A Nonintegrative Lentiviral Vector-Based Vaccine
Provides Long-Term Sterile Protection against Malaria
Fre ´de ´ric Coutant1¤a, Raul Yusef Sanchez David1¤b, Tristan Fe ´lix1, Aude Boulay1¤c, Laxmee Caleechurn1¤d,
Philippe Souque1, Catherine Thouvenot2, Catherine Bourgouin2, Anne-Sophie Beignon1*.,
1Unite ´ Virologie Mole ´culaire et Vaccinologie, Department of Virology, Institut Pasteur and CNRS URA3015, Institut Pasteur, Paris, France, 2Centre de Production et
d’Infection des Anophe `les (CEPIA), Department of Parasitology and Mycology, Institut Pasteur, Paris, France
Trials testing the RTS,S candidate malaria vaccine and radiation-attenuated sporozoites (RAS) have shown that protective
immunity against malaria can be induced and that an effective vaccine is not out of reach. However, longer-term protection
and higher protection rates are required to eradicate malaria from the endemic regions. It implies that there is still a need to
explore new vaccine strategies. Lentiviral vectors are very potent at inducing strong immunological memory. However their
integrative status challenges their safety profile. Eliminating the integration step obviates the risk of insertional
oncogenesis. Providing they confer sterile immunity, nonintegrative lentiviral vectors (NILV) hold promise as mass pediatric
vaccine by meeting high safety standards. In this study, we have assessed the protective efficacy of NILV against malaria in a
robust pre-clinical model. Mice were immunized with NILV encoding Plasmodium yoelii Circumsporozoite Protein (Py CSP)
and challenged with sporozoites one month later. In two independent protective efficacy studies, 50% (37.5–62.5) of the
animals were fully protected (p=0.0072 and p=0.0008 respectively when compared to naive mice). The remaining mice
with detectable parasitized red blood cells exhibited a prolonged patency and reduced parasitemia. Moreover, protection
was long-lasting with 42.8% sterile protection six months after the last immunization (p=0.0042). Post-challenge CD8+ T
cells to CSP, in contrast to anti-CSP antibodies, were associated with protection (r=20.6615 and p=0.0004 between the
frequency of IFN-g secreting specific T cells in spleen and parasitemia). However, while NILV and RAS immunizations elicited
comparable immunity to CSP, only RAS conferred 100% of sterile protection. Given that a better protection can be
anticipated from a multi-antigen vaccine and an optimized vector design, NILV appear as a promising malaria vaccine.
Citation: Coutant F, Sanchez David RY, Fe ´lix T, Boulay A, Caleechurn L, et al. (2012) A Nonintegrative Lentiviral Vector-Based Vaccine Provides Long-Term Sterile
Protection against Malaria. PLoS ONE 7(11): e48644. doi:10.1371/journal.pone.0048644
Editor: Thomas L. Richie, Naval Medical Research Center, United States of America
Received June 3, 2012; Accepted September 27, 2012; Published November 2, 2012
Copyright: ? 2012 Coutant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Institut Pasteur and the Centre national de la recherche scientifique (CNRS) supported this study. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: We have read the journal’s policy and have the following conflicts. PC is the founder of theravectys, a company whose goal is to develop
a therapeutic AIDS vaccine using lentiviral vectors. FC and PC are inventors of a patent titled ‘‘Lentiviral vector based immunological compounds against malaria’’
(international patent application W02011138251 filed on April 24, 2011) and which is owned by the Institut Pasteur. There are no further patents, products in
development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org (ASB); email@example.com (PC)
. These authors contributed equally to this work.
¤a Current address: Faculte ´ de me ´decine Paris Descartes, Paris, France
¤b Current address: Unite ´ de Ge ´nomique Virale et Vaccination, Department of Virology, Institut Pasteur and CNRS URA3015, Institut Pasteur, Paris, France
¤c Current address: Service d’Epide ´miologie, Institut Pasteur de Bangui, BP 923 Bangui, Re ´publique Centrafricaine
¤d Current address: Laboratoires Clarins S.A., BP147, 95304 Cergy Pontoise Cedex, France
Plasmodium is the causative agent of malaria, a life-threatening
disease affecting 216 million people worldwide and responsible for
655 000 deaths in 2010 according to the World malaria report 2011.
Repeated childhood exposure to Plasmodium naturally confers
specific immunity that protects against the most severe forms of
malaria, but does not confer sterile protection. Children remain at
risk until they have developed this partial immunity . Therefore
an ideal malaria vaccine should fully prevent infection from early
Plasmodium sporozoites are inoculated into the host’s skin by
bites from infected mosquitoes. After invading skin blood vessels,
they migrate to the liver where they invade hepatocytes and
develop. Infected hepatocytes then produce and release merozoites
into the blood circulation, which in turn invade red blood cells
[2,3,4]. The liver-stage is asymptomatic while the erythrocytic
stage is pathogenic. Immunizations with radiation-attenuated
sporozoites (RAS), which interrupt their development inside
hepatocytes, can confer sterile protection against malaria in
humans  and rodents . However, this strategy is not easily
applicable to large-scale approaches because of major technical
and logistical limitations, and was sub-optimally immunogenic and
protective in a recent phase I/IIa trial following subcutaneous and
intradermal injections . Several other candidate vaccines, such
as adenovirus or poxvirus vectorized Ag, have been or are being
evaluated for safety and immunogenicity and then for protection
using experimental challenges or in-field trials [8,9,10]. Malaria
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vaccine projects at advanced pre-clinical and clinical stages
globally are summarized by the WHO (the WHO.28_Nov_2011
vaccine_research/links/Rainbow/en/index.html). However, vac-
cine-induced immunity has so far failed to confer strong and long-
lasting protection against malaria [11,12]. The most advanced
candidate vaccine is the RTS,S, a sub-unit vaccine based on a
single pre-erythrocytic antigen (Ag), the Circumsporozoite protein
(CSP) from Plasmodium falciparum (Pf). It was shown to substantially
reduce clinical and severe Pf malaria episodes in infants from seven
countries in sub-Saharan Africa in a large phase III clinical trial
yet without completely preventing infection . Longer-term
protection needs to be documented and higher rates of protection
are likely required to achieve eradication of malaria in endemic
Thus, there is an urgent need to develop new vaccine strategies,
including new vectors. The liver stage, although clinically silent,
plays a key role in the parasite life cycle. A vaccine aiming to block
Plasmodium at the early steps of its cycle in the vertebrate host is
likely to be more successful than a vaccine based on erythrocytic
Ags only. A mosquito bite delivers about 100 sporozoites in the
skin. It results in the rapid invasion of few hepatocytes [15,16].
The liver-stage is completed in a few days, depending on the
parasite and host. Then, each infected hepatocyte releases about
30,000 merozoites into the blood stream . Infection of
hepatocytes renders parasites susceptible to recognition and
elimination by CD8+T cells . However, the low number of
sporozoites, the low frequency of infected hepatocytes and the
short duration of the liver stage make the task considerably difficult
for neutralizing Abs and/or effector T cells. It is anticipated that a
high frequency of CD8+ T cells with immediate effector functions
in the liver is required for protection against the disease
HIV-1 derived lentiviral vector (LV) are very potent at inducing
strong and broad cellular and humoral memory responses
[23,24,25,26,27]. They provide protective immunity against many
tumors and infectious diseases as shown in mice and monkeys
[28,29,30,31]. These properties are explained by their adjuvan-
ticity [32,33,34] and ability to efficiently transduce non-dividing
cells and in particular dendritic cells (DC), which are the most
effective antigen-presenting cells [35,36,37]. Despite these advan-
tages, their integrative status challenges their safety profile. The
risk of insertional mutagenesis likely precludes their large-scale use
as prophylactic and pediatric vaccines. Insertional mutagenesis
results from the presence of transcriptional enhancer sequences
within the vector construct [38,39,40], therefore the use of
promoters devoid of associated enhancer activity is a means to
improve the safety of integrative LV (ILV) . However, one of
the best strategies to obviate the risk of insertional oncogenesis is to
eliminate the integration step altogether by using a nonintegrative
LV (NILV) carrying a defective HIV-1 integrase . Double-
stranded episomal DNA circles, which accumulate in the nucleus
as a result of the integration defect, are highly competent for
transcription. Hence, transduction with NILV leads to the potent
and sustained expression of the gene of interest and effective gene
therapy for post-mitotic tissues such as ocular and brain tissues or
liver [43,44,45,46]. In contrast to ILV, NILV mediate stable gene
expression only in non-dividing cells, whereas expression is
transient in proliferating cells because of the partition of the
episomes between daughter cells and progressive dilution as cells
further divide. Since DC are non-dividing highly differentiated
cells, NILV should be immunogenic. We have shown that NILV
transduce conventional and plasmacytoid murine DC as efficiently
as ILV and that immunization with NILV encoding a secreted
form of the envelope of West Nile Virus protects mice against
lethal challenge through the induction of neutralizing antibodies
. It was also reported that mice immunized with NILV mount
potent CD8+ T cells against various antigens, such as HIV-1
gp120 and gag, HBsAg or OVA and hgp100, which mediate
effective tumor prophylaxis and therapy [48,49,50,51,52,53].
To explore the protective efficacy conferred by NILV against
malaria, we used the major pre-erythrocytic stage malaria vaccine
candidate Ag, CSP. CSP is the main protein of invading
sporozoites and it is highly immunogenic. It continues to be
transcribed in liver cells, and is very potently presented by infected
cells on their MHC class I molecules [18,54]. RTS,S, which is
composed of a single Ag, Pf CSP, provides some protection against
clinical and severe malaria .
We performed challenge experiments of BALB/c mice with
Plasmodium yoelii (Py). In this murine model of malaria, which is
widely used for the pre-clinical development of vaccines and drugs,
both antibodies to the central repeat domain of CSP and CSP-
specific CD8+T cells have been shown to mediate protection, by
inhibiting the migration of sporozoites from the skin to the liver as
well as hepatocyte invasion and by hindering the development of
parasites within hepatocytes, respectively, thus preventing blood-
stage malaria [55,56,57,58].
Here we report that a NILV encoding CSP induce protective
CD8+ T cell responses against malaria. After three immunizations,
50% (37.5–62.5) of the animals were fully protected when the
challenge with sporozoites was carried out one month after the last
immunization as demonstrated in two independent challenge
studies, while 42.8% of the mice did not develop parasitemia when
challenged 6 months after the last immunization. In addition to
this sterilizing and long-term protection, the remaining vaccinated
animals with detectable parasitized red blood cells exhibited a
delayed erythrocyte infection compared with naive animals and a
reduced parasitemia. Since immune control is exerted at the pre-
erythrocytic stage with CSP as the sole targeted Ag, this suggested
a reduced parasite burden in the liver.
To our knowledge, this is the first report of the use of LV as
malaria vaccine candidate. Data are encouraging. They provide a
proof-of-concept for the protective efficacy against malaria with a
basic NILV. Studies are ongoing to discover new protective Ags
and to improve the design of NILV to ensure stronger
immunogenicity and higher rates of protection.
Comparison of the Immunogenicity of Integrative and
Nonintegrative Lentiviral Vectors
We first compared the intensity of cellular immune responses
induced by NILV and ILV. Both types of vector particles are
produced by transient transfection of 293 T cells. They only differ
by the D64V substitution in the catalytic domain of the HIV-1
integrase encoded by Pol, blocking the DNA cleaving and joining
reactions of the integration step as previously described 
(Figure 1). The cellular immune responses directed against two
CD8+ T cells immunodominant epitopes present in Py CSP, S9I
and I10L [60,61], were assessed 10 days after a single injection of
various doses of vector particles (Figure 2). Immunization with
NILV resulted in lower frequencies of S9I-specific blood CD8+ T
cells (quantified by tetramer staining) and of S9I- and I10L-specific
IFNg secreting splenocytes (measured by IFNg elispot) as
(Figure 2B). The lower immunogenicity of NILV compared to
ILV was not dependent on CSP, as this was also observed with an
unrelated Ag, SIV GAG (Figure S2); and it could be
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(5E+08TU/mouse) (Figure 2B).
We next further characterized immune responses induced by
5E+08 TU/mouse of NILV versus 5E+07 TU/mouse of ILV
(Figure 2C). The kinetics of blood responses were quite similar
(Figure 2D). About one month after immunization, spleen
cellular immune responses induced by 10-times more NILV
particles could not be distinguished quantitatively and qualitatively
from those induced by ILV, as tested by tetramer staining
(Figure 2E), IFNg elispot (Figure 2F) and intracellular staining
of cytokines (ICS for IFNg, IL2 and TNFa) (Figure 2G). The
humoral response against CSP, measured 3 weeks post-immuni-
zation, was also found to be comparable between ILV and a 10-
times higher dose of NILV (Figure 2H).
In conclusion, an increased dose of vector particles could
overcome the relative defect of immunogenicity of NILV
compared to ILV, and a potent cellular and humoral immunity
against CSP could be elicited. The use of NILV was thus
validated. NILV offer the important advantage of circumventing
any fear about safety issues related to insertional mutagenesis.
by increasingthe doseof vectorparticles
Comparison of Immune Responses Directed against CSP
after Immunizations with Nonintegrative Lentiviral
Vector Particles and Radiation-attenuated Sporozoites
Multiple injections of RAS are known to induce potent
sterilizing immunity in mice and are rightly considered as the
gold standard of protection against Plasmodium infection. A prime/
boost strategy was designed to compare vaccine efficacy induced
by NILV and RAS. Vector particles were pseudotyped with non-
cross-reactive envelopes to allow efficient in vivo iterative admin-
istrations (Figure 1). Mice received three successive injections of
NILV particles pseudotyped with the glycoprotein of Vesicular
Stomatitis Virus (VSV-G) serotype Indiana (IND) first, then the
VSV-G serotype New Jersey (NJ) and finally the glycoprotein from
the Cocal Vesiculovirus. For the first injection, two doses were
tested, either 100 ng p24 or 1500 ng p24 (corresponding to
1.48E+07 or 2.22E+08 TU/mouse respectively for this batch of
vector). The first boost was performed 2 months after the prime
immunization with 1500 ng p24 (2.88E+08 TU/mouse), while the
second boost was done 5 months after the first one, also with
1500 ng p24 (3.33E+08 TU/mouse). For RAS immunization,
mice were immunized three times with 50,000 irradiated
sporozoites at monthly intervals (Figure 3A). The frequency of
blood specific T cells was followed longitudinally by tetramer
staining. For NILV, a priming dose lower than the boosting doses
(100–1500–1500 ng p24) led to more specific T cells than three
injections with 1500 ng p24 (Figure 3B). This prime/boost
protocol also induced as many blood CSP specific T cells as three
injections of RAS (Figure 3C). Thus it was selected for our
detailed comparative functional analysis.
One month after the last immunization, responses towards
S9I, I10L and S16I (a peptide containing a Py CSP CD4+
epitope and the S9I CD8+ T cell epitope) were measured in the
spleen by IFNg elispot (Figure 4A) and by ICS (Figure 4B).
They were of the same order of magnitude or even a bit higher
with NILV than with RAS immunizations. T cells were
multifunctional and able to produce simultaneously IL2, IFN-c
and TNF-a. When liver cells were re-stimulated with S9I, their
IFNg responses were similarly high (Figure 4C). When target
cells pulsed with S9I were injected in mice immunized with
NILV or RAS, they were promptly and equally well killed
in vivo (Figure 4D). Finally, both NILV and RAS immuniza-
tions led to an equivalent generation of antibodies to CSP
(Figure 4E). Collectively, this comparison study demonstrated
Figure 1. Nonintegrative lentiviral vector encoding Plasmodium yoelii CSP used in the study. Lentiviral vector particles were produced by
transient transfection of 293 T cells. The three plasmids used to generate particles are represented here (schematic representation not to scale). The
vector expression plasmid pTRIP encodes the vaccine antigen, Plasmodium yoelii CSP. The encapsidation plasmid, p8.74 or pD64V for ILV or NILV
respectively, codes for HIV-1 proteins required for particle formation and transduction. The envelope expression plasmid encodes non-crossreacting
glycoproteins from Vesiculoviruses used in a specific order to circumvent anti-vector particles antibodies generated after each immunization
(Vesicular Stomatitis Virus glycoprotein (VSV-G) Indiana (IND) serotype followed by VSV-G New Jersey (NJ) serotype followed by Cocal virus
glycoprotein). Genes coding for structural/enzymatic and regulatory HIV-1 proteins are in dark and light blue respectively, while HIV-1 cis-acting
sequences are in yellow and promoter sequences are in grey. The transferred gene, Py CSP, with a human codon-optimized sequence is in green. LTR,
long terminal repeat; Y, encapsidation signal; cPPT/CTS, central polypurine tract/central termination sequence responsible for the formation of the
DNA Flap structure during reverse-transcription which is a determinant of HIV-1 nuclear import; WPRE, Woodchuck hepatitis virus post-transcriptional
response element to enhance mRNA nuclear export on a Rev/RRE independent fashion.
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that three injections of NILV result in immune responses
directed against CSP, which were comparable in intensity and
quality with three injections of RAS.
Comparison of the Protective Efficacy of Nonintegrative
Lentiviral Vectors and Radiation-attenuated Sporozoites
One month after the last boost, animals immunized with
NILV (100–1500–1500ng p24) and RAS were challenged
intravenously with 500 live sporozoites. Protection was evaluated
by monitoring the duration of the pre-patent period (delay
between challenge and appearance of blood stage parasites) and
parasitemia (the percentage of parasitized red blood cells). Full
protection was defined as the complete absence of parasites in
blood after sporozoite challenge (sterile immunity). Immune
control is exerted at the pre-erythrocytic-stage of the life cycle of
the parasites since CSP is targeted. It implies that a delay in the
pre-patent period and onset of blood-stage infection results from
a reduced parasite burden in the liver. This was considered as
partial vaccine efficacy.
After 5 days, all naive mice exhibited patent parasitemia. By
contrast,some vaccinated animals
(Figure 5A). Sterile immunity was observed in 37.5% of the
mice immunized with NILV (3 out of 8 animals) and in 100% of
the mice immunized with RAS. Moreover, in the 5 remaining
NILV immunized mice with detectable parasitemia, there was a
delay in the course of erythrocyte invasion, as well as a 2.75 fold
Figure 2. NILV are as immunogenic as ILV when 10-times more particles are injected. BALB/c mice (n=5/group) were immunized by IM
injection with various doses (expressed as TU/mouse) of lentiviral vector particles encoding Py CSP, either NILV (%) or ILV (&). Ten days later, specific
cellular immune responses were assessed (Figure 2A). The frequency of S9I-specific blood CD8+ cells was assessed by S9I/Kdtetramer staining, and
the frequency of IFNg secreting splenocytes in response to overnight restimulation with S9I or I10L peptides was measured by IFNg elispot assay
(Figure 2B). Means + SD are shown. BALB/c mice (n=3/group) were IM immunized with NILV (%) or ILV (&) at the dose of 5E+08 or 5E+07 TU/
mouse respectively (Figure 2C). The frequency of S9I-specific blood CD8+ cells was followed over time by tetramer staining (Figure 2D). At day 24
post-immunization, spleen cellular response was analyzed by S9I/Kdtetramer staining (Figure 2E), by IFNg elispot in response to S9I and I10L
peptides (Figure 2F), and by intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (Figure 2G). Cells secreting individual
(green), 2 (blue) or 3 (red) cytokines are shown. Anti-(QGPGAP)2-specific IgG at day 21 post-immunization were quantified by ELISA and expressed as
titers (Figure 2H). Medians + range are shown.
Figure 3. NILV elicit as frequent blood CSP-specific T cells as RAS after 3 injections. BALB/c mice (n=6/group) were immunized 3 times by
IP injections of NILV. They were primed by administration of NILV particles encoding Py CSP and pseudotyped with VSV-G IND at the dose of 100 or
1500 ng p24/mouse. They were boosted 2 months later with 1500 ng p24 of NILV particles pseudotyped with VSV-G NJ, and boosted again 5 months
later with 1500 ng p24 of NILV particles pseudotyped with the glycoprotein from Cocal virus. Additionally, mice (n=6) from the same batch were
immunized 3 times by IV injection with RAS at monthly intervals (Figure 3A). The frequency of S9I-specific blood CD8+ cells was followed over time
by S9I/Kdtetramer staining after NILV (Figure 3B) and RAS immunizations (Figure 3C). Data from individual mice and means are shown. The Y-axis
uses a logarithmic scale.
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reduction in the level of parasitemia compared to naive animals at
day 9 post-challenge (Figures 5B–5E).
We next sought to elucidate immune correlates of protection.
We compared day 28 post challenge immune responses (day of
euthanasia) with day 9 parasitemia (peak of parasitemia for the
naive animals). Analysis of immune responses in challenged
animals revealed that CSP-specific CD8+ T cells correlated with
the levels of protection against infection but not with anti-CSP
Abs (Figure 6A). Among the various immune functions
directed against CSP that we analyzed, none allowed to
These data showing that NILV immunizations can provide a
potent immune control of the parasite liver stage were strength-
ened by a second independent study. In this trial, NILV afforded
an even stronger protection, with 62.5% sterile protection (5 out of
8 mice) (Figures 7A–7B). Partially protected mice also showed
two times less parasitized red blood cells in comparison with naive
animals (Figures 7C–7D). As expected three weeks post-
challenge, naive mice displayed a dramatic splenomegaly.
Moreover, their spleens and livers showed a dark pigmentation
likely resulting from the accumulation of hemozoin produced by
the parasite during the digestion of red blood cell hemoglobin. By
contrast, the capacity of 5 out of 8 vaccinated mice to mount a
sterile immune response coincided with the preservation of their
spleen size and liver pigmentation (Figure 7E).
Importantly, even when the challenge was performed 6 months
after the last immunization with NILV (Figure 8A), 42.8% of the
mice still failed to develop any detectable parasitemia, while the
remaining vaccinated mice succeeded in controlling parasitemia to
a lower level compared to naive animals (Figures 8B–8E). This
important result illustrates the long-lasting protection conferred by
our vaccine strategy.
Figure 4. Three immunizations with NILV and RAS induce comparable pre-challenge CSP-specific immune responses. Groups of
BALB/c mice (n=6/group) were immunized 3 times with NILV by IP injections (in red) or with RAS by IV injections (in black). Immune responses, both
cellular and humoral, were compared 28 days after the last immunization. The frequency of IFNg secreting splenocytes in response to restimulation
with S9I, I10L or S16I peptides was measured by IFNg elispot assay (Figure 4A). The quality of the S9I specific response was further studied by
intracellular staining of 3 cytokines, IFNg, IL2 and TNFa, in response to S9I (Figure 4B). The frequency of IFNg secreting liver cells after restimulation
with S9I was analyzed by IFNg elispot (Figure 4C). Additional mice (n=6/group) were immunized to compare the vaccine-induced in vivo killing
capacity of S9I-pulsed target cells (Figure 4D). The presence of IgG directed against (QGPGAP)2was assessed by ELISA (Figure 4E). Individual
responses, means and SD are shown.
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Our studies were designed to define the protective efficacy of a
LV based-candidate malaria vaccine. Because NILV resolve the
much-feared risk of integrase-mediated insertional mutagenesis,
they would preferentially be used over ILV as prophylactic and
pediatric vaccine. Integration events in cells transduced with
NILV are limited to illegitimate recombination . They are
expected to be as extremely rare as with other transient gene
delivery methods such as DNA vaccines, which have a good
clinical safety record.
We report here that a single injection of NILV encoding CSP
elicits potent and sustained specific T cells. However, it appears
that current NILV are not as good as their integrative counterpart
to induce T cell responses and 10-times more NILV particles were
required to obtain immune responses as strong as with ILV, as
described previously [49,51]. This might be due either to the
intensity of Ag expression, which would be insufficient with NILV
compared to ILV, and/or to the critical involvement of some
dividing cells in the induction of immunity by LV since mitotic
cells lose episomal DNA contrary to integrated DNA.
A prime injection with a low dose of NILV followed by two
boosts with a higher dose led to CSP-specific immune responses
similar to three injections of RAS. Of note, responses to CSP
induced by RAS are usually moderate compared to other CSP-
based strategies, such as adenovirus or poxvirus [8,62,63]. One
month after the last immunization, NILV induced IFN-g secreting
cells in the spleen and liver (7446137 and 8006259 S9I specific
Figure 5. NILV immunizations provide protection against parasitemia after sporozoites challenge, but not as strong as RAS. Groups
of BALB/c mice (n=8/group) were immunized 3 times with NILV by intraperitoneal injections (in red) or with RAS by intravenous injections (in green),
or not (in black) (Figure 5A). They were challenged with 500 spz injected IV 28 days after the last immunization. The protective efficacies of both
vaccines against malaria were compared. Parasitemia were followed every other day from day 5 to day 23 post-challenge by Giemsa-stained blood
smears. The longitudinal follow-up of individual parasitemia is shown (Figure 5B) as well as means + SD (Figure 5C) and parasitemia at day 9 post-
challenge (which corresponds to the peak of viremia in the group of naive animals) (Figure 5D). Among the NILV-immunized mice (in red), fully (N)
versus partially (#) protected animals were further distinguished (Figure 5E). The Kruskal-Wallis test was used to compare 3 or 4 groups (Figure 5D
and Figure 5E respectively), followed by a Dunn’s multiple comparison post-test. Asterisks denote significance for the post-test (*p,0.05, **p,0.01
and or ***p,0.001). When comparing NILV and naive mice with a Mann-Whitney test (Figure 5D), **p=0.0072.
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IFN-g SFC/million respectively). Cells were poly-functional
(simultaneous secretion of IL2, IFNg and TNFa in response to
(91.663.6% in vivo killing of S9I pulsed splenocytes). Antibodies
splenocytes), and cytotoxic
280061567). This NILV regimen afforded some protection
against malaria, with 37.5 and 62.5% sterile protection in two
independent trials. The experimental differences between the two
CSP werealso elicited (anti-(QGPGAP)2Abtiter of
Figure 6. Protection is associated with CSP-specific CD8+ + T cells responses. Immune correlates of protection against malaria were studied
by plotting day 28 post-challenge immunity and day 9 post-challenge parasitemia as X and Y variables and using the Spearman test (the rsand p
values are shown) and linear regression (r2is shown) (Figure 6A). Immune responses in challenged mice were compared 28 days post-challenged
between the vaccine candidates and their level of protection (fully (N) or partially (#) protected NILV immunized animals in red) by S9I/Kdtetramer
staining and IFNg elispot assay with splenocytes and liver cells and elisa (Figure 6B). Means and SD are shown. The Kruskal-Wallis test was used to
compare 3 or 4 groups, followed by a Dunn’s post-test.
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Figure 7. The protective efficacy of NILV was confirmed in a second independent study. Groups of BALB/c mice (n=8/group) were
immunized 3 times with NILV by intraperitoneal injections (in red) or not (in black). They were challenged with 500 spz injected IV one month after
the last immunization. The % of parasitized red blood cells was followed every other day from day 5 to day 16 post-challenge by Giemsa-stained
blood smears. Individual parasitemia are shown (Figure 7A) as well as means + SD (Figure 7B) and parasitemia at day 10 post-challenge
(Figure 7C). Among the NILV-immunized mice (red circles), fully (N) versus partially (#) protected animals were further distinguished (Figure 7D).
The Mann-Whitney test was used to compare NILV and naive and the Kruskal-Wallis test followed by a Dunn’s post-test were used to compare fully,
partially and naive. The gross morphology of spleens and livers from NILV-immunized and naive mice at necropsy were compared 3 weeks post-
challenge (Figure 7E).
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studies were (i) different strains of BALB/c mice, (ii) various
batches of vectors, which were primarily characterized for their
p24 content and might have slightly differed in quality and titer
and finally (iii) parasites from the same strain but from different
batches. The difficulty to standardize the infectivity of sporozoites
stocks used for challenge is most probably the main reason for the
low reproducibility in protection. Protection was long-lasting with
42.8% sterile protection afforded six months after the last
What were the differences between fully versus partially
protected mice (no versus delayed and reduced parasitemia)?
Analysis of post-challenge immunity revealed that the CD8+ T cell
responses to CSP (but not anti-CSP Abs) correlate with protection
against malaria. There is no doubt that the challenge itself boosted
vaccine-induced immune responses. Post-challenge immunity is
expected to reflect pre-challenge vaccine-induced responses, but it
is unlikely that cells measured one month post-challenge were
directly involved as effectors in the clearance of the infected
hepatocytes a few days after challenge. Fully protected mice
showed a stronger recalled immunity than partially protected mice
one month after challenge (although it was not significant). This is
consistent with a threshold of protective memory CD8+ T cells to
exceed [20,64,65]. Whether the cytolytic activity of NILV-induced
CD8+ T cells is key to protection and/or whether the secretion of
IFNg and/or TNFa plays a central role remains to be determined
There were no major quantitative differences in the immune
responses to CSP after NILV and RAS immunizations, but RAS
immunizations were more protective than NILV and led, as
expected after 3 injections, to 100% sterile protection. Differential
effector mechanisms could be involved in protection after RAS
and NILV immunizations. The discrepancy between immunity
and protection could also be related to the induction of protective
immunity to non-CSP Ags by RAS only. Although CSP-specific T
cells dominate , the importance of non-CSP Ags in protection
was highlighted in several recent studies. Some protection was
reported in mice transgenic for Py CSP and thus tolerant to it ,
as well as in mice immunized with Plasmodium berghei (Pb) RAS and
challenged with a recombinant chimeric Pb expressing Pf CSP
[69,70,71]. In addition, in humans, both the intensity of responses
and the frequency of responders among protected individuals were
reported to be no higher with CSP than with other tested Ags in
Figure 8. NILV immunizations elicit enduring protective memory responses against malaria. Groups of BALB/c mice were immunized 3
times with NILV by intraperitoneal injections (n=7) (in red) or not (n=9) (in black). They were challenged with 500 spz injected IV six months after
the last immunization (Figure 8A). The % of parasitized red blood cells was followed by Giemsa-stained blood smears. Individual parasitemia are
shown (Figure 8B) as well as means + SD (Figure 8C) and parasitemia at day 9 post-challenge (Figure 8D). Among the NILV-immunized mice (in
red), fully (N) versus partially (#) protected animals were further distinguished (Figure 8E).
Nonintegrative Lentiviral Vectors against Malaria
PLOS ONE | www.plosone.org10November 2012 | Volume 7 | Issue 11 | e48644
two complementary immunomic studies [11,72]. Finally, the
superior protection provided by late-liver stage arresting geneti-
cally attenuated parasites (GAP) compared to early-liver stage
GAP or RAS also underscores the importance of the breadth of
the response .
How did NILV perform in comparison with other CSP-based
vaccine candidates? Admittedly, it has been hard to generate high-
level protective efficacy with vaccines encoding a single pre-
erythrocytic Ag. Heterologous prime/boost strategies are generally
required. Protection levels against malaria are most of the time
assessed with an early challenge with sporozoites (two weeks after
the last immunization). It was reported that 40% of animals
immunized against Py CSP were protected after a single injection
of HuAd5 , 69% after a prime/boost with DNA/NYVAC ,
100% after a prime/boost with HuAd5/VV, 80% after a prime/
boost with YFV17D/MVA. In addition, using TRAP-ME as Ag
(TRAP from Pf fused to a multiepitope (ME) string with multiple B
cell, CD4+, and CD8+ T cell epitopes from Pb CSP), a prime/
boost with a Chimpanzee Ad63/MVA resulted in sterile
protection of 100% of the immunized animals . We conclude
that NILV encoding CSP elicited a good duration of protection
with a vaccine efficacy yet-to improve, by providing 50% (37.5–
62.5%) and 42.8% sterile protection one and six months after the
last immunization, respectively.
Importantly, our lentiviral vectors are derived from HIV.
Multiple restrictions of HIV replication in murine cells have been
described in the literature [76,77,78,79]. They include blocks in
the early steps of HIV replication, suggesting that LV transduction
might be impaired in murine cells as compared to human cells.
Thus mice might not be the best animals to assess LV
immunogenicity and predict human responsiveness to LV
vaccines. The use of similar doses of LV in mice and monkeys
 is an indirect piece of evidence for a reduced LV transduction
efficiency in murine cells.
Our goal was to provide a comprehensive assessment of the
protective efficacy of NILV against malaria. Our data are
promising. They prompt us to design a novel generation of
NILV, improved for their immunogenicity , as well as to
identify new protective Ags to be added to CSP in a multigenic
vaccine . With the upcoming improvements, up-scaling of
lentiviral vector production and their stable conservation at 4uC
after lyophilisation, we believe that this novel vaccine strategy
could impact public health in the malaria domain.
Six-week-old female Balb/c were purchased from Harlan
Laboratories (Gannat, France). Because of a shortage from the
breeder company, two strains of BALB/c mice with the same
origin were used. BALB/cOlaHsd were used for the immunoge-
nicity studies and for the protective efficacy study comparing
NILV and RAS (Figures 2, 3, 4, 5, 6), while BALB/cAnNHsd
were used to confirm the protective efficacy induced by NILV one
month after the last immunization (Figure 7) and assess the
duration of protection (Figure 8).
All animal experiments were conducted in accordance with
guidelines established by the French and European regulations for
the care and use of laboratory animals (De ´crets 87–848, 2001–
464, 2001–486 and 2011–131 and European Directive 2010/63/
UE). The Institut Pasteur is in compliance with Standards for
Human Care and Use of Laboratory Animals and is accredited by
the US National Institut of Health Office of Laboratory Animal
Welfare (OLAW) (Animal Welfare Assurance Number: A5476-
01). Every effort was made to minimize suffering, as described in
the Guide for the ethical evaluation of experiments using
laboratory animals edited by the GIRCOR (Groupe Interprofes-
sionnel de Re ´flexion et de Communication sur la Recherche). This
study was approved by the Regional Committee on Ethics and
Animal Experimentation (CREEA) Ile de France Paris 1 (protocol
#2011-0007) and ASB holds the authorization for animal
Vector Plasmid Construction
The vector plasmid carrying a synthetic Homo sapiens codon
optimized form of Py CSP (Geneart) (pTRIP.ieCMV. Py CSP
co.WPRE), with a Kozak consensus sequence and ATG start
codon at 59 flanking site and TGA stop codon at 39 flanking site,
was generated by replacing the eGFP sequence from pTRI-
P.ieCMV.eGFP.WPRE after Bgl2/XhoI digestion with the Py
CSP co sequence. The comparison between the wild-type and
codon-optimized sequences is shown in Figure S1.
Envelope Expression Plasmids Construction
Mammalian codon-optimized synthetic genes (GeneArt) encod-
ing glycoproteins from the following Vesiculovirus were cloned
into a pVAX1 plasmid (Invitrogen): Vesicular Stomatitis Virus
Indiana serotype (GenBank FW591952), New Jersey serotype
(GenBank FW591956) and Cocal virus (GenBank: AF045556.1).
Lentiviral Vector Particles Production
HIV-1 derived vector particles were produced by transient
calcium phosphate co-transfection of HEK 293 T cells (ATCC)
with the vector plasmid pTRIP, an envelope expression plasmid
(encoding the glycoprotein from VSV, serotype Indiana (IND) or
New Jersey (NJ), or the glycoprotein from Cocal virus) and the
p8.7 or pD64V encapsidation plasmid for the production of ILV
or NILV particles respectively (as shown in Figure 1). The p24
(encoded by HIV-1 Gag from the encapsidation plasmid) content
was quantified by ELISA and expressed as ng p24/mL (physical
characterization). Vector gene transfer capacity was determined
by quantitative PCR after transduction of P4-CCR5 cells (which
are CD4+ CXCR4+ and CCR5+ HeLa cells carrying the LacZ
gene under the control of the HIV-1 long terminal repeat (LTR)
promoter ) in the presence of aphidicolin (Sigma) as previously
described  and was expressed as transduction unit (TU)/mL of
vector (functional characterization).
Immunization and infection were performed with the non-lethal
strain Plasmodium yoelii (Py) 17XNL, which was maintained by
alternate cyclic passages in Anopheles stephensi and Balb/c mice.
Parasitized red blood cells were maintained as frozen stabilate.
Mosquitoes were reared at the Center for Production and
Infection of Anopheles (CEPIA) of the Institut Pasteur using
standard procedures. Sporozoites were prepared by the Ozaki
method . Radiation-attenuated sporozoites (RAS) were
prepared as described previously . They were irradiated at
the dose of 18,000 Rad on ice using a gamma irradiator (IBL637
Synthetic peptides (PolyPeptide Laboratories France) were used
as Ag. S9I (Py CSP:280–288, SYVPSAEQI) and I10L (Py
CSP:58–67, IYNRNIVNRL) contain a CD8+ T cell epitope
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PLOS ONE | www.plosone.org11 November 2012 | Volume 7 | Issue 11 | e48644
and S16I (Py CSP:280–296, SYVPSAEQILEFVKQI) contains
both a CD4+ and the S9I CD8+ T cell epitope [60,61] (Figure
S1). (QGPGAP)2corresponds to the major central repeat of Py
CSP which is targeted by neutralizing antibodies .
NILV and RAS Mice Immunizations and Challenge
BALB/c mice were immunized by intra-peritoneal (IP) or
intramuscular (IM) injection of NILV. Doses were expressed as ng
p24/mouse and/or TU/mouse. BALB/c mice were immunized
by intravenous (IV) injection of 50,000 RAS (in the retro-orbital
vein). RAS were injected immediately after irradiation. Several
groups of mice were immunized in parallel so as to follow the
specific B and T cells responses in blood over time in one group, to
study responses in spleen and liver in another group at necropsy
and to analyze the cytotoxic activity in vivo in a third group.
Animals included in the protective efficacy studies differed from
those included in the immunogenicity studies. They were
immunized and challenged but not used for pre-challenge immune
Challenge experiments consisted in the IV injection of 500 Py
17XNL sporozoites in the retro-orbital vein. Thin blood smears
were stained with Giemsa and screened for the presence of
parasites in red blood cells.
Single Cell Suspensions Preparation
After euthanasia with CO2, the liver was perfused in situ through
the portal vein with PBS to remove circulating blood. Liver was
then dissected out and transferred into HBSS complemented with
5% FCS and gently squished on a 100-mm cell strainer.
Parenchymal cells (pellet) were removed by centrifugation at
50 g for 5 min. After a single wash by centrifugation at 300 g, T
cells were further enriched using a 35% Percoll (Sigma) RPMI
solution and centrifugation at 1360 g for 25 minutes . Red
blood cells from spleen were lysed using IOTest 3 lysing solution
Whole blood for longitudinal follow-up and splenocytes or liver
cells at necropsy were stained with an anti-mouse CD8a mAb
conjugated to APC (clone 53-6.7, BD Biosciences) and the S9I-Kd
tetramer-PE (Class I iTAGTMMHC custom tetramer, Beckman
Coulter, Fullerton, USA).
Nitrocellulose microplates (MAHA S4510, Millipore) were
coated with capture antibody (Mouse IFNg Elispot pair, BD
Pharmingen) and blocked with complete medium. Cells were
cultured at the concentration of 0.2 million/well. They were
incubated with 2 mg/ml of S9I, I10L or S16I peptides. Eigtheen
hours later, spots were revealed with the biotine-conjugated
antibody (Mouse IFNg Elispot pair, BD Pharmingen) followed by
streptavidin-AP (Roche) and BCIP/NBT substrate solution
(Promega). Spots were counted using a Bioreader 2000 (Biosys,
Karben, Germany). Mean number of IFNg spots-forming-cells
(SCF) per million cells was calculated from triplicate wells after
substracting the one from control wells (cultured in medium
Intracellular Cytokines Staining (ICS)
Splenocytes (2 millions/well) were cultured in the presence of
the S9I peptide (2mg/ml final) and anti-CD28 NA/LE MAb (1mg/
ml final, clone 37.51, BD Biosciences) for 1 hour. Then Brefeldin
A from Penicillium brefeldianum (2mg/ml final, Sigma) was added for
5 hours culture and cells were surface-stained for CD8a expression
(anti-CD8a-PerCP, clone 53-6.7, BD Pharmingen) and intracel-
lular-stained for IFNg, (anti-IFNg-FITC, clone XMG1.2, BD
Pharmingen), IL2 (anti-IL2-PE clone JES6-5H4, eBiosciences) and
TNFa (anti-TNFa-APC, clone MP6-XT22, eBiosciences). Flow-
cytometry acquisition and analysis were done with a CyAn2 ADP
analyser (Beckman Coulter, UK) equipped with Summit2 and
with FlowJo respectively.
In vivo Cytotoxic Assay
For target cells preparation, splenocytes from naive mice were
labeled with two concentrations (5 and 1 mM) of CFSE
(carbosyfluorescein-diacetate succinimydel ester, Vybrant CFDA-
SE cell-tracer kit, Molecular Probes). Splenocytes labeled with the
high concentration of CFSE were also pulsed with 5 mg/ml of the
S9I peptide. Each mouse received a mix of 10E+07 CFSE-labeled
cells containing an equal number of S9I pulsed and unpulsed cells
through the retro-orbital vein. After 15 h, single-cell suspensions
from spleen were analyzed by flow cytometry. The disappearance
of S9I-pulsed cells was determined by comparing the ratio of
pulsed to unpulsed populations in immunized versus naive mice.
The percentage of specific killing was established according to the
following calculation: (1-((CFSElownaive/CFSEhighnaive)/(CFSE-
Anti-CSP Antibody Response
NUNC Maxisorps plates were coated with the (QGPGAP)2
peptide diluted in PBS as described previously. After incubation
with serial dilutions of serum from immunized animals, the
presence of specific Abs was revealed using a Peroxidase-
Conjugated AffiniPure Goat anti-mouse IgG (Jackson Immuno
Research Laboratory) and OPD substrate (Sigma). OD was
measured at 492 nm with a Victor (Perkin-Elmer). Specific
antibody titers were defined as the reciprocal serum dilution
giving an optical density equal to 2 times the background obtained
with a pool of serum samples from naive mice (2OD=0.126,
0.164 and 0.162 for Figures 2H, 4E and 6E respectively).
Non-parametric tests were used (Prism, GraphPad). To
compare two groups, the Mann-Whitney test was used. To
compare more than two groups, the Kruskal-Wallis test was used,
followed by a Dunn’s post-test to compare all pairs. The Spearman
test was used to study correlations between immunity and
parasitemia, and linear regression was used to find out whether
immune responses predict the % of parasitized red blood cells.
Only statistically significant p values are indicated.
thetic gene. The CSP synthetic Homo sapiens codon-optimized
DNA sequence (GeneArt) is shown in blue and compared with the
wild-type DNA sequence in red (GenBank: J02695.1), which is T
and A rich. There is 51% similarity between both DNA sequences.
The amino acid sequence is also shown in green (UniProtKB/
Swiss-Prot: P06914.1). The peptides containing a CD8+ T cells
epitope used in the study, Py CSP S9I and I10L, as well as the
major central repeat were underlined.
Sequence of the codon-optimized CSP syn-
compared to ILV is also true for an unrelated Ag, SIV
GAG. C57BL/6 mice (n=3/group) were immunized IP with
The lower immunogenicity of NILV as
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PLOS ONE | www.plosone.org12November 2012 | Volume 7 | Issue 11 | e48644
900 ng p24 of NILV or ILV carrying a wild-type form of the gene
encoding SIVmac239 GAG. T cell responses were evaluated
eleven days later by IFNg elispot after restimulation of splenocytes
with the AL11 peptide, which contains the CD8+T cell
immunodominant epitope. Means + SD are shown.
The authors thank Christophe Pellefigues, Nicolas Puchot (CEPIA) for
sporozoites preparation, the Animalerie Centrale and the Plateforme de
Cytometrie (PFC) (Institut Pasteur), as well as Nathalie Arhel (Ho ˆpital
Saint-Louis, Paris) and Rogerio Amino (Institut Pasteur, Paris) for helpful
Conceived and designed the experiments: FC CB ASB PC. Performed the
experiments: FC RYSD TF AB LC PS CT ASB. Analyzed the data: FC
CB ASB PC. Contributed reagents/materials/analysis tools: CB. Wrote
the paper: FC CB ASB PC.
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Nonintegrative Lentiviral Vectors against Malaria
PLOS ONE | www.plosone.org 14 November 2012 | Volume 7 | Issue 11 | e48644