
development of approaches to block this essential step in Plasmodium biology.
113
As blocking erythrocyte invasion strategies are part of the rational of several
114
vaccine based on merozoite antigens, approaches designed to elucidate the
115
invasion phenomenon might facilitate validation and identification of potential
116
antigens that could be used as vaccine targets.
117
In this study, we investigated the immunogenicity of the domain M2-
118
MAEBL of P. yoelii. We amplified, cloned and expressed M2-MAEBL as a
119
prokaryotic fusion protein (rPyM2-MAEBL). Mice received four doses of the
120
recombinant antigen emulsified in Freund´s adjuvant. Immunization induced
121
robust protection against a lethal challenge with asexual forms of P. yoelii YM.
122
Protection was dependent on CD4+, but not CD8+ T cells, towards Th1. By
123
adapting an ex vivo invasion assay we could show that sera from immunized
124
mice inhibited invasion of parasite blood-stage forms. These results
5
125
demonstrate that MAEBL can be used as antigen in anti-malarial vaccine
126
formulations.
127 128
Methods
129
Parasites and animals
130
Six to seven week-old C57BL/6J mice were purchased from the
131
University of Campinas Animal Center (CEMIB-UNICAMP). Animals were
132
kept in a mouse pathogen free facility. All experiments and procedures were
133
approved by the Ethical Committee for Animal Research of the University of
134
Campinas (Protocol No. 1437-1). Blood forms of P. yoelii were obtained after
135
intraperitoneal (i.p.) injection of thawed glycerin frozen stocks.
136 137
Generation of the recombinant P. yoelii M2-MAEBL
138
DNA fragments encoding M2-MAEBL were obtained by PCR
139
amplification using Platinum Taq high-fidelity DNA polymerase (Invitrogen),
140
using P. yoelii genomic DNA from infected mice as a template. For the
141
amplification of the sequence corresponding to the M2 domain of the MAEBL
142
antigen, we used synthetic oligonucleotides based on the sequence from
143
amino acid 589 to 992 from the P. yoelii yoelii MAEBL gene (GenBank,
144
Accession number AF031886). For this, the following oligonucleotides were
145
used: (I) pET28aM2F 5’-CGC GGA TCC CTT AAC AAA TAT ATG AAA TCT
146
AAT GTT GAA CTT -3' and (II) anti-sense pIgSPM2R 5’-CTC GAA TTC CTA
147
CGA TTC ATC GGT ATT TCT TGT AG-3’; containing BamHI and EcoRI
148
sites, purchased from Invitrogen, USA. The resulting PCR product was
149
purified and digested with both restriction enzymes and inserted into the
6
150
pET28a vector (Novagen). The resulting plasmid was designated pET28aM2.
151
To verify that the reading frame was correct, inserts were sequenced using
152
vector-specific oligonucleotides covering the entire cloned sequence.
153
The recombinant protein (rPyM2-MAEBL) was expressed and purified
154
as previously described (31) with slight modifications. E. coli BL21 DE3
155
(Novagen) were transformed with the pET28aM2 plasmid and cultivated at
156
37°C in Luria-Bertani (LB) broth in the presence of 30 μg/ml kanamycin
157
(Sigma). Protein expression was induced at OD600 with 0,1 mM IPTG
158
(Invitrogen) for 4 h. After centrifugation, the bacterial pellet was resuspended
159
and sonicated in phosphate-buffered saline (PBS) supplemented with 1 mg/ml
160
lysozyme (Sigma) and 100 mM of PMSF (Sigma). The sonicated material was
161
then centrifuged, and the supernatant was separated from the pellet. Under
162
these conditions, rPyM2-MAEBL was found in the insoluble fraction. For
163
solubilization and purification, the pellet was resuspended in 100 mM Tris-HCl,
164
8 M urea, and 100 mM DTT, pH 8.0. Complete solubilization was achieved by
165
the passage of the solution through a 21-gauge needle. The solution was then
166
centrifuged and the supernatant applied to a column with N2+-NTA-agarose
167
resin (Qiagen). After extensive washing the recombinant protein was eluted
168
with a buffer with pH 5.0, and the eluted protein was dialyzed against PBS
169
with 2 M urea. Protein expression and concentration was determined by SDS-
170
PAGE and immunoblotting analysis.
171 172 173
7
174
Generation of a pdisplay library containing fragments of the MAEBL
175
immunogen
176
Primers were designed to be in-frame, complementary and specific to
177
different regions of MAEBL, without self-annealing sequences using the
178
software Amplify3x. Three different regions (MAEBL-3, MAEBL-4 and
179
MAEBL-5) encompassing the M2, repeat and C-terminal cysteine regions
180
respectively were designed. Six smaller, overlapping regions of the MAEBL-3
181
region (which contains different segments of the M2 region, of the region
182
between inter-domain between M2 and/or the repeats or repeats) were
183
designed and these were subcloned into the pDisplay vector (Invitrogen)
184
together with MAEBL-4 and MAEBL-5 constructs. The primer sequences used
185
to clone these fragments of MAEBL are all reported in Table S1.
186
Genes-of-interest were amplified with Phusion Hot Start II polymerase
187
(Finnzymes) according to manufacturer’s instructions, using the Biorad Peltier
188
thermal cycler. The PCR products were ligated into the XmaI and SacII (NEB)
189
linearized pDisplay vector according to manufacturer’s recommendations (In-
190
Fusion cloning, Clontech) before heat shock transformation into XL10 gold
191
bacteria (Agilent). Bacteria clones were screened by colony PCR using T7
192
and BgH primers for positive transformants. They were then sent for
193
sequencing to ensure codons were in-frame. The plasmids were purified
194
using Nucleobond AX Endotoxin-Free Midiprep kit (Macherey-Nagel) before
195
transfection. CHO (Chinese hamster cell) cells were used for the expression
196
of recombinant MAEBL proteins. Cells were grown in F12 complete medium
197
containing 100 units/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and
198
10% heat-inactivated fetal bovine serum (FBS) (Gibco) in a 37oC incubator
8
199
with 5% CO2. Sterile ethylene-diamine-tetra-acetic acid (EDTA) diluted to a
200
final concentration of 2 mM in PBS was used to detach transfected
201
cells/stable cell lines. Endofectin (Genecopeia) was used to transfect the
202
recombinant pDisplay plasmid into CHO cells. CHO cells were seeded into 6-
203
cm cell culture dishes one day before transfection in a density that would give
204
approximately 70 - 80% confluency on the day of transfection. To transfect the
205
cells, 9 μl of Endofectin was added dropwise with vortexing to 3 μg of plasmid
206
diluted in F12 supplemented with 100 units/ml penicillin and 100 μg/ml
207
streptomycin for 6-cm dishes. The DNA-Endofectin complex was incubated at
208
room temperature for 25 min before it was added dropwise to the cells. The
209
plates or dishes were swirled gently to distribute the transfection mixture
210
before returning them to the 37oC incubator overnight. The transfection
211
medium was replaced with fresh F12 complete medium the following day.
212 213
Immunoblotting analysis
214
Recombinant protein samples were loaded on SDS-PAGE under
215
denaturating conditions (2-mercaptoethanol). Proteins were then transferred
216
onto a nitrocellulose membrane (GE Healthcare). Immunoblotting was
217
conducted with anti-His6 (Life Technologies) at a 1:1500 dilution. The antibody
218
binding was detected by incubation with peroxidase-coupled goat anti-mouse
219
IgG (Sigma) at a 1:2000 dilution. Bands were detected using a solution
220
containing 50 mM Tris-HCl pH 7.5, 0.6 mg/mL of 3,3’ diaminobenzidine and 1
221
μL/mL of 0,03% H2O2 (Sigma) solution.
222 223
9
224
Immunization regimen
225
Groups of six to ten C57BL/6J male mice (Six- to 7-week-old) were
226
injected subcutaneously four times at three-week intervals with 5 μg of
227
rPyM2-MAEBL 1:1 (vol/vol) emulsified in complete Freund’s adjuvant (Sigma)
228
(CFA) for the first dose or incomplete Freund’s adjuvant (IFA) in the
229
subsequent doses. As a control group, animals were injected with 1:1 (vol/vol)
230
adjuvant (CFA/IFA) in PBS. Sera from immunized mice were collected
231
immediately before each dose and 3 weeks after the last dose (Figure 2A).
232 233
ELISA
234
Anti-rPyM2-MAEBL antibodies titers were determined by ELISA as
235
previously described (32). Plates were coated with 200 ng/well of recombinant
236
protein. Mice sera were tested at serial dilutions starting from 1:100. A dilution
237
of 1:2000 peroxidase-coupled goat anti-mouse IgG conjugated (Sigma) was
238
applied as a secondary antibody. Specific anti-MAEBL titers were determined
239
as the highest dilution yielding an OD492 higher than 0.1. Detection of IgG
240
subclasses was performed as described above, except that the secondary
241
antibody used was specific for mouse IgG1, IgG2b and IgG2c (Southern
242
Technologies).
243 244
Epitope mapping
245
Transfected cells (150 000 per well) were transiently transfected and seeded
246
in 96 well plates (Nunc). They were stained with serum diluted in FACS buffer
247
(PBS+10% FBS) for 1 h with agitation at 4oC. The different MAEBL constructs
248
are flanked by a myc epitope on the N-terminus and HA epitope on the C-
10
249
terminus when expressed on the surface of CHO cells, and both epitopes
250
were utilized to assess for protein expression on CHO cells using rabbit anti-
251
myc IgG (Upstate) or rabbit anti-HA IgG (Sigma) diluted 1:100. Untransfected
252
parental CHO cells were stained using the same sera and used as negative
253
control. For determination of serum recognition, the different test sera from
254
mice immunized with MAEBL protein or control vehicle DMSO were diluted
255
1:100 and incubated with the cells for 1 h with agitation at 4oC and then
256
washed once with FACS buffer. Secondary antibody – either goat anti-rabbit
257
IgG-AlexaFluor488 (Molecular Probes, Invitrogen) diluted 1:500 for checking
258
of protein expression or goat anti-mouse IgM or IgG-AlexaFluor488
259
(Molecular Probes) diluted 1:500, mixed with propidium iodide diluted to 0.001
260
mg/ml for the serum screening assay were incubated for 30 min with agitation
261
at 4oC. Labeled samples were analyzed using the high throughput flow
262
cytometer Accuri (Becton Dickinson). Flowjo (Tree Star) was used for all flow
263
cytometry analysis. Serum response was determined as the percentage of
264
cells recognized by serum divided by cells expressing the construct of interest
265
as illustrated in this formula:
266
Serum response = [(% cells recognized by immune serum – % cells
267
recognized by naïve serum) / (% Protein of interest POI expressing cells)] x
268
100%.
269
The 20% Cut-off for positivity was determined as the mean + 6 standard
270
deviation of the quadruplicate serum recognition values obtained for sera from
271
naïve animals. This high threshold was uses to reduce the possibility of false
272
positives.
273
Cellular proliferation and cytokines quantification
11
274
Ten
million
splenic cells
collected
from
animals
of
different
275
immunization groups (n=3) were cultured in 96-well plate in a final volume of
276
200 μL. rPyM2-MAEBL protein was added to the culture at a final
277
concentration of 1 or 10 µg/mL. As a control, some wells received ConA (50
278
µg/ml). Lymphocyte proliferation was assessed by the addition of 0.5 mCi/well
279
of methyl-[3H] thymidine ([3H] TdR (GE Healthcare) to the splenocytes after
280
48 h of culture. Splenocytes were cultured for an additional 18 h, and plates
281
were frozen. For later analysis, plates were thawed, and the culture was
282
collected on filter paper using a cell harvester. Tritium incorporation was
283
measured as radioactivity using Beta 1450 Microscintillator (Perkin Elmer).
284
The results are expressed as the average of the triplicate culture.
285
In parallel, culture supernatants were collected after 120 h, and the
286
amount of secreted IFN-γ, TNF, IL-2, IL-4 and IL-5 in cell culture supernatant
287
were simultaneously detected using the Cytometric Bead Array (CBA) Mouse
288
th1/th2 Cytokine Kit (BD). Cytokine concentrations in each sample were
289
determined with standard curves of known concentration of all five
290
recombinant proteins. Samples were read in a FACS Calibur flow cytometer
291
(Becton Dickinson). The detection limit of the assay was 0.5 pg/ml.
292 293
Immunofluorescence assays
294
Slides were prepared by collecting blood from BALB/c mice infected
295
with P. yoelii YM on day 5 post-infection (p.i.). The selection of mature stages
296
of the parasite was performed using LS-MACS columns (Miltenyi Biotec), and
297
thin blood smears were made, air-dried and stored until immunofluorescence
298
(IF) preparation and microscopy analysis. Immunofluorescence assays were
12
299
performed after fixing the blood smears with ice-cold acetone for 20 min and
300
air-dried. Well diameters were established with the aid of a Dako-PenTM
301
(Dako), and blocking was performed by 30-min incubation at 37°C with PBS
302
containing 3% BSA (USB). Pooled sera from the different immunization
303
groups and P. yoelii hyperimmune sera were diluted 1:100 in PBS
304
supplemented with 3% BSA and applied to the slides for 1 h at 37°C. Slides
305
were washed 3 X in PBS and incubated with Alexa-568 goat anti-mouse IgG
306
(Invitrogen) for 1 h at 37°C in the dark, then washed 3 X in PBS and
307
incubated
308
(Invitrogen) diluted in ultrapure (Millipore) water for 10 min at room
309
temperature. After another round of washing, Fluorosave (Caliobiochem) was
310
added, and slides were sealed with coverslips. Parasites were visualized with
311
the aid of a Nikon TS100 epifluorescence microscope. P. yoelii hyperimmune
312
sera were obtained from BALB/c mice infected with P. yoelii 17XNL clone 1.1
313
(non-lethal strain) once a month for a total period of 7 months. In parallel, to
314
confirm specificity, we also conducted immunofluorescence assays using
315
Leishmania amazonensis promastigote forms (gently donated by Dr. Selma
316
Giorgio, UNICAMP) and anti-rPyM2-MAEBL antisera.
with
DAPI
(4′,6-diamidino-2-phenylindole,
dihydrochloride-
317 318
Mice challenge and parasitemia assessment
319
The protection levels against blood forms were determined by
320
immunized mice survival against lethal challenge after intraperitoneal injection
321
of 106 P. yoelii YM-infected erythrocytes. Parasitemia was monitored,
322
according to Figure 2A, between 3 and 10 days post infection and determined
13
323
by counting at least 1,000 erythrocytes from thin blood smears stained with
324
the Panótico Rápido Kit (Laborclin).
325 326
CD4+ and CD8+ T lymphocyte depletion
327
Groups of 15 C57BL/6J mice were immunized as detailed in Figure 6A.
328
Three weeks after the fourth dose, five animals were injected with 0.5
329
mg/animal (Bioxcell) anti-CD4 (clone GK1.5) or anti-CD8 (clone YTS169.4)
330
monoclonal antibodies for the depletion of the corresponding lymphocyte
331
populations. The efficacy of In vivo depletion efficacy for this dose of
332
antibodies was determined in a preliminary experiment, as assessed by flow
333
cytometry of CD8+T and CD4+T cell using specific monoclonal antibodies
334
recognizing different epitopes in the CD4 or CD8 molecules. As a control, a
335
group of five immunized animals were injected with 0.5 mg per animal of
336
purified unrelated mouse IgG (Sigma, USA). On the following day, animals
337
were challenged with 106 P. yoelii YM IEs (lethal strain). The survival of
338
animals was monitored and parasitemia was evaluated as described
339
previously.
340 341
Ex vivo invasion assays of P. yoelii YM merozoites
342
To obtain high levels of reticulocytes to be used as target cells in the
343
invasion assay, reticulocytemia was induced in groups of four to six naive
344
mice by repeated daily retro-orbital bleeding for 6 days. Leukocyte removal
345
from the reticulocyte-enriched blood was achieved after two cycles of CF11
346
filtration (33).
347
Infected erythrocytes were enriched and maturated to schizonts.
14
348
Enrichment was achieved by bleeding P. yoelii YM-infected mice (n= 3) on
349
day 5 post-infection (>60% parasitemia) and isolating mature forms (15-18 h
350
of development) by magnetic concentration as previously described (34). A
351
blood smear was made after the procedure to determine the concentration of
352
parasites and verify the complete elimination of leukocytes. Next, to
353
synchronize parasites to at least 80% at the schizont stage, the pellet
354
corresponding to the mature forms of P. yoelii was resuspended in McCoy's
355
5A medium (Sigma) supplemented with 2.4 g/l glucose, 40 mg/ml gentamycin
356
(Sigma) and 20% human AB serum (UNICAMP’s blood bank), inactivated and
357
adsorbed on murine red blood cells and cultivated 3-6 h in 5% CO2 and at
358
37°C for maturation.
359
Finally, invasion assays were adapted from an assay for P. vivax [38]
360
performed by mixing P. yoelii concentrated schizonts and target cells
361
(enriched reticulocyte rich blood) at a 1:10 ratio corresponding to
362
approximately 10% of the initial parasitemia. The mixture was diluted to a 4%
363
hematocrit in 100 µl of McCoy's 5A medium supplemented with 2.4 g/l
364
glucose, 40 mg/ml gentamycin and then cultured for approximately 20 h in a
365
candle jar (35).
366
Immunized sera used in the invasion assays were heat inactivated by
367
incubation at 56ºC for 40 min. Pool sera from animals immunized with four
368
doses of the rPyM2-MAEBL diluted 1:100 were added to the culture. As
369
controls, the pool sera of animals injected four times with CFA/IFA at the
370
same dilution were used, and E64 (Sigma) at 100 μM was used as a positive
371
inhibition control because it ensures no schizont rupture. As a negative control
372
of inhibition, the assay was performed without addition of any drug or serum.
15
373
Freshly infected erythrocytes were determined by light microscopy and
374
parasitemia of young forms (rings and young trophozoites) was determined as
375
previously described. The standardized methodology for the P. yoelii invasion
376
assays is depicted in detail in Figure 7.
377 378
Statistical analysis
379
Survival curves of immunized animals after lethal challenge was
380
evaluated by the Log-Rank test. Comparison of the means of three or more
381
groups was performed using one-way ANOVA, and significance between
382
every two groups was assessed by the Bonferroni multiple comparison test
383
when data assumed Gaussian distribution, as assessed by the D’Agostino
384
and Pearson normality test. For samples of no Gaussian distribution, group
385
comparison was analyzed using the Kruskal-Wallis test followed by Dunn’s
386
test.
387
Whitney U test was used. All tests were performed using Prism GraphPad
388
software version 5.0a. The results were considered significant when P
1
Immunization with the MAEBL M2 domain protects against
2
lethal Plasmodium yoelii infection
3 4
Running title: MAEBL M2 domain as a malaria vaccine candidate.
5 6
Juliana A Leite1; Daniel Y Bargieri2; Bruna O Carvalho1,3; Letusa Albrecht1,4;
7
Stefanie CP Lopes1,5; Ana Carolina AV Kayano1; Alessandro S Farias1;
8
Wan Ni Chia6; Carla Claser6; Benoit Malleret6; Bruce Russell6,#;
9
Catarina Castiñeiras1; Leonilda MB Santos1; Marcelo Brocchi1;
10
Gerhard Wunderlich2; Irene S Soares7; Mauricio M Rodrigues8;
11
Laurent Rénia6; Fabio TM Costa1*.
12 13 14
1
15
UNICAMP. Campinas, SP. Brazil.
16
2
17
Brazil.
18
3
19
RJ, Brazil.
20
4
21
Brazil.
22
5
23
Manaus, AM, Brazil.
24
6
25
Research (A*STAR), Singapore, Singapore.
Department of Genetics, Evolution and Bioagents. University of Campinas –
Department of Parasitology, University of São Paulo – USP. São Paulo, SP,
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz – FIOCRUZ. Rio de Janeiro,
Instituto Carlos Chagas, Fundação Oswaldo Cruz – FIOCRUZ. Curitiba, PR,
Instituto Leônidas e Maria Deane, Fundação Oswaldo Cruz – FIOCRUZ.
Singapore Immunology Network, Agency for Science, Technology and
1
26
7
27
University of São Paulo - USP, São Paulo, SP, Brazil.
28
8
29
São Paulo, São Paulo, SP, Brazil.
Department of Clinical and Toxicological Analysis, Pharmaceutical Sciences,
Center for Cellular and Molecular Therapy - CTCMol, Federal University of
30 31
Running head: MAEBL, M2 domain, malaria vaccine, Plasmodium yoelii.
32 33
#
34
Singapore – NUS, Singapore, Singapore.
Present address: Department of Microbiology, National University of
35 36
*Address correspondence to: Fabio T M. Costa, [email protected]
37 38
Abstract
39
Malaria remains a world threatening disease largely due to the lack of a
40
long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane
41
molecule with a chimeric cysteine-rich ectodomain homologous to regions of
42
DBL-EBP and AMA1 antigens. Although MAEBL does not appear to be
43
essential for the survival of blood-stage forms, the ectodomains M1 and M2,
44
homologous to AMA1, seem to be involved in parasite attachment to
45
erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of
46
mosquito salivary glands and is expressed in liver stages. Here, the
47
Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector.
48
C57BL/6J mice were immunized with doses of the P. yoelii recombinant
49
protein (rPyM2-MAEBL). High levels of antibodies, with balanced IgG1/IgG2c
50
subclasses, were achieved. rPyM2-MAEBL antisera were capable of
2
51
recognizing the native antigen. Anti-MAEBL antibodies recognized different
52
MAEBL fragments expressed in CHO cells showing stronger IgM and IgG
53
response against the M2 domain and repeat region, respectively. After
54
challenge with P. yoelii YM (lethal strain)-infected erythrocytes, up to 90% of
55
immunized animals survived, and a reduction of parasitemia was observed.
56
Moreover, splenocytes harvested from immunized animals proliferated in a
57
dose-dependent manner in the presence of rPyM2-MAEBL. Protection was
58
highly dependent on CD4+, but not CD8+ T cells, towards Th1. rPyM2-
59
MAEBL antisera were also able to significantly inhibit parasite development as
60
observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these
61
findings support the use of MAEBL as a vaccine candidate and open
62
perspectives to understand the mechanisms involved in protection.
63 64
Introduction
65
Malaria remains one of the most devastating infectious diseases in
66
inter-tropical countries, affecting mainly children under the age of five and
67
pregnant women. Approximately 600,000 deaths occur every year (1). People
68
repeatedly exposed to malarial infections in endemic areas develop immunity
69
against clinical disease and subsequently against parasitemia (2-5).
70
Antibodies have been shown to be responsible for naturally acquired immunity,
71
since passive transfer of immune IgG from adults can protect against
72
Plasmodium falciparum blood-stage infection (2, 6-8), suggesting that a
73
malaria vaccine based on asexual antigens is feasible. Unfortunately, none of
74
the vaccines currently tested achieved a convincing rate of protected
3
75
individuals (9-11) and the observed protection was often short–lived or highly
76
strain-specific (12-16).
77
The stakes for blood stage vaccines are even higher when malaria
78
eradication is the goal because the vaccines must not only reduce disease but
79
also reduce the parasitic burden to a degree that reduces the transmission
80
(17). Despite considerable efforts, none of the blood stage vaccine candidates
81
have exhibited satisfactory clinical and sterile protection in field tests (18, 19).
82
Many of the current vaccine candidates were encountered based on
83
the finding that partly immune individuals possess high titers of antibodies
84
against the tested antigens. Recently, the finding that antibodies against
85
PfRH5 are highly effective in blocking merozoite reinvasion but are rarely
86
detected in significant quantities in semi-immune carriers was reported (20).
87
This finding suggests that other merozoite-exposed antigens against which no
88
significant response is developed in natural infections may also be effective
89
as vaccines.
90
MAEBL is a 200-kDa type 1 membrane protein that belongs to the
91
erythrocyte binding protein (ebl) family (21-24). It has a carboxy cysteine-rich
92
region homologous to region IV of the DBL-EBP (Duffy binding like –
93
Erythrocyte binding protein). Additionally, the MAEBL amino cysteine-rich
94
domains (M1 and M2 ligand domains) exhibit partial similarity with the apical
95
membrane antigen 1 (AMA1) (23, 25). The M1 and M2 domains from P. yoelii
96
have been shown to be functionally equivalent to the DBL ligand domain, as
97
they bind to mouse erythrocytes (25).
98
MAEBL is essential for the development of the parasite during
99
sporozoite infection of mosquito salivary glands (26, 27) and is also
4
100
expressed in the salivary gland sporozoite and during the late liver stage (28).
101
Weak expression of MAEBL can also be detected in blood merozoite forms,
102
although maebl deletion has no impact on blood parasite development (26).
103
Coincidently, only few antibodies are found in naturally infected individuals
104
from areas with low transmission rates (29). MAEBL is a highly conserved
105
gene between evolutionarily distinct species of Plasmodium (25). Among the
106
clones of P. falciparum and field isolates, there is little amino acid sequence
107
variation on M1 and M2 domains (21). Because MAEBL is a well conserved
108
gene and expressed at different parasite stages, it is considered an
109
interesting potential vaccine candidate (30).
110
The current knowledge of the mechanisms and interactions during
111
Plasmodium invasion of the erythrocytes are still limited, which impairs the
112
development of approaches to block this essential step in Plasmodium biology.
113
As blocking erythrocyte invasion strategies are part of the rational of several
114
vaccine based on merozoite antigens, approaches designed to elucidate the
115
invasion phenomenon might facilitate validation and identification of potential
116
antigens that could be used as vaccine targets.
117
In this study, we investigated the immunogenicity of the domain M2-
118
MAEBL of P. yoelii. We amplified, cloned and expressed M2-MAEBL as a
119
prokaryotic fusion protein (rPyM2-MAEBL). Mice received four doses of the
120
recombinant antigen emulsified in Freund´s adjuvant. Immunization induced
121
robust protection against a lethal challenge with asexual forms of P. yoelii YM.
122
Protection was dependent on CD4+, but not CD8+ T cells, towards Th1. By
123
adapting an ex vivo invasion assay we could show that sera from immunized
124
mice inhibited invasion of parasite blood-stage forms. These results
5
125
demonstrate that MAEBL can be used as antigen in anti-malarial vaccine
126
formulations.
127 128
Methods
129
Parasites and animals
130
Six to seven week-old C57BL/6J mice were purchased from the
131
University of Campinas Animal Center (CEMIB-UNICAMP). Animals were
132
kept in a mouse pathogen free facility. All experiments and procedures were
133
approved by the Ethical Committee for Animal Research of the University of
134
Campinas (Protocol No. 1437-1). Blood forms of P. yoelii were obtained after
135
intraperitoneal (i.p.) injection of thawed glycerin frozen stocks.
136 137
Generation of the recombinant P. yoelii M2-MAEBL
138
DNA fragments encoding M2-MAEBL were obtained by PCR
139
amplification using Platinum Taq high-fidelity DNA polymerase (Invitrogen),
140
using P. yoelii genomic DNA from infected mice as a template. For the
141
amplification of the sequence corresponding to the M2 domain of the MAEBL
142
antigen, we used synthetic oligonucleotides based on the sequence from
143
amino acid 589 to 992 from the P. yoelii yoelii MAEBL gene (GenBank,
144
Accession number AF031886). For this, the following oligonucleotides were
145
used: (I) pET28aM2F 5’-CGC GGA TCC CTT AAC AAA TAT ATG AAA TCT
146
AAT GTT GAA CTT -3' and (II) anti-sense pIgSPM2R 5’-CTC GAA TTC CTA
147
CGA TTC ATC GGT ATT TCT TGT AG-3’; containing BamHI and EcoRI
148
sites, purchased from Invitrogen, USA. The resulting PCR product was
149
purified and digested with both restriction enzymes and inserted into the
6
150
pET28a vector (Novagen). The resulting plasmid was designated pET28aM2.
151
To verify that the reading frame was correct, inserts were sequenced using
152
vector-specific oligonucleotides covering the entire cloned sequence.
153
The recombinant protein (rPyM2-MAEBL) was expressed and purified
154
as previously described (31) with slight modifications. E. coli BL21 DE3
155
(Novagen) were transformed with the pET28aM2 plasmid and cultivated at
156
37°C in Luria-Bertani (LB) broth in the presence of 30 μg/ml kanamycin
157
(Sigma). Protein expression was induced at OD600 with 0,1 mM IPTG
158
(Invitrogen) for 4 h. After centrifugation, the bacterial pellet was resuspended
159
and sonicated in phosphate-buffered saline (PBS) supplemented with 1 mg/ml
160
lysozyme (Sigma) and 100 mM of PMSF (Sigma). The sonicated material was
161
then centrifuged, and the supernatant was separated from the pellet. Under
162
these conditions, rPyM2-MAEBL was found in the insoluble fraction. For
163
solubilization and purification, the pellet was resuspended in 100 mM Tris-HCl,
164
8 M urea, and 100 mM DTT, pH 8.0. Complete solubilization was achieved by
165
the passage of the solution through a 21-gauge needle. The solution was then
166
centrifuged and the supernatant applied to a column with N2+-NTA-agarose
167
resin (Qiagen). After extensive washing the recombinant protein was eluted
168
with a buffer with pH 5.0, and the eluted protein was dialyzed against PBS
169
with 2 M urea. Protein expression and concentration was determined by SDS-
170
PAGE and immunoblotting analysis.
171 172 173
7
174
Generation of a pdisplay library containing fragments of the MAEBL
175
immunogen
176
Primers were designed to be in-frame, complementary and specific to
177
different regions of MAEBL, without self-annealing sequences using the
178
software Amplify3x. Three different regions (MAEBL-3, MAEBL-4 and
179
MAEBL-5) encompassing the M2, repeat and C-terminal cysteine regions
180
respectively were designed. Six smaller, overlapping regions of the MAEBL-3
181
region (which contains different segments of the M2 region, of the region
182
between inter-domain between M2 and/or the repeats or repeats) were
183
designed and these were subcloned into the pDisplay vector (Invitrogen)
184
together with MAEBL-4 and MAEBL-5 constructs. The primer sequences used
185
to clone these fragments of MAEBL are all reported in Table S1.
186
Genes-of-interest were amplified with Phusion Hot Start II polymerase
187
(Finnzymes) according to manufacturer’s instructions, using the Biorad Peltier
188
thermal cycler. The PCR products were ligated into the XmaI and SacII (NEB)
189
linearized pDisplay vector according to manufacturer’s recommendations (In-
190
Fusion cloning, Clontech) before heat shock transformation into XL10 gold
191
bacteria (Agilent). Bacteria clones were screened by colony PCR using T7
192
and BgH primers for positive transformants. They were then sent for
193
sequencing to ensure codons were in-frame. The plasmids were purified
194
using Nucleobond AX Endotoxin-Free Midiprep kit (Macherey-Nagel) before
195
transfection. CHO (Chinese hamster cell) cells were used for the expression
196
of recombinant MAEBL proteins. Cells were grown in F12 complete medium
197
containing 100 units/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and
198
10% heat-inactivated fetal bovine serum (FBS) (Gibco) in a 37oC incubator
8
199
with 5% CO2. Sterile ethylene-diamine-tetra-acetic acid (EDTA) diluted to a
200
final concentration of 2 mM in PBS was used to detach transfected
201
cells/stable cell lines. Endofectin (Genecopeia) was used to transfect the
202
recombinant pDisplay plasmid into CHO cells. CHO cells were seeded into 6-
203
cm cell culture dishes one day before transfection in a density that would give
204
approximately 70 - 80% confluency on the day of transfection. To transfect the
205
cells, 9 μl of Endofectin was added dropwise with vortexing to 3 μg of plasmid
206
diluted in F12 supplemented with 100 units/ml penicillin and 100 μg/ml
207
streptomycin for 6-cm dishes. The DNA-Endofectin complex was incubated at
208
room temperature for 25 min before it was added dropwise to the cells. The
209
plates or dishes were swirled gently to distribute the transfection mixture
210
before returning them to the 37oC incubator overnight. The transfection
211
medium was replaced with fresh F12 complete medium the following day.
212 213
Immunoblotting analysis
214
Recombinant protein samples were loaded on SDS-PAGE under
215
denaturating conditions (2-mercaptoethanol). Proteins were then transferred
216
onto a nitrocellulose membrane (GE Healthcare). Immunoblotting was
217
conducted with anti-His6 (Life Technologies) at a 1:1500 dilution. The antibody
218
binding was detected by incubation with peroxidase-coupled goat anti-mouse
219
IgG (Sigma) at a 1:2000 dilution. Bands were detected using a solution
220
containing 50 mM Tris-HCl pH 7.5, 0.6 mg/mL of 3,3’ diaminobenzidine and 1
221
μL/mL of 0,03% H2O2 (Sigma) solution.
222 223
9
224
Immunization regimen
225
Groups of six to ten C57BL/6J male mice (Six- to 7-week-old) were
226
injected subcutaneously four times at three-week intervals with 5 μg of
227
rPyM2-MAEBL 1:1 (vol/vol) emulsified in complete Freund’s adjuvant (Sigma)
228
(CFA) for the first dose or incomplete Freund’s adjuvant (IFA) in the
229
subsequent doses. As a control group, animals were injected with 1:1 (vol/vol)
230
adjuvant (CFA/IFA) in PBS. Sera from immunized mice were collected
231
immediately before each dose and 3 weeks after the last dose (Figure 2A).
232 233
ELISA
234
Anti-rPyM2-MAEBL antibodies titers were determined by ELISA as
235
previously described (32). Plates were coated with 200 ng/well of recombinant
236
protein. Mice sera were tested at serial dilutions starting from 1:100. A dilution
237
of 1:2000 peroxidase-coupled goat anti-mouse IgG conjugated (Sigma) was
238
applied as a secondary antibody. Specific anti-MAEBL titers were determined
239
as the highest dilution yielding an OD492 higher than 0.1. Detection of IgG
240
subclasses was performed as described above, except that the secondary
241
antibody used was specific for mouse IgG1, IgG2b and IgG2c (Southern
242
Technologies).
243 244
Epitope mapping
245
Transfected cells (150 000 per well) were transiently transfected and seeded
246
in 96 well plates (Nunc). They were stained with serum diluted in FACS buffer
247
(PBS+10% FBS) for 1 h with agitation at 4oC. The different MAEBL constructs
248
are flanked by a myc epitope on the N-terminus and HA epitope on the C-
10
249
terminus when expressed on the surface of CHO cells, and both epitopes
250
were utilized to assess for protein expression on CHO cells using rabbit anti-
251
myc IgG (Upstate) or rabbit anti-HA IgG (Sigma) diluted 1:100. Untransfected
252
parental CHO cells were stained using the same sera and used as negative
253
control. For determination of serum recognition, the different test sera from
254
mice immunized with MAEBL protein or control vehicle DMSO were diluted
255
1:100 and incubated with the cells for 1 h with agitation at 4oC and then
256
washed once with FACS buffer. Secondary antibody – either goat anti-rabbit
257
IgG-AlexaFluor488 (Molecular Probes, Invitrogen) diluted 1:500 for checking
258
of protein expression or goat anti-mouse IgM or IgG-AlexaFluor488
259
(Molecular Probes) diluted 1:500, mixed with propidium iodide diluted to 0.001
260
mg/ml for the serum screening assay were incubated for 30 min with agitation
261
at 4oC. Labeled samples were analyzed using the high throughput flow
262
cytometer Accuri (Becton Dickinson). Flowjo (Tree Star) was used for all flow
263
cytometry analysis. Serum response was determined as the percentage of
264
cells recognized by serum divided by cells expressing the construct of interest
265
as illustrated in this formula:
266
Serum response = [(% cells recognized by immune serum – % cells
267
recognized by naïve serum) / (% Protein of interest POI expressing cells)] x
268
100%.
269
The 20% Cut-off for positivity was determined as the mean + 6 standard
270
deviation of the quadruplicate serum recognition values obtained for sera from
271
naïve animals. This high threshold was uses to reduce the possibility of false
272
positives.
273
Cellular proliferation and cytokines quantification
11
274
Ten
million
splenic cells
collected
from
animals
of
different
275
immunization groups (n=3) were cultured in 96-well plate in a final volume of
276
200 μL. rPyM2-MAEBL protein was added to the culture at a final
277
concentration of 1 or 10 µg/mL. As a control, some wells received ConA (50
278
µg/ml). Lymphocyte proliferation was assessed by the addition of 0.5 mCi/well
279
of methyl-[3H] thymidine ([3H] TdR (GE Healthcare) to the splenocytes after
280
48 h of culture. Splenocytes were cultured for an additional 18 h, and plates
281
were frozen. For later analysis, plates were thawed, and the culture was
282
collected on filter paper using a cell harvester. Tritium incorporation was
283
measured as radioactivity using Beta 1450 Microscintillator (Perkin Elmer).
284
The results are expressed as the average of the triplicate culture.
285
In parallel, culture supernatants were collected after 120 h, and the
286
amount of secreted IFN-γ, TNF, IL-2, IL-4 and IL-5 in cell culture supernatant
287
were simultaneously detected using the Cytometric Bead Array (CBA) Mouse
288
th1/th2 Cytokine Kit (BD). Cytokine concentrations in each sample were
289
determined with standard curves of known concentration of all five
290
recombinant proteins. Samples were read in a FACS Calibur flow cytometer
291
(Becton Dickinson). The detection limit of the assay was 0.5 pg/ml.
292 293
Immunofluorescence assays
294
Slides were prepared by collecting blood from BALB/c mice infected
295
with P. yoelii YM on day 5 post-infection (p.i.). The selection of mature stages
296
of the parasite was performed using LS-MACS columns (Miltenyi Biotec), and
297
thin blood smears were made, air-dried and stored until immunofluorescence
298
(IF) preparation and microscopy analysis. Immunofluorescence assays were
12
299
performed after fixing the blood smears with ice-cold acetone for 20 min and
300
air-dried. Well diameters were established with the aid of a Dako-PenTM
301
(Dako), and blocking was performed by 30-min incubation at 37°C with PBS
302
containing 3% BSA (USB). Pooled sera from the different immunization
303
groups and P. yoelii hyperimmune sera were diluted 1:100 in PBS
304
supplemented with 3% BSA and applied to the slides for 1 h at 37°C. Slides
305
were washed 3 X in PBS and incubated with Alexa-568 goat anti-mouse IgG
306
(Invitrogen) for 1 h at 37°C in the dark, then washed 3 X in PBS and
307
incubated
308
(Invitrogen) diluted in ultrapure (Millipore) water for 10 min at room
309
temperature. After another round of washing, Fluorosave (Caliobiochem) was
310
added, and slides were sealed with coverslips. Parasites were visualized with
311
the aid of a Nikon TS100 epifluorescence microscope. P. yoelii hyperimmune
312
sera were obtained from BALB/c mice infected with P. yoelii 17XNL clone 1.1
313
(non-lethal strain) once a month for a total period of 7 months. In parallel, to
314
confirm specificity, we also conducted immunofluorescence assays using
315
Leishmania amazonensis promastigote forms (gently donated by Dr. Selma
316
Giorgio, UNICAMP) and anti-rPyM2-MAEBL antisera.
with
DAPI
(4′,6-diamidino-2-phenylindole,
dihydrochloride-
317 318
Mice challenge and parasitemia assessment
319
The protection levels against blood forms were determined by
320
immunized mice survival against lethal challenge after intraperitoneal injection
321
of 106 P. yoelii YM-infected erythrocytes. Parasitemia was monitored,
322
according to Figure 2A, between 3 and 10 days post infection and determined
13
323
by counting at least 1,000 erythrocytes from thin blood smears stained with
324
the Panótico Rápido Kit (Laborclin).
325 326
CD4+ and CD8+ T lymphocyte depletion
327
Groups of 15 C57BL/6J mice were immunized as detailed in Figure 6A.
328
Three weeks after the fourth dose, five animals were injected with 0.5
329
mg/animal (Bioxcell) anti-CD4 (clone GK1.5) or anti-CD8 (clone YTS169.4)
330
monoclonal antibodies for the depletion of the corresponding lymphocyte
331
populations. The efficacy of In vivo depletion efficacy for this dose of
332
antibodies was determined in a preliminary experiment, as assessed by flow
333
cytometry of CD8+T and CD4+T cell using specific monoclonal antibodies
334
recognizing different epitopes in the CD4 or CD8 molecules. As a control, a
335
group of five immunized animals were injected with 0.5 mg per animal of
336
purified unrelated mouse IgG (Sigma, USA). On the following day, animals
337
were challenged with 106 P. yoelii YM IEs (lethal strain). The survival of
338
animals was monitored and parasitemia was evaluated as described
339
previously.
340 341
Ex vivo invasion assays of P. yoelii YM merozoites
342
To obtain high levels of reticulocytes to be used as target cells in the
343
invasion assay, reticulocytemia was induced in groups of four to six naive
344
mice by repeated daily retro-orbital bleeding for 6 days. Leukocyte removal
345
from the reticulocyte-enriched blood was achieved after two cycles of CF11
346
filtration (33).
347
Infected erythrocytes were enriched and maturated to schizonts.
14
348
Enrichment was achieved by bleeding P. yoelii YM-infected mice (n= 3) on
349
day 5 post-infection (>60% parasitemia) and isolating mature forms (15-18 h
350
of development) by magnetic concentration as previously described (34). A
351
blood smear was made after the procedure to determine the concentration of
352
parasites and verify the complete elimination of leukocytes. Next, to
353
synchronize parasites to at least 80% at the schizont stage, the pellet
354
corresponding to the mature forms of P. yoelii was resuspended in McCoy's
355
5A medium (Sigma) supplemented with 2.4 g/l glucose, 40 mg/ml gentamycin
356
(Sigma) and 20% human AB serum (UNICAMP’s blood bank), inactivated and
357
adsorbed on murine red blood cells and cultivated 3-6 h in 5% CO2 and at
358
37°C for maturation.
359
Finally, invasion assays were adapted from an assay for P. vivax [38]
360
performed by mixing P. yoelii concentrated schizonts and target cells
361
(enriched reticulocyte rich blood) at a 1:10 ratio corresponding to
362
approximately 10% of the initial parasitemia. The mixture was diluted to a 4%
363
hematocrit in 100 µl of McCoy's 5A medium supplemented with 2.4 g/l
364
glucose, 40 mg/ml gentamycin and then cultured for approximately 20 h in a
365
candle jar (35).
366
Immunized sera used in the invasion assays were heat inactivated by
367
incubation at 56ºC for 40 min. Pool sera from animals immunized with four
368
doses of the rPyM2-MAEBL diluted 1:100 were added to the culture. As
369
controls, the pool sera of animals injected four times with CFA/IFA at the
370
same dilution were used, and E64 (Sigma) at 100 μM was used as a positive
371
inhibition control because it ensures no schizont rupture. As a negative control
372
of inhibition, the assay was performed without addition of any drug or serum.
15
373
Freshly infected erythrocytes were determined by light microscopy and
374
parasitemia of young forms (rings and young trophozoites) was determined as
375
previously described. The standardized methodology for the P. yoelii invasion
376
assays is depicted in detail in Figure 7.
377 378
Statistical analysis
379
Survival curves of immunized animals after lethal challenge was
380
evaluated by the Log-Rank test. Comparison of the means of three or more
381
groups was performed using one-way ANOVA, and significance between
382
every two groups was assessed by the Bonferroni multiple comparison test
383
when data assumed Gaussian distribution, as assessed by the D’Agostino
384
and Pearson normality test. For samples of no Gaussian distribution, group
385
comparison was analyzed using the Kruskal-Wallis test followed by Dunn’s
386
test.
387
Whitney U test was used. All tests were performed using Prism GraphPad
388
software version 5.0a. The results were considered significant when P
