New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.
ABSTRACT Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.
- SourceAvailable from: Susanne H Hodgson[Show abstract] [Hide abstract]
ABSTRACT: The development of protective vaccines against many difficult infectious pathogens will necessitate the induction of effective antibody responses. Here we assess humoral immune responses against two antigens from the blood-stage merozoite of the Plasmodium falciparum human malaria parasite - MSP1 and AMA1. These antigens were delivered to healthy malaria-naïve adult volunteers in Phase Ia clinical trials using recombinant replication-deficient viral vectors - ChAd63 to prime the immune response and MVA to boost. In subsequent Phase IIa clinical trials, immunized volunteers underwent controlled human malaria infection (CHMI) with P. falciparum to assess vaccine efficacy, whereby all but one volunteer developed low-density blood-stage parasitemia. Here we assess serum antibody responses against both the MSP1 and AMA1 antigens following i) ChAd63-MVA immunization, ii) immunization and CHMI, and iii) primary malaria exposure in the context of CHMI in unimmunized control volunteers. Responses were also assessed in a cohort of naturally-immune Kenyan adults to provide comparison with those induced by a lifetime of natural malaria exposure. Serum antibody responses against MSP1 and AMA1 were characterized in terms of i) total IgG responses before and after CHMI, ii) responses to allelic variants of MSP1 and AMA1, iii) functional growth inhibitory activity (GIA), iv) IgG avidity, and v) isotype responses (IgG1-4, IgA and IgM). These data provide the first in-depth assessment of the quality of adenovirus-MVA vaccine-induced antibody responses in humans, along with assessment of how these responses are modulated by subsequent low-density parasite exposure. Notable differences were observed in qualitative aspects of the human antibody responses against these malaria antigens depending on the means of their induction and/or exposure of the host to the malaria parasite. Given the continued clinical development of viral vectored vaccines for malaria and a range of other diseases targets, these data should help to guide further immuno-monitoring studies of vaccine-induced human antibody responses.PLoS ONE 09/2014; 9(9):e107903. · 3.53 Impact Factor
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ABSTRACT: Substantial effort has been placed in developing efficacious recombinant attenuated adenovirus-based vaccines. However induction of immunity to the vector is a significant obstacle to its repeated use. Here we demonstrate that skin-based delivery of an adenovirus-based malaria vaccine, HAdV5-PyMSP1 42 , to mice using silicon microneedles induces equivalent or enhanced antibody responses to the encoded antigen, however it results in decreased anti-vector responses, compared to intradermal delivery. Microneedle-mediated vaccine priming and resultant induction of low anti-vector antibody titres permitted repeated use of the same adenovirus vaccine vector. This resulted in significantly increased antigen-specific antibody responses in these mice compared to ID-treated mice. Boosting with a heterologous vaccine; MVA-PyMSP1 42 also resulted in significantly greater antibody responses in mice primed with HAdV5-PyMSP1 42 using MN compared to the ID route. The highest protection against blood-stage malaria challenge was observed when a heterologous route of immunization (MN/ID) was used. Therefore, microneedle-mediated immunization has potential to both overcome some of the logistic obstacles surrounding needle-and-syringe-based immunization as well as to facilitate the repeated use of the same adenovirus vaccine thereby potentially reducing manufacturing costs of multiple vaccines. This could have important benefits in the clinical ease of use of adenovirus-based immunization strategies. I mmunization is the most successful strategy to combat infectious diseases. The creation of an effective Plasmodium falciparum malaria vaccine has been a much sought after goal for the vaccine community, however development of an efficacious malaria vaccine has been clinically challenging 1 . Recombinant rep-lication-defective adenoviral vectored vaccines were initially developed as candidate vaccines for induction of T cell responses against HIV-1, liver-stage malaria parasites and other intercellular pathogens 2Scientific Reports 08/2014; 4. · 5.08 Impact Factor
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ABSTRACT: The development of a highly effective and deployable malaria vaccine remains an urgent priority for improving global public health. Despite recent strides in disease prevention and control, the Plasmodium falciparum human malaria parasite continues to exert a huge toll in terms of morbidity and mortality (Murray et al., 2012). The most advanced malaria subunit vaccine, a virus-like particle known as RTS,S, has shown only modest efficacy in young children in Phase III clinical trials (Agnandji et al., 2012), and thus new approaches are urgently needed (Moorthy et al., 2013).Cell Host & Microbe. 01/2015; 367.
INFECTION AND IMMUNITY, Nov. 2010, p. 4601–4612
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 11
New Candidate Vaccines against Blood-Stage Plasmodium falciparum
Malaria: Prime-Boost Immunization Regimens Incorporating
Human and Simian Adenoviral Vectors and Poxviral
Vectors Expressing an Optimized Antigen Based
on Merozoite Surface Protein 1?†
Anna L. Goodman,1* C. Epp,2D. Moss,3A. A. Holder,3J. M. Wilson,4G. P. Gao,4‡ C. A. Long,5
E. J. Remarque,6A. W. Thomas,6V. Ammendola,7S. Colloca,7M. D. J. Dicks,1S. Biswas,1
D. Seibel,2L. M. van Duivenvoorde,6§ S. C. Gilbert,1A. V. S. Hill,1and S. J. Draper1
The Jenner Institute, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, United Kingdom1; Hygiene Institut,
Abteilung Parasitologie, Universita ¨tsklinikum Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany2; Division of
Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom3;
Gene Therapy Program, Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Suite 2000 Translational Research Labs, 125 S. 31st Street, Philadelphia,
Pennsylvania 19104-34034; Laboratory of Malaria and Vector Research, National Institute of
Allergy and Infectious Disease, National Institutes of Health, Rockville, Maryland 208525;
Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, Netherlands6;
and Okairo `s AG, Via dei Castelli Romani 22, 00040 Pomezia, Rome, Italy7
Received 29 March 2010/Returned for modification 1 June 2010/Accepted 5 August 2010
Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage
malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the
inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel
vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic
insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent
both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and
poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in
an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines
induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies
induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These
vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as
well as cross-strain humoral immunity to P. falciparum MSP-1.
Attempts to generate protective blood-stage immunity to
Plasmodium falciparum by vaccination in humans have met
with limited success to date (18). The focus for most vaccine
candidates has been on the induction of antibodies against
merozoite antigens and merozoite surface protein 1 (MSP-1) in
particular (24). Antibodies against the blood stage of P. falcipa-
rum are known to contribute to protective immunity in humans
(40). However, the induction of antibodies to the 42-kDa portion
of MSP-1 (MSP-142) appeared to be insufficient to provide pro-
tective immunity in humans in one study (39). Evidence from
both animal models and humans (detailed below) suggests that
cell-mediated immune responses to MSP-1 could be additionally
required to induce protective immune responses.
During the process of merozoite invasion into erythrocytes,
MSP-1 undergoes two proteolytic processing steps; following
the first step, only MSP-142remains membrane bound, and a
second cleavage of MSP-142into 33-kDa (MSP-133) and 19-
kDa (MSP-119) portions is then required for erythrocyte inva-
sion (4). MSP-119is a major target of protective antibodies,
and MSP-133is a target of both CD8?T cells and CD4?helper
T cells (11, 21, 25). Antibodies to MSP-119are thought to act
though the direct inhibition of merozoite invasion into the red
blood cell and via cytophilic antibody-mediated antibody-de-
pendent cellular inhibition (24, 33). CD4?T cells specific to
MSP-133are able to partially protect nude mice from lethal
Plasmodium chabaudi and Plasmodium yoelii infections (53,
57), while transferred antibodies to MSP-119alone are unable
to protect nude mice against P. yoelii (22). CD4?T cells
against MSP-133play an important role in providing help for
priming MSP-119-specific B cells in vaccine-induced protection
against murine malaria (11), and depletion of CD4?T cells has
been shown to reduce protection against P. yoelii (23).
* Corresponding author. Mailing address: The Jenner Institute, Uni-
versity of Oxford, Old Road Campus Research Building, Oxford OX3
7DQ, United Kingdom. Phone: 44-1865-617616. Fax: 44-1865-617608.
† Supplemental material for this article may be found at http://iai
‡ Present address: Molecular Genetics & Microbiology, University
of Massachusetts Medical School, 381 Plantation Street, Suite 250,
Worcester, MA 01605.
§ Present address: Department of Clinical Immunology and Rheu-
matology, Amsterdam Medical Center, Amsterdam, Netherlands.
?Published ahead of print on 16 August 2010.
Following the discovery that MSP-1 is also expressed late in
the liver stage (49), CD8?T cells directed against MSP-133
have been shown to protect against P. yoelii in the preerythro-
cytic stage (11, 27). In addition, immune responses induced by
immunization with nonlethal blood-stage parasites of P. yoelii
have been shown to protect against sporozoite challenge,
through CD4?and CD8?T cell mechanisms and at least partly
through release of gamma interferon (IFN-?) (2). This discov-
ery that CD8?T cells mediate significant antiparasitic activity
against the liver stage of P. yoelii provides an argument that
similar mechanisms may occur in human P. falciparum malaria.
Further suggestion of the role of cellular immunity in protec-
tion against P. falciparum comes from those studies in humans
in which protective immunity has been associated with signif-
icant cellular immune responses to blood-stage parasites, in
the absence of strong blood-stage antibody responses (42, 47).
In the first study, the secretion of IFN-? appeared to be asso-
ciated with protection against blood-stage P. falciparum ma-
laria (42), and in the second, the presence of polyfunctional T
cells, secreting tumor necrosis factor alpha (TNF-?) and inter-
leukin-2 (IL-2) in combination with IFN-? when stimulated by
blood-stage parasites, was shown to be associated with protec-
tion against P. falciparum (47). We therefore sought to develop
a vaccine targeting MSP-1, which would induce strong cellular
immune responses in conjunction with high antibody titers.
While inhibitory antibodies prevent MSP-119processing and
erythrocyte invasion and appear to be beneficial to the human
host, blocking antibodies act to inhibit the action of these
beneficial antibodies (19). Enzyme-linked immunosorbent as-
says (ELISAs) and immunofluorescence assays (IFAs) are rou-
tinely used to quantify the responses to vaccination but give no
functional information as to levels of invasion-inhibitory anti-
bodies. Growth-inhibitory activity (GIA) assays measure the
growth of P. falciparum in the presence of immune sera in vitro,
and such assays may be of greater clinical relevance when
assessing vaccines targeting regions of MSP-1. The definition
of epitopes for inhibitory and blocking monoclonal antibodies
has enabled the design of vaccines that aim to induce inhibi-
tory, in preference to blocking, antibodies against MSP-119(13,
16). We investigated the inclusion of this approach in the
design of MSP-1 antigens in this study.
Regions of MSP-1, such as MSP-133, show extensive poly-
morphism, and this divides those sequences into two sets of
allelic families that demonstrate extensive diversification (55).
Allelic variation has been shown to reduce antibody produc-
tion by single-allele vaccine constructs to heterologous strains
in humans (37) and to reduce protection against heterologous
strain challenge in nonhuman primates (31). Moreover human
T cell responses against some peptides representing these two
major allelic types of MSP-1 have been found to be mutually
inhibitory in in vitro assays, a phenomenon termed altered
peptide ligand antagonism (28). Within the ?190-kDa protein
sequence of MSP-1, blocks have been defined on the basis of
their extensive, more limited, and minimal genetic diversity
(55). For example in block 8 there is only 10% homology at the
amino acid level, while in block 17, encoding MSP-119, there is
90% homology (55). Blocks 16 (MSP-133) and 17 (MSP-119)
together encode MSP-142.
Virus-vectored vaccines are becoming established as rela-
tively inexpensive, effective, and safe alternative vaccine plat-
forms to conjugate protein-in-adjuvant vaccines (12, 20). We
have previously demonstrated the induction of high-titer pro-
tective antibodies against P. yoelii rodent malaria by using a
recombinant replication-incompetent form of human adenovi-
rus serotype 5 (AdHu5) and modified vaccinia virus Ankara
(MVA) as vaccine vectors in a heterologous prime-boost reg-
imen (13). A heterologous human adenoviral regimen has re-
cently been shown to induce a strong, polyfunctional, and pro-
tective T cell response against simian immunodeficiency virus
(SIV) in rhesus macaques (30), and a homologous AdHu5
regimen expressing MSP-142has been shown to induce GIA in
rabbits (5). Experience in the HIV vaccine field has empha-
sized the importance of avoiding preexisting antivector immu-
nity when developing vectored vaccines (6). The immunoge-
nicity of virus-vectored vaccines is reduced in the presence of
preexisting vector-neutralizing antibodies against AdHu5 (6–
8), but such antibodies do not reduce the immunogenicity of
simian adenoviral vectors in humans (K. Ewer et al. unpub-
lished data). We hypothesized that a heterologous chimpanzee
adenoviral vector regimen may induce strong immune re-
sponses and be more suitable for clinical use. The simian
adenoviruses C6, C7, and C9 have structural similarity and
sequences close to those of human adenovirus 4 in subgroup E.
These vaccine vectors have shown promise in preclinical vac-
cines against infections such as rabies and HIV (45, 59). An
alternative simian adenovirus, AdCh63, which is closely related
to C6, C7, and C9, has recently been found to be safe, immu-
nogenic, and efficacious for human use when used to express
the well-studied preerythrocytic vaccine candidate antigen
ME-TRAP (Ewer et al., submitted for publication). We there-
fore assessed our MSP-1 vaccine constructs in simian adeno-
To develop an MSP-1 vaccine for clinical trials, we investi-
gated the possibility of including in vectors (i) the conserved
blocks of MSP-1, (ii) both allelic forms of MSP-142, and (iii) a
modified MSP-1 sequence to improve antibody fine specificity
and of using a variety of DNA-based virus-vectored vaccines,
including several simian adenoviral serotypes. We show in an-
imal models that both cellular and humoral immune responses
can be generated by this approach. We propose that such
responses may help to overcome some of the limitations of
previous generations of vaccines against MSP-1.
MATERIALS AND METHODS
Antigen inserts. PfM115 and PfM128 were based on the structure of MSP-1.
A schematic representation of the structures of the composite antigen inserts
“PfM115” and “PfM128” is shown in Fig. 1. PfM128 is identical to PfM115 with
the exception of the addition of the FVO block 17 (MSP-119). Full sequence
details can be found in the supplemental material. The rationale for this novel
antigen structure was an effort to maximize inclusion of conserved antigen
sequences, include both alleles of MSP-142, and minimize polymorphic regions,
glycosylation targets, and epitopes in block 17 that are thought to induce block-
Vaccines. The final antigen inserts were codon optimized for mammalian
expression and synthesized by GeneArt GmbH (Regensburg, Germany). Viral
vaccine vectors included modified vaccinia virus Ankara (MVA), human adeno-
virus serotype 5 (AdHu5), and the simian adenoviruses (SAd) serotypes 6 (C6),
7 (C7), 9 (C9), and 63 (AdCh63). PfM115 was cloned into SAd viral vectors C6,
C7, and C9 using methods described previously (48). Insertion of PfM115 and
PfM128 into AdHu5 and MVA and of PfM128 into AdCh63 was performed
using methods previously described for other antigen inserts (13, 44). As the
majority of experiments were performed using PfM115, all vaccines express
PfM115 unless stated to express PfM128.
4602 GOODMAN ET AL.INFECT. IMMUN.
Animals and immunizations. Groups of ?6 female BALB/c and C57BL/6
mice (BMSU, Oxford University, United Kingdom) were 6 to 8 weeks of age at
the start of the experiments. All procedures were performed in accordance with
the terms of the United Kingdom Animals (Scientific Procedures) Act Project
License and were approved by the University of Oxford Animal Care and Ethical
Review Committee. Mice were immunized intradermally (i.d.) into both ears.
Doses of vaccine used were 5 ? 1010viral particles (VP) of adenoviral vaccines
and 5 ? 107PFU of MVA PfM115 unless otherwise stated. Doses of PfM128
vaccines were 1010VP of adenoviral vaccines and 107PFU of MVA PfM128
unless otherwise stated. Groups of ?3 New Zealand White rabbits were immu-
nized, and serum collection was performed by Agrobio (France). Doses of
vaccine used were 5 ? 1010VP of adenoviral vaccines and 1 ? 108PFU (MVA
PfM115) or 5 ? 107PFU (MVA PfM128) of orthopoxviral vaccines. All vaccines
were administered i.d. unless otherwise stated. Viral vector vaccines were pre-
pared in sterile, endotoxin-free phosphate-buffered saline (PBS). For compari-
son, two groups of six mice were immunized with a modified MSP-119protein
(FVO allele) in PBS, complete Freund’s adjuvant (CFA), or incomplete
Freund’s adjuvant. This was conjugated at the C terminus to the core domain of
murine C4 binding protein (C4BP) (IMX108) (38) and was a kind gift from F.
Hill (Imaxio, France). Three protein immunizations were administered subcu-
taneously (s.c.) at 2-week intervals. Three doses were administered. The first
dose was formulated in PBS or CFA. Subsequent doses were formulated in PBS
or incomplete Freund’s adjuvant. All mouse data shown are representative of
two or three experiments. Rabbit experiments were performed once only and
data shown are from a single data set, with assays repeated to ensure technical
In vitro GIA assay. A standardized in vitro growth inhibition activity (GIA)
assay was performed at the GIA Reference Center (LMVR, NIH) as previously
described (32). Rabbit IgGs from individual animals vaccinated with all PfM115
vaccines were tested against 3D7 and FVO parasites at 2.5 mg/ml and against
3D7 parasites at 10 mg/ml. Rabbit IgGs from animals vaccinated with AdCh63
PfM128, AdHu5 PfM128, and MVA PfM128 vaccines were tested against 3D7
parasites at 10 mg/ml.
ELISA. Recombinant glutathione S-transferase (GST) fusions of P. falciparum
ETSR MSP-119and QKNG MSP-119were prepared as described previously (13).
Protein was applied at an optimized concentration (2 ?g/ml ETSR and 5 ?g/ml
QKNG) in PBS. Sera were diluted to 1:100 (preboost) or 1:1,000 (postboost),
added in duplicate wells, and serially diluted. For monoclonal antibody (MAb)
ELISAs, MAbs at a concentration of 5 ?g/ml were applied in place of sera. The
endpoint titers were taken as the x axis intercept of the dilution curve at an
absorbance value three standard deviations greater than the optical density at
405 nm (OD405) for naïve mouse or rabbit sera. Recombinant full-length His6-
tagged MSP-1 (a heterodimeric complex reconstituted from MSP-183/30and
MSP-138/42) and the 83-, 38-, and 42-kDa subunits of MSP-1 were prepared and
purified as described elsewhere (15, 26). MSP-142ELISAs for correlation with
GIA were performed on purified IgG using methods described elsewhere (35).
Recombinant full-length MSP-1 was kindly provided by H. Bujard (ZMBH,
Germany). All ELISAs were performed using previously published methods (13,
Immunofluorescence assay (IFA). Slides were prepared with a thin smear of P.
falciparum schizonts from culture and fixed with 4% formaldehyde and 1%
NP-40 for 15 min at room temperature. Rabbit sera were diluted 1:1,000 in PBS
and incubated on slides for 45 min. Slides were washed with PBS and incubated
with Alexa 488 goat anti-rabbit IgG for 30 min. Slides were then washed in PBS,
DAPI (4?,6?-diamidino-2-phenylindole) was applied, and the slides were viewed
under a fluorescence microscope.
Intracellular cytokine staining. Flow cytometry analysis of T cell phenotype
was performed according to a protocol detailed in the supplemental material and
based on methods published elsewhere (11). Data were analyzed using a Cyan
ADP flow cytometer, FloJo (version 9), and SPICE (Mario Roederer). Back-
ground responses in unstimulated cells were subtracted from the stimulated
responses prior to analysis.
Immunostaining. Chicken embryo fibroblasts were infected with MVA
PfM115 or MVA PfM128 and incubated for 3 days. Infected cells were then fixed
and permeabilized. Cells were incubated with inhibitory (12.8, 12.10, and 1E1),
neutral (2F10), or blocking (111.4, 2.2) monoclonal antibody at 1:1,000 (5 ?g/ml)
for 1 h. Goat anti-mouse–horseradish peroxidase (HRP) (Amersham Bio-
sciences) was applied and detected using chromogenic diaminobenzidine (DAB).
Plaques were visible by eye and were imaged using a gel camera. Binding of
antibody to PfM115 was determined by the presence (?) or absence (?) of
visible stained MVA plaques.
Statistics. Statistical significance was analyzed using Prism version 5 (Graph-
Pad Software Inc., CA) and STATA. Details of the statistical tests used for each
analysis are listed in the supplemental material.
Antigen design: PfM115 and PfM128. A first-generation
MSP-1 vaccine insert, termed PfM115, was designed to include
the four relatively conserved blocks of MSP-1 (blocks 1, 3, 5,
and 12) from the 3D7 strain of P. falciparum. These were
encoded from the N to the C terminus, followed by block 16
from the Wellcome strain (FVO) and then blocks 16 and 17
from the 3D7 strain (Fig. 1). A flexible linker was encoded
between the two forms of block 16. The FVO and 3D7 strains
represent the alternative dimorphic families for block 16,
which encodes MSP-133. Immunization with MSP-133has been
shown to induce CD8?and CD4?T cells and provide essential
T cell help for antibody responses to MSP-119, the target of
protective antibodies in mice (11). Three amino acid substitu-
tions were made in the latter fragment, as described elsewhere,
to enhance inhibitory antibody induction and to attempt to
reduce induction of blocking antibodies (16). A second-gener-
ation construct, termed PfM128, has previously been reported
(13). PfM128 differs from PfM115 in the inclusion of the 19-
kDa fragment (block 17) from the FVO strain of P. falciparum
immediately C terminal to the FVO strain 33-kDa fragment in
the insert (Fig. 1).
Cellular immune responses to vaccination. T cell responses
have been associated with the control of blood-stage malaria in
mice and humans (17, 42). IFN-? secretion and T cell-medi-
ated responses to blood-stage antigens are thought to be of
importance in mediating protection against P. falciparum and
P. berghei (42, 47, 60). We hypothesized that polyfunctional
CD8?and CD4?T cell responses would be induced by immu-
nization with heterologous prime-boost virus-vectored vac-
cines and that the use of simian adenoviral vectors in place of
human adenoviral vectors would not compromise vaccine im-
munogenicity. We sought to characterize the T cell responses
using polychromatic flow cytometry.
In order to establish whether simian adenovirus-vectored
vaccines would induce T cell responses to MSP-1, mice were
FIG. 1. A schematic representation of the design of the composite
MSP-1 antigen inserts. The design of PfM115 and PfM128 was based
on the structure of merozoite surface protein 1 and included the four
more-conserved blocks (blocks 1, 3, 5, and 12) and FVO and 3D7
allelic variants of blocks 16 and 17 as shown.
VOL. 78, 2010 SIMIAN ADENOVIRUS-VECTORED VACCINES AGAINST PfMSP-14603
immunized with a priming dose of adenovirus-vectored vaccine
that expressed PfM115. A boost immunization of MVA (M),
AdHu5, C6, or C7 expressing the PfM115 insert was admin-
istered 8 weeks later. Vector administration is denoted by
prime_boost (e.g., AdHu5_M means AdHu5 PfM115 prime
and MVA PfM115 boost). Where PfM128 was expressed as
the antigen, this is additionally denoted (e.g., AdHu5_M
PfM128). C9 has been demonstrated to cross-react with C7
neutralizing antibodies and was therefore not used as a boost-
ing agent (48). Overlapping peptide pools were used to map T
cell epitopes from PfM115 in mice (see Fig. S2 in the supple-
mental material). We identified a single immunodominant
CD8?T cell epitope in the FVO MSP-133region of PfM115 in
BALB/c mice and several weaker epitopes in C57BL/6 mice
(see Table S1 in the supplemental material). The BALB/c
epitope was consistent with H-2dclass I epitope prediction
(http://www.syfpeithi.de) and with previous literature demon-
strating T cell epitopes in the 33-kDa region of MSP-1 in other
murine models (52). No CD4?T cell epitope was identified in
BALB/c mice. A single CD4?T cell epitope was found in
C57BL/6 mice (see Table S1 in the supplemental material).
All prime-boost regimens expressing PfM115 were found to
induce T cell responses in mice (Fig. 2). CD4?T cell responses
were measured in C57BL/6 mice, and no significant differences
in CD4?IFN-??, TNF-??, or IL-2?T cell responses were
found between groups receiving PfM115 in different vaccine
vectors (Fig. 2A). Heterologous adenoviral regimens showed a
trend toward increased CD8?TNF-??and IFN-??T cell
responses, while a C7 simian adenoviral prime induced stron-
ger CD8?IL-2?T cell responses in C57BL/6 mice (P ? 0.05)
(Fig. 2B). However, though these differences were replicated
on repetition of the experiment in C57BL/6 mice, when the
vaccines were administered to BALB/c mice, this effect was not
seen (Fig. 2C). Overall, the replacement of a human adenovi-
ral vector with a simian adenoviral vector did not appear to
alter cellular immune responses. A proportion of T cells were
polyfunctional and capable of coexpressing the cytokines
TNF-?, IFN-?, and IL-2 (Fig. 2D). The majority of these T
cells were capable of producing both IFN-? and TNF-? (rep-
resented as blue in pie charts), with a smaller number of poly-
functional triple-positive, IL-2-producing cells (shown in red).
Mice were immunized with vaccines expressing the sec-
ond-generation antigen PfM128. AdHu5 and AdCh63 viral
vectors expressing PfM128 were administered to mice and
boosted 8 weeks later by immunization with MVA (M) ex-
pressing PfM128. CD8?and CD4?IFN-??, TNF-??, and
IL-2?responses were not compromised by the replacement of
AdHu5 with AdCh63 PfM128 (Fig. 3). CD8?cytokine re-
sponses tended to increase with AdCh63_M PfM128 compared
with AdHu5_M PfM128; these differences were significant
only in the case of IL-2 (P ? 0.05, Mann-Whitney test) (Fig.
3B). Polyfunctional analysis demonstrated a small but signifi-
cant increase in both polyfunctional CD4?and CD8?T cells
with the AdCh63 prime (P ? 0.01, Mann-Whitney test) (Fig.
3C and D). However, once again neither the qualitative nor the
quantitative improvement in CD8?T cell responses with
AdCh63 was replicated in BALB/c mice (data not shown),
suggesting that overall responses are not altered by replace-
ment of AdHu5 with AdCh63.
Humoral responses against MSP-119following a single im-
munization. In order to establish the level of the antibody
response to MSP-1 induced by simian adenovirus-vectored vac-
FIG. 2. T cell responses following immunization with viral vectors expressing PfM115. MSP-1-specific cytokine production from splenocytes of
mice previously immunized with AdHu5 or C7 expressing PfM115, followed by a boost immunization at 8 weeks with C6, C7, or MVA
(M) expressing PfM115 (prime_boost), was assessed at 2 weeks after the final immunization. Multiparameter flow cytometry was used to determine
the total frequencies of IFN-?-, TNF-?-, and IL-2-producing T cells. CD4?(A) and CD8?(B to D) T cell responses to vaccination are shown.
The murine strains used are shown in the figure. In panel D The fraction of the total response comprising cells expressing one (?), two (??)
or, three (???) cytokines in the experiment for panel B is shown. Adenoviral vectors and poxviral vectors were given at doses of 5 ? 1010
VP or 5 ? 107PFU, respectively. Data are means ? standard errors of the means (SEM) (n ? 6).
4604 GOODMAN ET AL.INFECT. IMMUN.
cines, BALB/c mice were immunized with a variety of vaccine
regimens. The BALB/c strain of mouse was chosen because
antibody responses were higher than those seen in C57BL/6
mice, despite the absence of a detectable CD4?T cell epitope
in this strain. This appears to be a feature of the antibody
responses of BALB/c mice in general and was not particular to
this antigen (13). It appears that CD4?T cell responses were
induced in BALB/c mice, as antibody class switching occurred
as evidenced by the presence of IgG, but these responses were
below the detection limit of our cellular assays. The immuno-
genicity of AdHu5 was compared with those of the simian
adenoviral vectors, C6, C7 C9, and AdCh63. MSP-119is known
to be the target of IgG-mediated immunity in mice and hu-
mans (1, 11), and ELISAs were therefore performed against
this region of MSP-1 unless otherwise stated. Antibody titers
were monitored following a single immunization and were
found to increase over time, as seen previously with P. yoelii
MSP-142vaccines (13). The replacement of a single dose of
AdHu5 with the simian vector C6, C7, or C9 did not lead to a
significant difference in the total IgG antibody titers to MSP-
119that were achieved following immunization, though there
was a trend to a weaker antibody response when immunization
was with C9 (Fig. 4A). Similarly, when mice were immunized
with AdHu5 PfM128 or AdCh63 PfM128, no difference was
seen between groups (Fig. 4B). This result differs from previ-
ous work with the P. berghei circumsporozoite malaria antigen
in which significantly weaker antibody responses were found
when C6 was used as a priming vector (46). The trend toward
a weaker response with C9 (also known as C68) has been
documented previously with rabies vaccines administered
subcutaneously (59). These data demonstrate that total IgG
immune responses to P. falciparum MSP-119are mostly pre-
served when AdHu5 is replaced with simian adenoviral vac-
Humoral responses against MSP-119following heterologous
prime-boost immunization. We have previously shown that a
heterologous poxviral boost can improve antibody responses to
P. yoelii MSP-142(13). We also reported that adenoviral vec-
tors prime stronger antibody responses than the orthopoxviral
vector MVA (13). We hypothesized that a heterologous ad-
enoviral boost might induce a stronger antibody response than
a poxviral boost. A boost immunization was administered, and
antibody responses to MSP-119for all groups combined were
compared to preboost titers. We found a significant increase in
the antibody titers following a heterologous boost immuniza-
tion (Fig. 4C); however, the replacement of MVA with a het-
erologous adenoviral vector did not increase total IgG re-
sponses. In order to confirm that the absence of differences
between the vaccine regimens was not due to the relatively
high vaccine doses, this experiment was repeated using a
lower-dose regimen in order to induce suboptimal titers. There
were still no significant differences between antibody responses
to different viral vector regimens (see Fig. S3 in the supple-
mental material). At the peak of the response, which was at
week 10, all mouse IgG isotypes were detected with all vaccine
regimens (Fig. 4D). Heterologous simian adenoviral vectors
and MVA are thus equally effectively at boosting antibody
responses to MSP-119in a prime-boost vaccine regimen. As
proof of principal, one regimen (AdHu5_M PfM115) was com-
pared to three doses of MSP-119protein conjugated to murine
C4BP core domain (IMX108) (13, 38). AdHu5_PfM115 was
found to be as immunogenic as protein in Freund’s adjuvant
and significantly more immunogenic than protein in PBS alone
(see Fig. S1 in the supplemental material).
FIG. 3. T cell responses following immunization with viral vectors expressing PfM128. MSP-1-specific cytokine production from splenocytes of
C57BL/6 mice previously immunized with AdHu5 or AdCh63 expressing PfM128, followed by a boost immunization at 8 weeks with MVA
(M) expressing PfM128 (prime_boost), was assessed at 2 weeks after the final immunization. Multiparameter flow cytometry was used to determine
the total frequencies of IFN-?-, TNF-?-, and IL-2-producing T cells. CD4?(A and C) and CD8?(B and D) T cell responses to vaccination are
shown. Panels C and D show the fraction of the total response comprising cells expressing one (?), two (??), or three (???) cytokines.
Adenoviral vectors and poxviral vectors were given at doses of 1010VP or 107PFU, respectively. Data are means ? SEM (n ? 6).
VOL. 78, 2010SIMIAN ADENOVIRUS-VECTORED VACCINES AGAINST PfMSP-1 4605
Humoral responses against MSP-1 in rabbits. In order to
obtain sufficient sera for assays of parasite growth inhibition,
the prime-boost immunization regimens described above were
administered to New Zealand White rabbits. Strong antibody
responses to MSP-119were found in all groups following a
heterologous adenoviral or orthopoxviral boost (see Fig. S4A
in the supplemental material). Antibody responses were in-
creased significantly by the boost immunization. No significant
differences were found between immunization regimens at any
individual time point or overall. Antibodies were analyzed at
week 10 and were detected to all regions of MSP-1 contained
in the vaccine insert, with no significant differences between
regimens (see Fig. S4C in the supplemental material). MSP-183
incorporates blocks 1, 3, and 5 while MSP-138incorporates
block 12 and MSP-142incorporates blocks 16 and 17. The
proteins covering these three regions were based on the 3D7
allelic form. Antibody responses to MSP-130were not assessed,
as this block was not included in the vaccine. Responses to the
entire full-length MSP-1 molecule (see Fig. S4B in the supple-
mental material) did also not differ significantly between the
Cross-strain humoral immunogenicity against MSP-119.
Four amino acids have been found to differ between the dif-
ferent allelic variants of MSP-119and are thought to be im-
portant in antigen processing (34). These changes are E 3 Q
at amino acid (aa) 1644 and TSR 3 KNG at aa 1691, 1700, and
1701. The allelic variants are therefore conventionally referred
to as “ETSR” (3D7/Mad20 strain) or “QKNG” (FVO/Well-
come/K1/FCR3 strain). We first investigated whether antibod-
ies produced in response to PfM115 immunization were spe-
cific to the allelic variant of the vaccine (3D7 [ETSR]) or
whether they also had activity against the heterologous allelic
variant (FVO [QKNG]). ELISAs were performed on sera col-
lected at week 10 from mice immunized with all prime-boost
PfM115 combinations in order to determine the total IgG
titers against MSP-119ETSR and MSP-119QKNG. Antibody
responses to the alternative allelic variant of MSP-119(QKNG)
correlated with the homologous allelic antibody response
(ETSR) in mice, and a stronger correlation was seen in rabbits
(Fig. 5A and B). Such differences in cross-reactive antibody
responses, dependent on the species immunized, have been
reported previously for protein vaccines based on one or two
alleles on the apical membrane antigen 1 (AMA-1) vaccine
candidate (36). We hypothesized that the QKNG responses
could be further improved with PfM128, which includes both
allelic variants of MSP-119. Following immunization with
AdHu5 PfM128 and MVA PfM128, total IgG responses to
QKNG MSP-119in mice were significantly increased, while
responses to ETSR MSP-119were maintained (Fig. 6). Mice
immunized with this regimen showed a stronger correlation
between ETSR and QKNG MSP-119ELISA titers than those
immunized with PfM115 (Fig. 5A and C). A strong correlation
in rabbits was maintained, similar to that seen with PfM115
(Fig. 5B and D).
Humoral responses to P. falciparum. Immunofluorescence
assays (IFAs) were performed in order to determine if these
FIG. 4. Vaccine-induced antibody responses to MSP-119. BALB/c mice were immunized with adenoviral vectors (AdHu5, C6, C7, and C9), and
total IgG titers to GST-MSP-119(ETSR) were measured by ELISA (unless stated otherwise). (A) Comparison of antibody responses at week 8
following a single immunization with 5 ? 1010adenoviral particles (VP) expressing PfM115. (B) Comparison of antibody responses at week 2
following a single immunization with 1010VP expressing PfM128. (C) Total IgG responses (geometric mean titer [GMT]) over time following
immunization as for panel A, followed at week 8 by a boost with 5 ? 107PFU MVA PfM115 (M) or 5 ? 1010VP AdHu5 or C6 PfM115 as shown
(prime_boost). (D) Isotype ELISAs were performed at week 10 on samples from panel C. Total IgG titers to GST–MSP-119ETSR (A to D) and
GST-MSP-119QKNG (C) were measured. Geometric mean titers (? 95% confidence intervals [CIs]) are shown.***, Different from week 8 GMT
(P ? 0.001).
4606 GOODMAN ET AL.INFECT. IMMUN.
antibodies were able to bind MSP-1 in its native form in P.
falciparum. Sera were taken preimmunization and at 2 weeks
after the boost immunization from all those rabbits immunized
with vaccines expressing PfM115, and IgG was purified. Sera
from immunized rabbits from all groups bound P. falciparum at
a dilution factor of 1:1,000 (Fig. 7A), while preimmunization
rabbit IgG did not. Sera were also taken at 2 weeks after the
boost immunization from all those rabbits immunized with
AdHu5_M PfM128, and a further IFA was performed. Sera
were tested directly on slides from 3D7 and FCR3 parasites,
and the IFA titer was found to be 1:6,400 against both parasite
strains (median; see Fig. S5 in the supplemental material).
The activity of antibodies against P. falciparum in vitro was
established using a standard growth inhibition activity (GIA)
assay (32). AdHu5_M PfM128 immunization of BALB/c mice
has previously been shown to induce GIAs of 70% against 3D7
strain and 85% against FVO strain P. falciparum (13). BALB/c
mice were immunized with AdHu5_M PfM115 as previously,
and the GIAs were 46% against 3D7 parasites and 52% against
heterologous FVO parasites. In rabbits, PfM115 vaccine regi-
mens demonstrated efficacy against the in vitro growth of P.
falciparum (Fig. 7C and D). Antibody titers to recombinant
3D7 MSP-142were found to have a sigmoidal relationship
with the 3D7 GIA of purified IgG from rabbits (Fig. 7B).
AdHu5_M PfM115 was administered intramuscularly (i.m.)
(group A§) and i.d. (group B), and there was no statistical
difference by the Mann-Whitney U test, but there was less
variation in GIA at 10 mg/ml with i.m. administration (coeffi-
cient of variation ? 6%) than with intradermal administration
(coefficient of variation ? 50%) (Fig. 7C). Vectors were there-
fore administered i.m. to subsequent groups of rabbits (group
C§, AdHu5_M PfM128; group D§, AdCh63_M PfM128). At
10 mg/ml no significant differences in GIA were found between
different viral vector combinations, or between those express-
ing PfM115 or PfM128 by the Mann-Whitney U test or one-
way analysis of variance (ANOVA) (Fig. 7C). GIA was also
FIG. 5. Antibody responses to MSP-1 allelic variants. BALB/c mice (A and C) and New Zealand White rabbits (B and D) were immunized with
vaccines incorporating the 3D7 allelic variant in PfM115 (A and B) or both allelic variants in PfM128 (C and D) of MSP-119. Mice were immunized
as described for Fig. 4 and 6. Rabbits were immunized as described for Fig. 7. Animals received a range of prime_boost immunization regimens
expressing PfM115 or AdHu5 PfM128 and a boost immunization of MVA PfM128 (C and D). Sera were taken at week 10. Total IgG ELISAs were
performed for GST–MSP-119ETSR (3D7 allelic variant) and GST–MSP-119QKNG (FVO allelic variant). Antibody titers from individual animals
and linear regression lines are shown, along with Pearson rank correlations (r2) and P values.
FIG. 6. Vaccine-induced antibody responses to MSP-119following
AdHu5_M PfM115 or AdHu5_M PfM128. BALB/c mice were immu-
nized with 1010adenoviral particles of AdHu5 expressing either
PfM115 or PfM128 as indicated and were boosted at week 8 by im-
munization with 107PFU of MVA expressing the same antigen. Sera
were taken at 2 weeks following the final immunization, and total IgG
titers to GST–MSP-119(ETSR or QKNG, as shown) were measured by
ELISA.*, different from PfM115 QKNG (P ? 0.05).
VOL. 78, 2010 SIMIAN ADENOVIRUS-VECTORED VACCINES AGAINST PfMSP-14607
tested at 2.5 mg/ml using sera from rabbits vaccinated with
PfM115 regimens (group A, AdHu5_M PfM115 i.m.; group B,
AdHu5_M PfM115 i.d.; group E, C7_M PfM115 i.d.; group F,
C7_C6 PfM115 i.d.; group G, C7_AdHu5 PfM115 i.d.). There
was no significant difference in GIA by the Mann-Whitney U
test when homologous or heterologous parasite strains were
used in this assay, and there was no significant difference be-
tween groups receiving different regimens when assessed by
one-way ANOVA (Fig. 7D [circles represent 3D7 parasites
and squares FVO]). Although GIA has been reported to be
induced by antibodies targeting multiple regions of MSP-1 (58)
and despite the induction of antibodies against MSP-183and
MSP-138(see Fig. S4C in the supplemental material), the func-
tional antibody responses appeared to be directed to the MSP-
119region of the vaccine, as demonstrated by the complete
reversal of GIA when recombinant MSP-119protein was ap-
plied to wells containing a mixture of parasites and immunized
rabbit sera (see Fig. S6 in the supplemental material).
Aiming to minimize the induction of blocking antibodies. It
has previously been suggested that a successful vaccine might
be one designed in such a way as to optimize inhibitory anti-
bodies while at the same time minimizing blocking antibodies
by using specific amino acid substitutions (19). These have
been previously incorporated into a protein-in-adjuvant vac-
cine and been found to improve immune responses and GIA
(16). In PfM115 and PfM128, two cysteine residues in the
sequence of MSP-119were replaced (in both alleles in PfM128)
in order to disrupt a disulfide bond and attempt to minimize
the induction of blocking antibodies and enhance antigen pro-
cessing. These were the C12I and C28W changes (16). A fur-
ther amino acid change, S3A, was also included in order to
remove a potential N glycosylation site. N glycosylation of P.
falciparum blood-stage proteins is rare, and the removal of
glycosylation sites has been shown to improve antibody titers,
antibody avidity, and protective efficacy of a recombinant
MSP-142vaccine in nonhuman primates (54). We applied a
panel of murine monoclonal antibodies specific for epitopes
within the MSP-119domain to the poxviral vaccine viruses
(MVA PfM115 and MVA PfM128) in a plaque immunoassay.
We found that the MSP-1 protein expressed by MVA PfM115
and MVA PfM128 bound the inhibitory antibodies 12.10 and
12.8, the neutral antibody 2F10, and the blocking antibodies
7.5 and 1E1. The PfM115 antigen expressed by MVA failed to
bind the blocking antibody 111.4. Proteins expressed by both
MVA PfM115 and MVA PfM128 failed to bind the blocking
antibody 2.2 (see Table S2 in the supplemental material). The
binding to 111.4 by MSP-1 antigen expressed by MVA PfM128
but not MVA PfM115 was as expected because this antibody
binds to an epitope present only in the QKNG allele (but not
ETSR) of MSP-119which is not present in PfM115 but is
present in PfM128 (9). Overall, the abolition of binding to
MAb 2.2 showed that this combination of amino acid sub-
stitutions can in part remove binding to some, but not all,
FIG. 7. Functional antibodies against whole P. falciparum. Rabbits (n ? 3/group) were immunized with AdHu5 or C7 expressing PfM115 and
boosted 8 weeks later with MVA PfM115 (M), AdHu5, or C6. Alternatively, rabbits were immunized with AdHu5 or AdCh63 expressing PfM128
and boosted with MVA expressing PfM128. Doses used were 5 ? 1010VP (adenoviruses) or 108PFU (MVA). Both vaccines were administered
i.d. or i.m. (§). Groups were as follows: A§, AdHu5_M PfM115 i.m.; B, AdHu5_M PfM115 i.d.; C§, AdHu5_M PfM128; D§, AdCh63_M PfM128;
E, C7_M PfM115 i.d.; F, C7_C6 PfM115 i.d.; G, C7_AdHu5 PfM115 i.d. Purified polyclonal IgG for GIA was obtained from sera collected at 2
weeks after the final immunization. (A) IFAs were performed with sera from immunized rabbits. Areas in green are stained with anti-rabbit IgG
secondary antibody, while areas in blue are stained with DAPI. No significant green staining was seen with naïve sera. A representative slide from
a rabbit from group A is shown (1:1,000). (B) GIA responses were correlated with total IgG ELISA titer to 3D7 MSP-142. (C) GIA against 3D7
strains in vitro at 10 mg/ml IgG was determined. (D) GIA against 3D7 (circles) and FVO (squares) at 2.5 mg/ml IgG was determined. Individual
and median values are shown.
4608GOODMAN ET AL.INFECT. IMMUN.
known blocking antibody epitopes while maintaining inhib-
We have shown here the induction of T cell- and anti-
body-mediated immunogenicity against P. falciparum MSP-1
with viral vector vaccines. In the field of vaccine research it
is challenging and probably unreliable to compare directly
data generated in different laboratories, but in a direct com-
parison in our laboratory we found our initial regimen
(AdHu5_M PfM115) to be equivalent to a protein vaccine
administered in Freund’s adjuvant—a formulation which, al-
though highly immunogenic, is unsuitable for human use. The
field of virus-vectored vaccines is now providing a platform for
inducing strong cellular and humoral immune responses with-
out the need for potentially reactogenic chemical adjuvants. To
generate vectored vaccine candidates suitable for clinical as-
sessment in the challenging field of blood-stage malaria vac-
cines, we have assessed the suitability of four new vaccine
design features for MSP-1. These are a detailed assessment of
simian adenoviral vectors as alternatives to the widely used
AdHu5 serotype; the use of the four N-terminal conserved
regions to try to generate T cell responses to more conserved
rather than very variable regions of MSP-1; the inclusion of
two allelic variants of the C-terminal MSP-142arrayed in tan-
dem in a vectored insert; and the introduction of recently
described point mutations in the 19-kDa fragment(s), a major
target of protective antibodies, to enhance overall immunoge-
nicity and reduce the likelihood of developing unwanted block-
We have demonstrated here that replacement of AdHu5
with a simian adenoviral vector compromises neither antibody
nor T cell responses in animal models. We have also shown
that MSP-1-specific CD4?T cells can be induced in C57BL/6
mice by all vaccination regimens tested. The proportion of
CD4?T cells that are polyfunctional and express the cytokines
IFN-?, TNF-?, and IL-2 has been found to be a marker of the
protective efficacy of the CD4?T cell response in murine
leishmaniasis and human malaria (10, 47), and vaccine-induced
polyfunctional T cells appear to be more durable than less-
polyfunctional T cells in murine models of preerythrocytic ma-
laria (44). We found little difference in the magnitude of the
response, defined by these cytokines, for CD4?T cells induced
by diverse vector-based heterologous prime-boost immuniza-
tion regimens, but in some cases (e.g., AdCh63_M PfM128) a
more polyfunctional profile of CD4?T cells was observed with
simian than with human adenoviral vectors.
Studies in the P. yoelii model have demonstrated the impor-
tance of immunity to MSP-1, not only at the blood stage but
also in preerythrocytic stages of parasite development (27). In
agreement with other murine data (2, 27), we recently dem-
onstrated the importance of CD8?T cell responses against P.
yoelii MSP-133and their ability to partially control parasite
growth at the liver stage of infection, indicating that the induc-
tion of such T cell responses by clinically relevant vaccine
vectors may be of protective importance (11). The importance
of polyfunctional CD8?T cells has been suggested in the field
of HIV research, where the heterologous adenovirus-vectored
vaccine combination of recombinant AdHu26 and AdHu5 was
found to induce stronger, more polyfunctional CD8?T cell
responses than a homologous AdHu5 regimen. These re-
sponses were associated with increased protection against an
SIV challenge in rhesus macaques (30). In addition, recent
work has shown evidence of GIA and T cell responses using a
homologous AdHu5 vaccine expressing MSP-142(5). However,
the continued clinical development of AdHu5 may be prob-
lematic, and a heterologous simian adenoviral (SAd_SAd) or
simian adenoviral and MVA (SAd_M) regimen could there-
fore have an advantage. T cell responses have rarely been
reported in preclinical studies of MSP-1 protein-in-adjuvant
vaccines but have been shown following AdHu5 vector immu-
nization (5). Here we show that induction of polyfunctional
CD8?T cell responses to P. falciparum MSP-1 is not compro-
mised by replacement of AdHu5 with a simian adenoviral
vector, and some responses even appeared to be enhanced in
C57BL/6 mice. Antibody titers and inhibition of parasite
growth were also maintained with simian adenovirus-vectored
vaccines. The median magnitude of GIA against 3D7 parasites
using 10 mg/ml IgG was 74% (interquartile range [IQR], 64.5
to 81.5%) across all four groups tested. Other studies have
previously shown induction of comparable levels of GIA using
sera from rabbits immunized with MSP-142protein in Freund’s
adjuvant (complete and incomplete) (3) and following viral
vector immunization (5).
The concentration of T cell epitopes in the MSP-133region
is consistent with published data suggesting that T cell epitopes
are often found in this region in P. falciparum in humans and
P. yoelii in mice (13, 25, 52). It was on the basis of such
published data that we originally decided to include both al-
leles of MSP-133in the composite antigen constructs. These
vaccines differ from previous MSP-1 protein-based vaccines,
usually based on MSP-119or MSP-142, by the inclusion of the
four relatively conserved blocks 1, 3, 5, and 12. These were
included in an attempt to induce conserved T cell responses
that would transcend allelic differences. Despite the induction
of antibodies to the four conserved blocks, we observed no T
cell epitopes in BALB/c or C57BL/6 mice in these regions (see
Table S1 in the supplemental material). This was despite the
presence of documented T cell epitopes in blocks 1 and 3 in
humans (29, 43) as well as in both allelic forms of block 16 (14).
However, this probably simply reflects the lesser capacity of
inbred mice strains to recognize multiple peptide epitopes than
outbred humans with a more diverse repertoire of antigen-
presenting HLA molecules.
There was a sigmoidal relationship between the ELISA titer
to MSP-142and the GIA of purified IgG. This suggests that the
functional activity of the sera was related to responses to the
42-kDa C terminus. Using a reversal-of-inhibition assay, we
have found the growth-inhibiting activity of the sera to be
related to the presence of antibodies to the 19-kDa C terminus
of MSP-1 (see Fig. S6 in the supplemental material). It has
been shown previously that antibodies against all subunits of
MSP-1 are capable of preventing parasite growth in vitro (58).
Despite induction of antibodies to the conserved regions of
MSP-1 by these virus-vectored vaccines, it was only the anti-
bodies to MSP-119that appeared to be essential for growth-
inhibitory activity in this study. Although PfM115 includes only
the 3D7 allele of MSP-119, the antibodies induced by these
vaccines demonstrated activity against the alternative allelic
VOL. 78, 2010 SIMIAN ADENOVIRUS-VECTORED VACCINES AGAINST PfMSP-14609
FVO strain of P. falciparum as measured by ELISA and GIA
assays. The inclusion of the second allele of MSP-119in
PfM128 in mice led to stronger antibody induction in mice
against the QKNG allelic variant and may therefore be of
benefit for human vaccination.
Previous vaccines based on MSP-1 have utilized a panel of
monoclonal antibodies in order to confirm vaccine antigen
conformation and identity (41, 50). However, only one previ-
ous attempt has been made to alter the structure of the antigen
to minimize the induction of blocking antibodies (16). We
found that these amino acid changes abolished binding of one
blocking monoclonal antibody (MAb 2.2) without abolishing
inhibitory MAb binding or GIA activity. The abolishment of
binding to blocking MAb 2.2 with the modified protein pro-
duced by the virus-vectored vaccines may or may not have been
associated with a reduction in the induction of blocking anti-
bodies in general following in vivo immunization. Further work
is necessary to determine whether such changes will have a
significantly beneficial effect in the development of effective
vaccines based on MSP-1, given that such blocking antibodies
appear to interfere with protection against malaria in humans
(19). In C57BL/6 mice a CD8?T cell epitope was also detected
within MSP-119, which may indicate that the removal of the
disulfide bond aided antigen processing and presentation.
However, we did not develop viral vaccines expressing the
antigen PfM115 without the amino acid alterations and were
thus unable to compare directly the response of this vaccine to
that of a wild-type antigen construct. We also did not develop
a viral vaccine based on the MSP-142region alone in order to
determine whether inclusion of the conserved blocks was of
benefit to overall immunogenicity. Testing of this construct in
outbred populations with a greater repertoire of major histo-
compatibility complex (MHC) molecules will be of benefit in
addressing this question.
Overall, the vectors and immunization approach described
here should now provide an opportunity to assess the protec-
tive efficacy of strong T cell responses combined with high-level
antibody responses against MSP-1 in humans. This could be
assessed by blood-stage challenge (51) or by sporozoite chal-
lenge using infectious mosquito bites (56). The former allows
lower-dose parasite challenges to be employed, but the latter
may allow an added protective effect of T cells against MSP-1
expressed during merozoite development in the liver stage of
the life cycle to be measured. The approach described here
represents the beginning of a new P. falciparum blood-stage
malaria vaccine strategy and also encourages the clinical as-
sessment of both simian adenovirus-MVA and heterologous
simian adenoviral vector regimens for other, diverse disease
Statistics were analyzed with the assistance of N. Alder (University
of Oxford). We thank J. M. Reuter (Agrobio), S. Moretz (NIH), N.
Edwards, E. Forbes, J. Furze, A. Reyes-Sandoval, A. Spencer, and D.
Worth for their assistance with this work.
This work was supported primarily by grant G0600424 and in part by
funding agreement U117532067 from the Medical Research Council.
In addition, this work was supported in part by the Division of Intra-
mural Research, National Institutes of Allergy and Infectious Dis-
eases, National Institutes of Health, and also by the PATH/Malaria
Vaccine Initiative for support of the GIA Reference Center and by the
European Commission through the European Malaria Vaccine Devel-
opment Association, contract LSH-2005-037506. This work was also
funded by NIDDK grant P30 DK-47757 (J.M.W.). S.J.D. is a Junior
Research Fellow of Merton College, Oxford. A.V.S.H. is a Wellcome
Trust Principal Research Fellow. A.L.G. was an MRC clinical training
fellow while undertaking this research.
A.L.G., S.C.G., A.V.S.H., and S.J.D. are named inventors on patent
applications covering vectored malaria vaccines and immunization reg-
imens. Authors from Okairo `s are employees of and/or shareholders in
Okairo `s, which is developing vectored malaria vaccines. J.M.W. is a
consultant to ReGenX Holdings and is a founder of, holds equity in,
and receives a grant from affiliates of ReGenX Holdings; in addition,
he is an inventor on patents licensed to various biopharmaceutical
companies, including affiliates of ReGenX Holdings.
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4612 GOODMAN ET AL.INFECT. IMMUN.