Utilization of genomic sequence information to develop malaria vaccines
1 Malaria Program, Naval Medical Research Center, Silver Spring, MD
20910-7500, USA
2 Department of Molecular Microbiology and Immunology, School of Hygiene and
Public Health, Johns Hopkins University, Baltimore, MD 21205-2179,
USA
3 Pan American Health Organization, Washington, DC 20910, USA
4 Henry M. Jackson Foundation, Rockville, MD 20852, USA
5 La Jolla Institute for Allergy and Immunology, San Diego, CA 92121,
USA
6 University of California Irvine, Irvine, CA 92697, USA
7 The Scripps Research Institute, La Jolla, CA 92037, USA
* Author for correspondence (e-mail: dooland{at}nmrc.navy.mil)
Accepted 21 July 2003
![]() |
Summary |
---|
Key words: Plasmodium, P. falciparum, vaccine, genomics, proteomics, molecular immunology, immune screening, multi-epitope
![]() |
Challenges in malaria vaccine development |
---|
The development of a malaria vaccine is, however, a formidable challenge.
Despite a relatively intense and systematic research effort conducted since
the 1960s and clinical trials of a large number of candidate vaccines with a
range of delivery systems designed to induce protective antibody or
cell-mediated immune responses against the sporozoite in circulation, the
infected hepatocyte or the parasitized erythrocyte, few humans have been
protected (reviewed in Richie and Saul,
2002). Even in animal models, vaccines have not been optimal. As
compared to developing vaccines against viruses and bacteria, developing a
vaccine against malaria is complicated by the complexity of the parasite as
well as the complexity of the host's response to the parasite. Challenges
include the complex multi-stage parasite life cycle, a large 23 Mb genome
encoding more than 5300 proteins, distinct stage-specific expression of the
proteins, the requirement for distinct immune mechanisms targeting these
different stages, the poor understanding of the protective immune mechanisms,
allelic heterogeneity of parasite antigens between strains, antigenic
variation within a single strain, sequence polymorphism of critical target
epitopes, parasite evasion of host immune responses, and variant disease
expression based on epidemiology, transmission dynamics and the genetic
background and age of the host (Hoffman et
al., in press
).
![]() |
Models demonstrating the feasibility of developing a malaria vaccine |
---|
Model 1. Sterile protective immunity
One model, immunization with radiation-attenuated sporozoites, is for the
design of a vaccine to prevent all clinical manifestations of malaria
(reviewed in Hoffman et al.,
2002a). In 1967, it was demonstrated that mice immunized with
Plasmodium berghei sporozoites, attenuated by exposure to xray or
gamma-radiation such that they could invade the host hepatocyte and undergo
limited development but could not mature into blood-stage parasites, were
protected against challenge with infectious sporozoites
(Nussenzweig et al., 1967
).
Since the parasite was unable to mature to the erythrocytic stage, clinical
symptoms of disease and transmission of malaria did not occur. Immunization
with heatkilled, formalin-inactivated or lysed sporozoites was not effective,
but protective immunity could be induced in mice immunized with infectious
sporozoites and treated with chloroquine to prevent erythrocyte infection.
These data emphasize the requirement for live sporozoites targeting the liver.
In 1973, it was demonstrated that human volunteers could be protected against
P. falciparum sporozoite challenge by immunization with
radiation-attenuated P. falciparum sporozoites (Clyde et al.,
1973a,b). It is now well established that immunization of mice or humans with
radiation-attenuated Plasmodium sporozoites confers sterile
protective immunity against challenge with infectious, non-attenuated
sporozoites in virtually all recipients (reviewed in
Nussenzweig and Nussenzweig,
1989
; Hoffman et al.,
2002a
). This protection is effective against challenge with
massive doses of infectious sporozoites, is species-specific but not
strain-specific, is efficacious in outbred and inbred mouse strains differing
in genetic background, as well as major histocompatibility complex
(MHC)-diverse humans, and persists for at least 9 months in humans. The
importance of immune mechanisms that are active in the irradiated sporozoite
model is highlighted by the fact that infection-blocking immunity in humans
rarely, if ever, occurs under natural conditions.
The Plasmodium spp. parasite developing within the host hepatocyte
is the major target of protective immune responses induced by immunization
with irradiated sporozoites. CD8+ T cells specific for peptide
epitopes from proteins expressed by irradiated sporozoites in the hepatocyte
are considered the primary immune effectors, and protection is mediated by
interferon (IFN-
) released by these CD8+ T cells (as
well as other cells) rather than by direct cytotoxic T cell-mediated lysis
(Doolan and Hoffman, 1999
,
2000
;
Good and Doolan, 1999
;
Plebanski and Hill, 2000
).
CD4+ T cells that recognize parasite-derived peptide/class II MHC
molecule complexes on the hepatocyte, as well as antibodies against sporozoite
surface proteins that neutralize the infectivity of sporozoites for
hepatocytes, may also play a role (reviewed in
Sinnis and Nussenzweig, 1996
;
Good and Doolan, 1999
). The
targets of cellular immunity (both at the CD8+ and CD4+
T cell level) are largely unknown, however, and correlates of protection after
sporozoite immunization are unclear. Also, it is not established whether the
protective immune responses induced by immunization with irradiated
sporozoites are narrowly focused on a few immunodominant antigens and epitopes
or, alternatively, are broadly dispersed on a relatively large number of
parasite antigens. Recently, we have demonstrated that 16 of 27 putative
P. falciparum proteins identified by multidimensional protein
identification technology (MudPIT) were recognized by volunteers immunized
with irradiated sporozoites; nine proteins were highly antigenic, three were
of intermediate reactivity, and four were weakly antigenic
(Doolan et al., 2003
). These
data support our contention that protective immune responses induced by
immunization with irradiated sporozoites are probably directed against
multiple antigens, and against multiple epitopes on those antigens, with
variable potency. Thus, a multi-antigen vaccine that induces CD8+
as well as CD4+ T cell responses against liver-stage antigens may
be required to mimic the breadth and complexity of the irradiated
sporozoite-induced protection.
The induction of sterile immunity in humans by immunization with radiation-attenuated P. falciparum sporozoites provides proof-of-principle regarding the feasibility of a malaria vaccine that prevents blood-stage infection and clinical disease. This model also suggests a logical approach for making such a vaccine: identify the antigenic targets of irradiated sporozoite-induced immunity (those antigens expressed by irradiated sporozoites within hepatocytes that are recognized by sporozoite-induced T cell responses) and package these antigens or their critical minimal epitopes in a vaccine formulation that is immunogenic and suitable for manufacture and administration.
Model 2. Anti-disease immunity
The second model is that of naturally acquired immunity, for the design of
a vaccine to prevent death and severe disease. In areas where malaria is
transmitted, individuals that survive past a certain age will become
reinfected and will become clinically ill but will not develop severe disease
or die. In areas with annual, stable transmission, there is little to no
severe disease or malaria-associated deaths after the age of 7-10 years; in
areas with very intense transmission, this transition may occur as early as
the second or third year of life. The decrease in the incidence of P.
falciparum infections, the prevalence and density of parasitemia, and the
morbidity and mortality associated with Plasmodium spp. infection
with natural exposure is consistent with acquisition of anti-malarial immunity
in humans (Baird, 1995,
1998
;
Snow et al., 1998
). Moreover,
passive transfer of purified immunoglobulin derived from adults with naturally
acquired immunity following lifelong exposure to endemic malaria results in a
marked decrease in P. falciparum blood-stage parasitemia and
resolution of symptoms in the recipients
(Cohen et al., 1961
;
McGregor and Carrington, 1963
;
Sabchareon et al., 1991
). A
clinically important degree of erythrocytic stage immunity can be also induced
by repeated experimental exposure to blood-stage infection (reviewed in
Jeffery, 1966
; Collins and
Jeffery,
1999a
,b
).
In naturally acquired immunity, all arms of the immune system are probably
activated against all stages of the parasite life cycle. However, most
malariologists believe that the most important effectors in naturally acquired
immunity are antibodies directed against parasite proteins expressed on the
surface of erythrocytes that prevent sequestration in the microcirculation
(Duffy et al., 2001),
antibodies directed against parasite proteins expressed on the surface of
merozoites that prevent invasion of erythrocytes
(Sim et al., 2001
), and
antibodies expressed against either type of parasite protein that are capable
of mediating antibody-dependent cellular inhibition
(Bouharoun-Tayoun et al.,
1995
), whereby biologically active molecules, including cytokines,
nitric oxide and free oxygen intermediates, are released from
reticuloendothelial or other cells after activation through the Fc component
of the bound antibody molecule. Furthermore, pathogenesis of the clinical
disease (Miller et al., 1994
;
Marsh et al., 1996
;
Clark and Schofield, 2000
) may
be mediated by these same host-derived biologically active molecules or by
putative toxins released from the infected erythrocytes
(Playfair, 1996
;
Clark and Schofield, 2000
;
Schofield et al., 2002
), and
neutralization of these via antibodies may play a role in protection.
Finally, antibody responses against sporozoites as well as T cell responses
against parasite proteins expressed within infected hepatocytes probably also
contribute to naturally acquired disease modulating immunity. By reducing the
number of Plasmodium spp. parasites maturing within the host
hepatocyte, these pre-erythrocytic stage immune responses would be expected to
dramatically reduce the initial blood-stage parasite burden and consequently
the magnitude of the subsequent asexual stage amplification. In both
hospitalized populations and semi-immune populations, most investigators have
demonstrated a direct correlation between P. falciparum parasite
density and morbidity and mortality associated with P. falciparum
infection (McElroy et al.,
1994
,
1997
;
Mbogo et al., 1995
;
Vounatsou et al., 2000
). The
critical antigens targeted by naturally acquired immune responses are yet to
be defined. Given the complexity of the parasite, and the host, it is likely
that tens, hundreds or even thousands of parasite proteins may be targeted.
This breadth of response may be further expanded by exposure to many
heterogeneous parasite strains.
The existence of naturally acquired immunity and the demonstrations that a
clinically important degree of erythrocytic stage immunity can be induced by
experimental (Jeffery, 1966;
Collins and Jeffery,
1999a
,b
)
or natural (Baird, 1995
,
1998
) exposure to repeated
blood-stage infection provide a strong rationale for the identification of the
antigenic targets of naturally acquired immunity (those antigens expressed on
the surface of merozoites or infected erythrocytes or in apical organelles
that are recognized by antibodies induced in the context of naturally acquired
immunity) and the development of vaccines designed to induce high levels of
antibody responses against these antigens.
![]() |
Current approaches to malaria vaccine development |
---|
Our hypothesis is that by reducing the number of parasites emerging from the liver (via T cell immune responses directed against those antigens expressed by irradiated sporozoites in hepatocytes) and priming the immune system to erythrocytic stage antigens that will be boosted by infection from natural exposure (via antibody responses directed against parasite proteins expressed on the surface of merozoites or infected erythrocytes or apical organelles), one will reduce the severity and mortality of P. falciparum malaria.
Most current candidate malaria vaccines are designed to induce protective immune responses against pre-erythrocytic and/or erythrocytic stage antigens. Another type of vaccine being developed, a transmission-blocking vaccine, is designed to protect the entire community rather than the immunized individual, by inducing protective antibodies against sexual stage antigens and thereby reducing the intensity of malaria transmission.
Two main approaches to malaria vaccine development are currently being
pursued worldwide. The most work has been done, and progress achieved, on an
approach focused on maximizing the magnitude and quality of immune responses
to a single or a few key antigens, such as the P. falciparum
circumsporozoite protein (CSP) or merozoite surface protein 1 (MSP1), by
immunizing with synthetic peptides or recombinant proteins in an adjuvant
(Mahanty et al., 2003). These
vaccines are being designed to primarily induce antibody and CD4+ T
cell responses, but there is also interest in eliciting CD8+ T cell
responses. Researchers focusing on this approach consider that the subset of
parasite antigens currently identified as potential vaccine targets are
adequate, that any one given antigen is likely to be as good as another
provided that it is expressed in an appropriate context and that the major
obstacle to fielding an effective vaccine lies with optimizing the induction
of the desired immune response by vaccination. Some success with this approach
has been shown with the demonstration that a CSP-based vaccine formulated in a
strong adjuvant can provide short-term protection of malaria-naïve
volunteers against experimental challenge and of semi-immune adults against
naturally transmitted malaria. However, the transient nature of this
protection and the inability of the vaccine to induce the class I-restricted
responses considered important for pre-erythrocytic stage protection
(Lalvani et al., 1999
) show
that the vaccine is far from adequate as currently formulated. The questions
of whether all antigens are the same and it is the vaccine delivery system
that matters, whether the single `key' antigen has already been identified or
whether there is a single `key' antigen that has not yet been identified
remain.
The second approach to malaria vaccine development is to focus on all of
the currently known, promising candidate antigens and to induce good immune
responses against them; for example, by priming with plasmid DNA and then
boosting with DNA, recombinant viruses or recombinant proteins. The goal is to
elicit CD8+ and CD4+ T cell as well as antibody
responses. Researchers focusing on this multi-valent, multi-immune response
approach are skeptical about the ability of a vaccine based on a single
antigen to protect against a parasite as complex as Plasmodium that,
as evidenced by its ability to establish a chronic, recrudescing infection,
has evolved mechanisms for resisting all arms of the host immune response.
Furthermore, genetic restriction of the host immune response and parasite
variation of target antigens and epitopes pose enormous obstacles for vaccine
development. That a multivalent approach may be more successful than an
approach based on a single or few antigens is supported by the experimental
demonstration that immunization of mice with a mixture of DNA vaccines
encoding two pre-erythrocytic stage antigens could circumvent the genetic
restriction of protection seen with each vaccine alone and could confer
additive protection in some genetically distinct mouse strains not protected
or poorly protected by either of the individual vaccines
(Doolan et al., 1996).
Additional support is provided by recent data demonstrating enhanced
protection with a tetravalent Plasmodium knowlesi vaccine as compared
with vaccines based on only one or two of the four antigens (W. R. Weiss,
unpublished results). Thus, researchers pursuing this second approach are
focusing their efforts on the development of a multi-valent, multi-stage and
multi-immune response vaccine (Doolan and
Hoffman, 1997
) structured around the two extremes of vaccine
design. Efforts to date have focused primarily on the technology of DNA-based
vaccines (Doolan and Hoffman,
2001
; Moorthy and Hill,
2002
). First-generation DNA vaccines have proved suboptimal, but a
number of immune-enhancement strategies show promise, at least in animal
models. Most impressive are heterologous prime/boost approaches with DNA,
recombinant viruses or recombinant protein in adjuvant, which show great
potential for the induction of high levels of T cell or antibody responses,
and protection (reviewed in Schneider et
al., 1999
; McConkey et al.,
2003
; Dunachie and Hill,
2003
).
A major shortcoming of this approach is that it is based on a limited panel
of already well-characterized antigens that may or may not be the most optimal
targets of protective immunity (if, in fact, all antigens are not created
equal). Additional, and potentially more promising, antigens could be included
once identified. Regardless, in order for the vaccine to be effective in
genetically diverse host populations and against all antigenically distinct
P. falciparum strains, sufficient diversity must be represented by
the panel of antigens to ensure that multiple arms of the immune system are
activated and to allow for overcoming or circumventing the genetic restriction
of the host immune response and the polymorphism of critical target epitopes.
Finally, this vaccine approach and the number of antigens that can be targeted
will be limited by logistical considerations regarding the size of the insert
that can be included in a given vaccine delivery system and the number of
antigens that can be formulated or administered simultaneously in the absence
of antigenic competition (Sedegah et al.,
in press), as well as by manufacturing considerations.
![]() |
An alternative approach, designed to mimic whole-organism-induced protective immunity |
---|
We believe that the sterile immunity achieved after immunization with
irradiated sporozoites is probably directed against a large number of proteins
expressed by irradiated sporozoites in hepatocytes and against a number of
epitopes on those proteins and that the naturally acquired immunity
experienced by those living in endemic areas is probably directed against a
large number of proteins expressed on the surface of merozoites or infected
erythrocytes or in apical organelles. Small or modest immune response against
tens, hundreds or thousands of parasite proteins may be additive or
synergistic. We are working, therefore, towards the development of a new
generation vaccine, based on the presumption that duplicating and sustaining
the protective immunity induced by whole-organism vaccination may require a
vaccine that mimics the complexity of the organism itself, incorporating
antigens from multiple stages and accounting for the extraordinary diversity
of natural parasite populations. This approach requires the identification of
an unprecedented number of parasite-derived proteins, the minimal
CD8+ and CD4+ T cell epitopes on those antigens, and
development of a vaccine delivery system that reproduces the breadth and
multiplicity of the whole-organism-induced protective immunity. Our strategy
is to focus on the two human models of whole-organism-induced immunity -
irradiated sporozoite immunization and naturally acquired immunity - and to
systematically identify and prioritize the antigens in the Plasmodium
parasite targeted by the different immune responses. The central assumption is
that, of the 5300 potential proteins in the P. falciparum genome
(Gardner et al., 2002
), there
will be one subset that is the target of protective T cell responses directed
against the proteins expressed by the liver-stage parasite (those antigens
expressed by irradiated sporozoites in hepatocytes) and another subset that is
the target of protective antibody responses (those antigens expressed on
merozoites, on infected erythrocytes and in apical organelles). We also
anticipate that results of genome-based studies will suggest a prioritization
amongst these antigens, according to their relative magnitude of immune
reactivity and presumed protective capacity.
![]() |
The P. falciparum genome and proteome - the foundation for a `Genomes-to-Vaccines' approach |
---|
The genomic sequence of P. falciparum was completed and published
in October 2002 (Gardner et al.,
2002). The P. falciparum proteome represented by
stage-specific sporozoites, merozoites, trophozoites and gametocytes was also
elucidated using MudPIT, which combines in-line high-resolution liquid
chromatography and tandem mass spectroscopy
(Washburn et al., 2002
), and
was published simultaneously with the P. falciparum genome
(Florens et al., 2002
;
Lasonder et al., 2002
). Since
then, additional erythrocyte-surface-expressed parasite proteins have been
identified using high-throughput proteomics (L. Florens, X. Liu, Y. Wang, O.
Yang, O. Schwartz, M. Peglar, D. J. Carucci, J. R. Yates, III, Jr and Y. Wu,
manuscript submitted). More recently, the gene expression profile of the
P. falciparum parasite during the different stages of the parasite
life cycle has been completed (LeRoch et
al., 2003
). Putative hepatic-stage-specific proteins have also
been identified through EST sequencing, additional proteomics studies and
bioinformatic methods (J. Aguiar, unpublished; P. L. Blair, unpublished; L.
Florens, unpublished). In total, these data provide a set of open reading
frames (ORFs) corresponding to potential P. falciparum target
antigens and evidence for expression of these genes in different stages of the
parasite life cycle. This foundation can be exploited for the identification
and prioritization of novel antigens and epitopes that may be targets of
anti-malarial protective immunity.
The genomic sequences of a number of other human, monkey or rodent
Plasmodium spp. used in vaccine or drug development research,
including a P. falciparum clinical isolate, P. vivax, P.
knowlesi, P. reichanowi, P. yoelii
(Carlton et al., 2002), P.
chabaudi and P. berghei, have been also determined or are in
progress (Hoffman et al.,
2002b
;
http://www.tigr.org).
The complete genomic sequence of the human host
(Venter et al., 2001
;
Lander et al., 2001
) and the
Anopheles gambiae mosquito vector (the most important vector of
P. falciparum in sub-Saharan Africa;
Holt et al., 2002
) are now
also available. The hope is that these data sets will facilitate additional
characterization of those P. falciparum antigens for which a monkey
or mouse orthologue is identified and will allow for comparative genomics
(Thompson et al., 2001
;
Waters, 2002
) and other
studies designed to characterize Plasmodium spp. parasite antigens
and their biological function relative to the insect or vertebrate host or to
identify potential avenues for the development of novel interventions
(Wirth, 2002
). However, the
promise of these multiple genomic data sets cannot be fully realized without
developing appropriate technologies for systematically converting genomic data
into protective vaccines, drugs or diagnostics.
![]() |
Platform technologies for P. falciparum Genomes-to-Vaccines |
---|
Recombinatorial cloning (GatewayTM)
Traditional methods of cloning are too inefficient, laborious and costly to
be applied to P. falciparum. Moreover, the high adenine/thymine
content of the P. falciparum genome results in a large proportion of
clones containing internal deletions and rearrangements and large numbers of
non-recombinant clones. There is also a need to clone selected ORFs into
multiple vectors, depending on the intended application (DNA vaccination,
protein expression, transfection studies, etc.). Accordingly, we have
evaluated the potential utility of a highly efficient directional
recombinatorial cloning system. The GatewayTM system (InVitrogen Inc.,
Carlsbad, CA, USA) is designed to rapidly and efficiently clone large numbers
of genes into plasmid vectors by exploiting the well-characterized
site-specific recombination between bacteriophage lambda and Escherichia
coli. The recombination process is designed such that a `suicide' gene in
the entry vector (and expression vectors), which is not able to support
bacterial growth on standard E. coli host cells, must be replaced by
the gene of interest to permit survival, making the plasmid-to-plasmid cloning
essentially 100% effective. Dr Joshua Labaer and colleagues at the Harvard
Institute of Proteomics (Harvard Medical School, Boston, MA, USA) provided
invaluable assistance in adapting the GatewayTM system for use with
P. falciparum; these researchers have produced a complete set of more
than 7000 GatewayTM clones from Saccharomyces cerevisiae and a
set of clones from the human genome
(Brizuela et al., 2001).
Initially, a set of GatewayTM entry clones and destination clones in a customized DNA vaccine plasmid (J. C. Aguiar, J. LaBaer, V. Y. Shamailova, M. Koundinya, J. A. Russrl, P. L. Blair, F. Huang, K. Strang, W. Mar, R. Anthony et al., manuscript in preparation) and in a recombinant protein expression vector are generated for a prioritized subset of P. falciparum ORFs. Then, antisera against each of the gene products are generated in mice and used to establish stage specificity and subcellular localization (see below; stage-specific expression) and perhaps inhibitory activity using functional assays such as the inhibition of sporozoite invasion, inhibition of liver-stage development or growth inhibition assays. Capacity of the putative proteins to be recognized by immune sera from individuals residing in malaria-endemic areas or T cells from irradiated-sporozoite-immunized volunteers can also be evaluated (see below; immune screening).
Proof-of-principle studies with a subset of 111 full-length single exon genes (J. C. Aguiar, J. LaBaer, V. Y. Shamailova, M. Koundinya, J. A. Russrl, P. L. Blair, F. Huang, K. Strang, W. Mar, R. Anthony et al., manuscript in preparation) demonstrated that more than 86% of ORFs were cloned into entry and DNA vaccine vectors, in the absence of sequence deletions or rearrangements, and that antibodies that recognized P. falciparum merozoites, sporozoites and gametocytes could be generated by in vivo immunization with the GatewayTM-compatible DNA vaccines. These data establish the GatewayTM as a platform technology for exploiting P. falciparum genomic data for vaccine development.
In a parallel project, we are applying the GatewayTM system to the
Plasmodium yoelii rodent model. The goal is to identify and
characterize P. yoelii antigens that can protect against P.
yoelii sporozoite challenge. The underlying assumption is that P.
falciparum orthologues of such antigens would represent good candidate
vaccine antigens. This project focuses on data from the recently completed
genomic sequence of P. yoelii yoelii (17XNL;
Carlton et al., 2002). Selected
ORFs, predicted to be expressed in the sporozoite/liver stage, are cloned
using approaches analogous to those established for P. falciparum.
Inbred BALB/c mice are immunized with pools of plasmids and challenged with
infectious P. yoelii sporozoites. The capacity of the gene to reduce
liver-stage parasite burden is quantified using Taqman® RT-PCR
(Witney et al., 2001
). Plasmid
pools associated with protection are then deconvoluted to identify the
individual protective genes. The advantage to the pooled plasmid approach is
that it offers a higher probability of a positive outcome, given that DNA
vaccines based on single antigens may not necessarily be capable of conferring
a high degree of protection against parasite challenge and that the protection
induced by the multi-antigenic Plasmodium sporozoite may reflect the
summation of immune responses directed against a relatively large number of
parasite antigens. Preliminary studies with 192 single exon ORFs selected from
a P. yoelii sporozoite EST library
(Kappe et al., 2001
), with 19
of the 192 being evaluated in vivo to date, have established
proof-of-principle for this approach (D. Haddad, E. Bilcikova and W. R. Weiss,
manuscript in preparation).
The primary outcome of the P. yoelii studies is protection rather
than immunogenicity (although we recognize the value in correlating results of
in vitro immunological screening assays with in vivo
protection). This is a major advantage in the context of vaccine development,
since in vivo identification of protective antigens is not feasible
in the P. falciparum system prior to clinical testing, and
potentially protective P. falciparum antigens may be identified by
extrapolation from the P. yoelii system. It should be recognized,
however, that the demonstration that a P. yoelii antigen is capable
of protecting against P. yoelii sporozoite challenge in a mouse model
does not necessarily mean that its P. falciparum orthologue would
protect against P. falciparum parasite challenge in humans. An
additional confounder is that the P. yoelii studies are carried out
in a single genetically homogeneous inbred mouse strain (BALB/c; H-2d) whereas
the target population for a P. falciparum vaccine is a genetically
heterogeneous outbred human population. Nonetheless, mouse models are
generally considered as suitable animal models for malaria vaccine development
(Renia et al., 2002;
Sanni et al., 2002
), and
P. falciparum orthologues of all characterized and protective P.
yoelii antigens are currently considered high priority vaccine
candidates.
Transcriptionally active PCR (TAP)
With the GatewayTM system, transcriptionally active genes are created
by cloning the gene of interest into a replication-competent expression
vector, transforming and growing bacteria and then purifying the plasmid. Even
given the efficient nature of the recombinatorial approach as compared with
traditional cloning methods, this approach is time, labor and cost prohibitive
if a large number of genes needs to be analyzed. Genome-wide screening is also
limited by the ability to validate that the appropriate recall immune
responses can be induced against identified targets.
One solution is to render PCR products transcriptionally active
(Sykes and Johnston, 1999). In
response to this need, Dr Philip Felgner and colleagues (Gene Therapy Systems
Inc., San Diego, CA, USA) developed a technology called transcriptionally
active PCR, which allows for rapid and efficient generation of hundreds or
thousands of genes in a form that is transcriptionally active in
vitro and in vivo and that therefore can be used for
high-throughput functional screening on a genome-wide basis
(Liang et al., 2002
). The
technology uses nested PCR, in which two or more DNA fragments can be joined
in a desired orientation, to introduce functional promoter and terminator
sequences onto the gene of interest. Additionally, the technology can be
modified by adding either epitope-tags (influenza-HA) or mammalian
[cytomegalovirus (CMV)] or prokaryotic (bacterial phage T7) promoters.
Epitope-tagging facilitates detection and characterization of the protein by
western blot, enzyme-linked immunosorbent assay (ELISA), immunocytochemistry
and fluorescence-activated cell sorting (FACS). Adding a T7 promoter allows
for the generation of gene-specific mRNAs in a cell-free system and the
generation of proteins in a cell-free transcription/translation system. The
TAP system therefore offers enormous potential for genome-wide screening. To
assess immunological potency, for example, TAP fragments or TAP proteins can
be screened in novel cellular or humoral assays to quantify the capacity of
each antigen to induce recall cellular or humoral immune responses from
malaria-immune individuals (see below; immune screening assays).
Using a panel of already well-characterized P. falciparum and P. yoelii antigens, as well as a limited panel of ORFs selected from the P. falciparum genomic sequence database, we have evaluated the potential of TAP in the context of Plasmodium (D. A. Regis, P. Quinones-Casas and D. L, Doolan, manuscript in preparation). We have identified conditions for the efficient amplification of all P. falciparum and P. yoelii genes from either genomic DNA or plasmid DNA template and have established that Plasmodium-derived TAP fragments can be expressed in vitro and in vivo and are immunogenic in mice. We are currently developing and validating cellular and humoral immune screening assays based on TAP technology (see below; immune screening).
Protein arrays
Recognition of the less than perfect correlation between RNA and protein
expression (Gygi et al., 1999)
and the influence of post-translational modification of proteins on phenotypic
and functional outcome, including recognition by the host immune system,
highlights the importance of genome-wide screening based on the translated
products of the gene sequences. Cell-free gene transcription and translation
systems, which couple T7 RNA polymerase-driven transcription with translation,
offer the potential for high-throughput protein production. Dr Philip Felgner
and colleagues at the University of California, Irvine (Irvine, CA, USA) have
adapted the cell-free transcription and translation system to a
high-throughput protein expression platform using a robotics workstation,
enabling 384 different purified proteins to be produced and purified in a
single day. In collaboration with Dr Felgner, we are applying protein array
technology for functional screening of the P. falciparum genome.
The P. falciparum proteome will be prepared in two forms. The first form will be on protein `chips', and the chips will be used to quantify serum antibody titers from immune or semi-immune humans against each of the target ORFs. The arrays will be screened for capacity of the target antigens to be recognized by sera collected from clinically distinct cohorts of individuals naturally exposed to malaria. For the second proteome format, each individual protein will be purified and stored in 96-well microtiter plates in a form that will enable them to be delivered to, and processed by, antigen-presenting cells (APCs). These APCs will then be used as targets for in vitro cellular assays to evaluate the capacity of each putative protein to be recognized by recall T cell responses from irradiated-sporozoite-immunized volunteers. It is anticipated that this quantitative array-based humoral and cellular immune response scan will provide a profile of immune responses against P. falciparum in humans.
ImmunoSense
The approaches detailed above are aimed at identifying the complement of
P. falciparum antigens targeted by protective T cell and/or antibody
responses. The ImmunoSense approach offers the opportunity to simultaneously
identify the target antigens as well as the minimal epitopes on those
antigens. It is an integrated approach (executed in collaboration with Dr
Alessandro Sette and colleagues, La Jolla Institute of Allergy and Immunology,
San Diego, CA, USA) that incorporates bioinformatic predictions, human
leukocyte antigen (HLA) supertype considerations, high-throughput binding
assays and cellular assays. In essence, it represents a process in which
epitope predictions are utilized in `reverse', as a tool to identify new
antigens; epitope identification has thus far been seen only as a means to
identify the epitopes contained within a protein considered as a target of
cellular immunity.
A detailed description of the ImmunoSense approach, and its application in
the malaria model, has been reported elsewhere
(Doolan et al., 2003). In
brief, amino acid sequences corresponding to the subset of Plasmodium
sporozoite/liver-stage ORFs are scanned using allele-specific computerized
algorithms for peptide epitopes predicted to bind with high affinity to Class
I and Class II HLA supertypes. HLA supertypes are characterized by largely
overlapping peptide repertoires and are expressed at high frequencies in all
major ethnicities (Southwood et al.,
1998
; Sette and Sidney,
1998
,
1999
). Pools of peptides, each
pool representing a putative antigen, are screened for immune reactivity by
assays such as IFN-
ELISPOT (enzyme-linked immunospot), using
peripheral blood mononuclear cells (PBMCs) from individuals immunized with
radiation-attenuated P. falciparum sporozoites or mock-immunized
controls. Peptide pools that recall strong T cell responses are deconvoluted
to identify the individual peptide epitopes that are recognized. These
antigens and epitopes can be prioritized according to the magnitude of the
recall response and their capacity to be preferentially recognized by
protected versus non-protected volunteers, as well as by their
ability to bind strongly to multiple members of the relevant superfamily.
We have validated this approach in the context of P. falciparum
(Doolan et al., 2003).
Starting with 27 ORFs thought to be expressed in the sporozoite proteome, we
identified 16 novel proteins reproducibly recognized by
irradiatedsporozoite-immunized volunteers. Nine antigens were highly antigenic
(recognized by >50% of volunteers in >25% of assays), three antigens
were of intermediate reactivity, and four were of low reactivity
(Doolan et al., 2003
).
Significantly, a number of antigens identified using this strategy were more
antigenic than well-characterized antigens currently considered the best
vaccine candidate antigens. These experiments provide proof-of-concept for the
Immunosense approach, thereby allowing for more comprehensive genome-wide
analysis.
The anticipated outcome of these studies will be (1) the identification, from the complete P. falciparum proteome, of those proteins that correspond to immunodominant antigens recognized by volunteers immunized with radiation-attenuated P. falciparum sporozoites and their minimal target epitopes and (2) the prioritization of those antigens and epitopes on the basis of immune reactivity and according to their potential association with protection against P. falciparum sporozoite challenge. Additionally, it is expected that the studies will establish whether protective immune responses in humans immunized with irradiated P. falciparum sporozoites are narrowly focused on a few immunodominant antigens and epitopes or, alternatively, are broadly dispersed on a relatively large number of parasite antigens.
![]() |
Selection and prioritization of P. falciparum ORFs for genome-wide screening by Genomes-to-Vaccines approaches |
---|
Other experimentally derived data can also be considered, such as the
relative level of expression of the gene or the corresponding protein or the
degree of polymorphism as indicated by SNP analysis. For example, one outcome
of the MudPIT analysis of P. falciparum sporozoites
(Florens et al., 2002) is a
database detailing the number of MudPIT runs in which corresponding peptides
were identified, the number of peptide hits per protein, the percentage of the
protein sequence covered by those peptides, and the unique locus identifier of
the corresponding genomic sequence. ORFs within this data set can therefore be
ranked according to their relative level of expression in the sporozoite
proteome. Similarly, information regarding stage-specific gene expression is
also available, as a result of AffyMatrix gene chip studies
(LeRoch et al., 2003
).
Correlation between protein expression and gene expression data sets is
currently in progress (J. R. Yates and E. Winzeler, personal
communication).
Despite this wealth of information, and in the absence of genome-wide validation, it is not obvious which tools are the most appropriate and which criteria are the most valid for ORF selection in the context of vaccine development. We have elected to use a combination of bioinformatics and comparative genomics approaches based on a number of genomic, proteomic, gene expression and other large-scale Plasmodium functional genomic data sets to select and prioritize P. falciparum ORFs for genome-wide screening.
One approach (based on the irradiated sporozoite model) has been to combine
transcription and proteome analyses, using sequences derived from a P.
yoelii sporozoite EST database (Kappe
et al., 2001), P. falciparum sporozoite EST database (J.
C. Aguiar, J. LaBaer, V. Y. Shamailova, M. Koundinya, J. A. Russrl, P. L.
Blair, F. Huang, K. Strang, W. Mar, R. Anthony et al., manuscript in
preparation), complete P. falciparum genome expression profiling
using gene chips (Le Roch et al.,
2003
) and proteome analyses
(Florens et al., 2002
;
Lasonder et al., 2002
).
Potential Plasmodium liver-stage-specific expressed genes were down
selected by comparing P. falciparum orthologues of a P.
yoelii laser capture microdissected liver-stage library
(Sacci et al., 2002
) with
annotated genes not present in current EST libraries and not possessing
peptide matches in P. falciparum proteome analyses. Selected antigens
were further examined and ranked using the PlasmoDB web-based database for
predicted signal sequences and transmembrane domains (for sporozoite antigens
only), Pfam and GO assignments, sequence similarities and exon/intron gene
structure. These criteria led to a manageable set (
250 annotated genes)
of putative vaccine targets for study (representing
5% of the P.
falciparum genome; P. L. Blair, unpublished results). Selected ORFs can
then be subjected to an automated primer design algorithm, such as a Primer3
(White Head Institute, Cambridge, MA, USA), for predicting oligonucleotide
primer pairs suitable for high-throughput cloning using the GatewayTM
technology or other Genomes-to-Vaccines approaches.
![]() |
Target credentialing |
---|
Stage-specific expression and subcellular localization
Because the requirement for inducing different types of immune responses by
vaccination depends on the stage during the parasite's life cycle at which the
protein is expressed, identification of the stage-specific expression of
putative antigens is critical. MudPIT analysis of different preparations of
parasite material (e.g. sporozoites, merozoites, trophozoites, gametocytes;
Florens et al., 2002;
Lasonder et al., 2002
)
provides evidence for expression of putative proteins in the different stages
of the parasite's life cycle but not relative expression between the different
stages. Comprehensive gene expression data sets
(Le Roch et al., 2003
) provide
information on expression of all ORFs within any given stage as well as
between stages. P. falciparum DNA microarray studies (Hayward et al.,
2003; Rathod et al., 2002
)
also provide information regarding stage-specific expression, but such data
sets are currently limited to only a subset of the P. falciparum
genome. Finally, proteomic analysis and protein structure prediction
algorithms can suggest which proteins are likely to be membrane-associated
surface proteins, but the accuracy of such predictions has yet to be validated
on a genome-wide scale.
More specific information regarding the stage-specific expression of
putative P. falciparum proteins can be obtained by screening
antigen-specific sera against parasite preparations
(Hoffman et al., 1998). These
sera will be generated for those ORFs evaluated in the Gateway project.
However, financial and logistical reasons preclude comprehensive in
vivo immunization studies for the thousands of putative P.
falciparum proteins in the proteome. Antisera are evaluated by
immunofluoresence antibody tests (IFAT) against stage-specific slides prepared
from P. falciparum sporozoites, cultured hepatoma cells infected with
irradiated or nonirradiated P. falciparum sporozoites,
erythrocytic-stage P. falciparum parasites from carefully
synchronized cultures corresponding to ring trophozoites, mature trophozoites,
and schizonts or gametocytes.
Information on the subcellular localization of putative proteins can also be obtained from the IFAT studies. Although IFAT does not have the resolution of immunoelectron microscopy, it is nonetheless possible to distinguish a number of subcellular localization patterns; for example, in sporozoites, predominant surface expression can be distinguished from internal expression associated with micronemes, a secretory organelle, and, in the erythrocytic-stage parasites, expression on the surface of infected erythrocytes, on the surface of parasites within the red cell, in the apical organelles (micronemes and rhoptries) and within the parasite cytoplasm can be reliably distinguished.
Immune screening
In our Genomes-to-Vaccines program, we propose to identify, from the
complete P. falciparum proteome, (1) the subset of antigens expressed
by irradiated sporozoites in hepatocytes that are recognized by protective T
cell responses and (2) the subset of antigens expressed on the surface of
merozoites or infected erythrocytes or apical organelles that are recognized
by protective antibody responses. A critical component of our strategy is the
evaluation of immune responses. Standard immunological assays are not suitable
for high-throughput screening with plasmid DNA, PCR fragments or recombinant
proteins. Therefore, we are developing and optimizing novel cellular and
humoral immune assays that can be applied to genome-wide screening (P.
Quinones-Cases and D. L. Doolan, unpublished results; J. Aguiar and G. T.
Brice, unpublished results).
To identify targets of protective T cell responses, we are utilizing
specimens from irradiated-sporozoite-immunized volunteers (both protected and
not protected against sporozoite challenge), presuming that the entire
repertoire of sporozoite-induced T cell specificities will be represented in
those individuals. We are focusing on T cell-derived IFN- as the
primary marker of cellular immunogenicity, since this is considered to be the
most appropriate in vitro marker of preerythrocytic stage protection
identified to date (Good and Doolan,
1999
; Plebanski and Hill,
2000
). However, where possible, we will apply information from
other studies designed to identify robust and predictive in vitro
marker(s) of anti-malarial protective immunity; e.g. immunologically relevant
DNA microarray studies with specimens from irradiated-sporozoite-immunized
volunteers (C. Dobano, P. Quinones-Casas and D. L. Doolan, unpublished
results).
To identify the subset of P. falciparum proteins expressed on the surface of merozoites or infected erythrocytes or in apical organelles, we are assaying sera from adults with naturally acquired immunity, since it is presumed that the entire repertoire of antibody specificities is represented in those individuals. However, given the complexity of host-parasite interactions and the variant disease expression based on epidemiology, transmission dynamics and the genetic background and age of the host, we also propose to ultimately analyze the responses of well-characterized cohorts of naturally exposed individuals with distinct categories of clinical disease (e.g. severe malaria, mild malaria, asymptomatic and symptomatic), as well as individuals of different ages (neonates, infants, children and adults). Sera from volunteers immunized with radiation-attenuated P. falciparum sporozoites and collected at different time points during the immunization and challenge process will also be evaluated, to provide a kinetic profile of antibody responses to pre-erythrocytic stage antigens.
Biological function
One indication as to the value of a putative antigen for inclusion in a
vaccine relates to its biological function, at least in the case of protective
antibody responses where the target antigen or the critical linear or
conformational B cell epitope(s) on the antigen must be accessible to the
antibodies (e.g. surface or secreted molecules). The biological function of
the target protein is less important in the case of T cell responses, where
the presence of MHC-binding epitope(s) in the sequence and appropriate
processing of the antigen and presentation of the T cell epitope in the
context of the MHC Class I and/or Class II molecules for recognition by the
host immune system is critical.
Unfortunately, there are no algorithms to predict how a given antigen will
function in vivo, and there are not yet established methods to
accurately assess biological function in vivo, particularly in a
high-throughput manner. Some approaches, such as gene-knockout studies
(Wickham et al., 2003), are
potentially useful in this regard but are currently not adapted for
genome-wide application. For those putative proteins where the desired immune
effector mechanism is an inhibitory antibody, however, it is possible to
assess biological activity in vitro. Specifically, the capacity of
antigen-specific antisera to inhibit parasite development or invasion could be
evaluated by the inhibition of sporozoite invasion assay, inhibition of
liver-stage development assay, or growth inhibition assay.
Ultimately, the capacity of an individual or pool of erythrocytic-stage
proteins to induce protective antibodies in vivo could be assessed by
immunizing Aotus monkeys with plasmid DNA or recombinant proteins and
challenging with P. falciparum blood-stage parasites
(Collins, 2002). Demonstration
of pre-erythrocytic protection against P. falciparum 3D7 strain is
currently not feasible in animal models, since a reproducible
Aotus-P. falciparum 3D7 sporozoite challenge model is not
available, and mice cannot be infected with P. falciparum. However,
reproducible infection of intact Aotus lemurinus griseimembra monkeys
by intravenous inoculation with sporozoites from a monkey-adapted P.
falciparum strain (Santa Lucia) and a wild-type P. falciparum
strain (Cali-Colombia-4, FCC-4) has been recently reported
(Zapata et al., 2002
),
allowing for the possibility of evaluating protection with heterologous P.
falciparum sequences of a given antigen of interest. Also, a reproducible
sporozoite challenge model is available for P. knowlesi in rhesus
monkeys (Collins, 2002
;
Rogers et al., 2001
) and for
P. yoelii in mice (Sedegah et
al., 1982
), so protection could be evaluated for those P.
falciparum antigens that have identified as P. knowlesi or
P. yoelii orthologues.
![]() |
The next step: multi-epitope vaccine development |
---|
In other systems, induction of simultaneous responses against multiple
epitopes derived from multiple antigens has already been demonstrated. The
immunogenicity of multi-epitope constructs appears to be strongly influenced
by a number of different variables, and the immunogenicity (or antigenicity)
of the same epitope expressed in the context of different vaccine constructs
can vary over several orders of magnitude. This situation underscores the
necessity of a systematic study of different variables in order to establish
clear criteria for the optimal design of multi-epitope vaccines (reviewed in
Sette et al., 2001,
2002
). To address this in the
context of malaria, in collaboration with Jeff Alexander, Brian Livingston,
Mark Newman and colleagues (Epimmune Inc., San Diego, CA, USA), we are
designing and optimizing multi-epitope vaccines comprising a panel of
CD8+ and CD4+ T cell epitopes derived from four P.
falciparum pre-erythrocytic stage antigens. These epitopes were
identified by Class I and Class II algorithm predictions and peptide
binding/recognition strategies similar to those described above for the
ImmunoSense approach and were recognized by recall immune responses from
volunteers immunized with irradiated sporozoites or naturally exposed to
malaria (Doolan et al., 1997
,
2000
). Studies are aimed at
optimizing vaccine design by eliminating junctional epitopes, optimizing
spacers between epitopes and considering the order of epitopes, effect of
flanking regions, and cellular targeting to antigen processing and
presentation pathways. Recognition of individual epitopes is demonstrated by
immunogenicity assays utilizing HLA transgenic mice and/or antigenicity assays
using human APCs transfected in vitro with the prototype vaccine. The
simplest vaccine configuration capable of effective delivery of the selected
sets of epitopes will also be determined. Subsequent studies will identify the
optimal vaccine delivery strategy for simultaneous induction of immune
responses against multiple epitopes, and the appropriate vaccine formulation
studies. Overall, it is anticipated that these studies will define operational
rules for the design and optimization of multi-epitope-based vaccines.
Finally, we propose to compare the efficacy of the multi-epitope vaccine with the whole-organism-irradiated sporozoite vaccine, as well as whole antigen subunit vaccines (DNA and viral vectored), in vivo in HLA transgenic mice and in vitro in antigenicity assays using humans APCs. It is anticipated that these studies will validate the multi-epitope approach beyond the level of antigen and epitope identification and will provide important information regarding the potential of multi-epitope-based approaches to mimic whole-organism-induced immunity.
![]() |
Conclusion |
---|
We gratefully acknowledge the many individuals who have contributed intellectually or technically to the efforts described here. Special thanks are due to Daniel Freilich, Lolita Bebris, Mara Berzins and other members of the NMRC Malaria Program Clinical Trials team for acquiring the human specimens which form the foundation of this program; Xiaowu Liang (Gene Therapy Systems, InVitrogen) for developing the TAP and protein array platforms; John Sidney (La Jolla Institute for Allergy and Infectious Diseases) for developing the algorithms core to the ImmunoSense platform and for HLA expertise; Jeff Alexander, Brian Livingston, Scott Southwood and colleagues (Epimmune) for epitope identification and multi-epitope vaccine development; Leonardo Brizuela, Pascal Braun and colleagues at the Harvard Institute of Proteomics for assistance and expertise with the Gateway system; Diana Haddad and Erika Bilcikova for implementing the P. yoelli Gateway project; and Bill Rogers for challenging and expanding our concepts regarding Genomes-to-Vaccines. The studies reported herein were conducted in accordance with US Navy regulations governing the protection of human subjects in medical research. All protocols involving human subjects were reviewed and approved by the Naval Medical Research Center's IRB in accordance with the US Navy regulations (SECNAVINST 3900.39B) governing the use of human subjects in medical research. The opinions and assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the US Navy or the Department of Defense. Work was supported by funds allocated to the Naval Medical Research and Development Center by the US Army Medical Research Material Command (work units 61102A.S13.F.A0009 and 62787A.870.F.A0228).
![]() |
Footnotes |
---|
![]() |
References |
---|
Bahl, A., Brunk, B., Crabtree, J., Fraunholz, M. J., Gajria, B.,
Grant, G. R., Ginsburg, H., Gupta, D., Kissinger, J. C., Labo, P. et al.
(2003). PlasmoDB: the Plasmodium genome resource. A
database integrating experimental and computational data. Nucleic
Acids Res. 31,212
-215.
Baird, J. (1995). Host age as a determinant of naturally acquired immunity to Plasmodium falciparum. Parasitol. Today 11,105 -111.[CrossRef]
Baird, J. K. (1998). Age-dependent characteristics of protection versus susceptibility to Plasmodium falciparum. Ann. Trop. Med. Parasitol. 92,367 -390.[CrossRef][Medline]
Berzins, K. and Perlmann, P. (1996). Malaria vaccines: attacking infected erythrocytes. In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L Hoffman), pp. 105-144. Washington, DC: ASM Press.
Bouharoun-Tayoun, H., Oeuvray, C., Lunel, F. and Druilhe, P. (1995). Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages. J. Exp. Med. 182,409 -418.[Abstract]
Breman, J. G. (2001). Ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. Am. J. Trop. Med. Hyg. 64, 1-11.
Brizuela, L., Braun, P. and Labaer, J. (2001). FLEXGene repository: from sequenced genomes to gene repositories for high-throughput functional biology and proteomics. Mol. Biochem. Parasit. 118,155 -165.[CrossRef][Medline]
Brown, G. and Rogerson, S. J. (1996). Preventing cytoadherence of infected erythrocytes to endothelial cells and noninfected erythrocytes. In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L Hoffman), pp.145 -166. Washington, DC: ASM Press.
Carlton, J. M., Angiuoli, S. V., Suh, B. B., Kooij, T. W., Pertea, M., Silva, J. C., Ermolaeva, M. D., Allen, J. E., Selengut, J. D., Koo, H. L. et al. (2002). Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii. Nature 419,512 -519.[CrossRef][Medline]
Clark, I. A. and Schofield, L. (2000). Pathogenesis of malaria. Parasitol. Today 16,451 -454.[CrossRef][Medline]
Clyde, D. F., McCarthy, V. C., Miller, R. M. and Hornick, R. B. (1973). Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 266,398 -401.[Medline]
Clyde, D. F., Most, H., McCarthy, V. C. and Vanderberg, J. P. (1973). Immunization of man against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 266,169 -177.[Medline]
Cohen, S., McGregor, I. A. and Carrington, S. (1961). Gamma-globulin and acquired immunity to human malaria. Nature 192,733 -737.
Collins, W. E. (2002). Nonhuman primate models. I. Nonhuman primate host-parasite combinations. Methods Mol. Med. 72,77 -84.[Medline]
Collins, W. E. and Jeffery, G. M. (1999a). A
retrospective examination of sporozoite- and trophozoite-induced infections
with Plasmodium falciparum: development of parasitologic and clinical
immunity during primary infection. Am. J. Trop. Med.
Hyg. 61,4
-19.
Collins, W. E. and Jeffery, G. M. (1999b). A
retrospective examination of secondary sporozoite- and trophozoite-induced
infections with Plasmodium falciparum: development of parasitologic
and clinical immunity following secondary infection. Am. J. Trop.
Med. Hyg. 61,20
-35.
Doolan, D. L. and Hoffman, S. L. (1997). Multi-gene vaccination against malaria: a multi-stage, multi-immune response approach. Parasitol. Today 13,171 -178.[CrossRef]
Doolan, D. L. and Hoffman, S. L. (1999). NK
cells and IL-12 are required for antigen-specific adaptive immunity against
malaria initiated by CD8+ T cells. J.
Immunol. 163,884
-892.
Doolan, D. L. and Hoffman, S. L. (2000). The
complexity of protective immunity against liver-stage malaria. J.
Immunol. 165,1453
-1462.
Doolan, D. L. and Hoffman, S. L. (2001). DNA-based vaccines against malaria: status and promise of the Multi-Stage Malaria DNA Vaccine Operation. Int. J. Parasitol. 31,753 -762.[CrossRef][Medline]
Doolan, D. L., Hoffman, S. L., Southwood, S., Wentworth, P. A., Sidney, J., Chesnut, R. W., Keogh, E., Appella, E., Nutman, T. B., Lal, A. A. et al. (1997). Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7,97 -112.[Medline]
Doolan, D. L., Sedegah, M., Hedstrom, R. C., Hobart, P.,
Charoenvit, Y. and Hoffman, S. L. (1996). Circumventing
genetic restriction of protection against malaria with multi-gene DNA
immunization: CD8+ T cell, interferon-, nitric oxide
dependent immunity. J. Exp. Med.
183,1739
-1746.[Abstract]
Doolan, D. L., Southwood, S., Chesnut, R., Appella, E., Gomez,
E., Richards, A., Higashimoto, Y. I., Maewal, A., Sidney, J., Gramzinski, R.
A. et al. (2000). HLA-DR-promiscuous T cell epitopes from
Plasmodium falciparum pre-erythrocytic-stage antigens restricted by
multiple HLA class II alleles. J. Immunol.
165,1123
-1137.
Doolan, D. L., Southwood, S., Freilich, D. A., Sidney, J.,
Graber, N. L., Shatney, L., Bebris, L., Florens, L., Dobano, C., Witney, A. A.
et al. (2003). Identification of new P. falciparum
antigens by antigenic analysis of genomic and proteomic data. Proc.
Natl. Acad. Sci. USA 100,9952
-9957.
Duffy, P. E., Craig, A. G. and Baruch, D. I. (2001). Variant proteins on the surface of malaria-infected erythrocytes - developing vaccines. Trends Parasitol. 17,354 -356.[CrossRef][Medline]
Dunachie, S. J. and Hill, A. V. S. (2003).
Prime-boost strategies for malaria vaccine development. J. Exp.
Biol. 206,3771
-3779.
Florens, L., Washburn, M. P., Raine, J. D., Anthony, R. M., Grainger, M., Haynes, J. D., Moch, J. K., Muster, N., Sacci, J. B., Tabb, D. L. et al. (2002). A proteomic view of the Plasmodium falciparum life cycle. Nature 419,520 -526.[CrossRef][Medline]
Gallup, J. L. and Sachs, J. D. (2001). The
economic burden of malaria. Am. J. Trop. Med. Hyg.
64, 85-96.
Gardner, M. J., Shallom, S. J., Carlton, J. M., Salzberg, S. L., Nene, V., Shoaibi, A., Ciecko, A., Lynn, J., Rizzo, M., Weaver, B. et al. (2002). Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14. Nature 419,531 -534.[CrossRef][Medline]
Good, M. F. and Doolan, D. L. (1999). Immune effector mechanisms in malaria. Curr. Opin. Immunol. 11,412 -419.[CrossRef][Medline]
Gygi, S. P., Rochon, Y., Franza, B. R. and Aebersold, R.
(1999). Correlation between protein and mRNA abundance in
yeast.Mol. Cell. Biol.
19,1720
-1730.
Hayward, R. E., Derisi, J. L., Alfadhli, S., Kaslow, D. C., Brown, P. O. and Rathod, P. K. (2000). Shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria. Mol. Microbiol. 35, 6-14.[CrossRef][Medline]
Hoffman, S. L., Rogers, W. O., Carucci, D. J. and Venter, J. C. (1998). From genomics to vaccines: malaria as a model system. Nat. Med. 4,1351 -1353.[CrossRef][Medline]
Hoffman, S. L., Goh, L. M., Luke, T. C., Schneider, I., Le, T. P., Doolan, D. L., Sacci, J., de la Vega, P., Dowler, M., Paul, C. et al. (2002a). Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185,1155 -1164.[CrossRef][Medline]
Hoffman, S. L., Subramanian, G. M., Collins, F. H. and Venter, J. C. (2002b). Plasmodium, human and Anopheles genomics and malaria. Nature 415,702 -709.[CrossRef][Medline]
Hoffman, S. L., Doolan, D. L. and Richie, T. L. (in press). Malaria: a complex disease that may require a complex vaccine. In New Generation Vaccines (ed. M. M. Levine, J. B. Kaper, R. Rappuoli, M. Liu and M. F. Good). New York, NY: Marcel Dekker, Inc.
Holder, A. A. (1996). Preventing merozoite invasion of erythrocytes. In Malaria Vaccine Development (ed. S. L. Hoffman), pp.77 -104. Washington, DC: ASM Press.
Holt, R. A., Subramanian, G. M., Halpern, A., Sutton, G. G.,
Charlab, R., Nusskern, D. R., Wincker, P., Clark, A. G., Ribeiro, J. M.,
Wides, R. et al. (2002). The genome sequence of the malaria
mosquito Anopheles gambiae. Science
298,129
-149.
Jeffery, G. M. (1966). Epidemiological significance of repeated infections with homologous and heterologous strains and species of Plasmodium. Bull. World Health Org. 35,873 -882.[Medline]
Kappe, S. H., Gardner, M. J., Brown, S. M., Ross, J.,
Matuschewski, K., Ribeiro, J. M., Adams, J. H., Quackenbush, J., Cho, J.,
Carucci, D. J. et al. (2001). Exploring the transcriptome of
the malaria sporozoite stage. Proc. Natl. Acad. Sci.
USA 98,9895
-9900.
Lalvani, A., Moris, P., Voss, G., Pathan, A. A., Kester, K. E., Brookes, R., Lee, E., Koutsoukos, M., Plebanski, M., Delchambre, M. et al., (1999). Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine. J. Infect. Dis. 180,1656 -1664.[CrossRef][Medline]
Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., Devon, K., Dewar, K., Doyle, M., FitzHugh, W. et al. (2001). Initial sequencing and analysis of the human genome. Nature 409,860 -921.[CrossRef][Medline]
Lasonder, E., Ishihama, Y., Andersen, J. S., Vermunt, A. M., Pain, A., Sauerwein, R. W., Eling, W. M., Hall, N., Waters, A. P., Stunnenberg, H. G. et al., (2002). Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature 419,537 -542.[CrossRef][Medline]
Le Roch, K. G., Zhou, Y., Blair, P. L., Grainger, M., Moch, J. K., Haynes, J. D., De La Vega, P., Holder, A. A., Batalov, S., Carucci, D. J. et al. (2003). Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 31 July (Epub ahead of print).
Liang, X., Teng, A., Braun, D. M., Felgner, J., Wang, Y., Baker,
S. I., Chen, S., Zelphati, O. and Felgner, P. L. (2002).
Transcriptionally active polymerase chain reaction (TAP): high throughput gene
expression using genome sequence data. J. Biol. Chem.
277,3593
-3598.
Mahanty, S., Saul, A. and Miller, L. H. (2003).
Progress in the development of recombinant and synthetic blood-stage malaria
vaccines. J. Exp. Biol.
206,3781
-3788.
Marsh, K., English, M., Crawley, J. and Peshu, N. (1996). The pathogenesis of severe malaria in African children. Ann. Trop. Med. Parasitol. 90,395 -402.[Medline]
Mbogo, C. N., Snow, R. W., Khamala, C. P., Kabiru, E. W., Ouma, J. H., Githure, J. I., Marsh, K. and Beier, J. C. (1995). Relationships between Plasmodium falciparum transmission by vector populations and the incidence of severe disease at nine sites on the Kenyan coast. Am. J. Trop. Med. Hyg. 52,201 -206.[Medline]
McConkey, S. J., Reece, W. H., Moorthy, V. S., Webster, D., Dunachie, S., Butcher, G., Vuola, J. M., Blanchard, T. J., Gothard, P., Watkins, K. et al. (2003). Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat. Med. 9,729 -735.[CrossRef][Medline]
McElroy, P. D., Beier, J. C., Oster, C. N., Beadle, C., Sherwood, J. A., Oloo, A. J. and Hoffman, S. L. (1994). Predicting outcome in malaria: correlation between rate of exposure to infected mosquitoes and level of Plasmodium falciparum parasitemia. Am. J. Trop. Med. Hyg. 51,523 -532.[Medline]
McElroy, P. D., Beier, J. C., Oster, C. N., Onyango, F. K., Oloo, A. J., Lin, X., Beadle, C. and Hoffman, S. L. (1997). Dose- and time-dependent relations between infective Anopheles inoculation and outcomes of Plasmodium falciparum parasitemia among children in western Kenya. Am. J. Epidemiol. 145,945 -956.[Abstract]
McGregor, I. A. and Carrington, S. P. (1963).
Treatment of East African P. falciparum malaria with West African
human -globulin. Trans. R. Soc. Trop. Med. Hyg.
57,170
-175.
Miller, L. H., Good, M. F. and Milon, G. (1994). Malaria pathogenesis. Science 264,1878 -1883.[Medline]
Moorthy, V. and Hill, A. V. (2002). Malaria
vaccines. Br. Med. Bull.
62, 59-72.
Nussenzweig, V. and Nussenzweig, R. S. (1989). Rationale for the development of an engineered sporozoite malaria vaccine. Adv. Immunol. 45,283 -334.[Medline]
Nussenzweig, R. S., Vanderberg, J., Most, H. and Orton, C. (1967). Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei. Nature 216,160 -162.[Medline]
Playfair, J. H. L. (1996). An antitoxic vaccine for malaria? In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L Hoffman), pp.167 -180. Washington, DC: ASM Press.
Plebanski, M. and Hill, A. V. (2000). The immunology of malaria infection. Curr. Opin. Immunol. 12,437 -441.[CrossRef][Medline]
Rathod, P. K., Ganesan, K., Hayward, R. E., Bozdech, Z. and DeRisi, J. L. (2002). DNA microarrays for malaria. Trends Parasitol. 18,39 -45.[CrossRef][Medline]
Renia, L., Belnoue, E. and Landau, I. (2002). Mouse models for preerythrocytic-stage malaria. Methods Mol. Med 72,41 -55.[Medline]
Richie, T. L. and Saul, A. (2002). Progress and challenges for malaria vaccines. Nature 415,694 -701.[CrossRef][Medline]
Rogers, W. O., Baird, J. K., Kumar, A., Tine, J. A., Weiss, W.,
Aguiar, J. C., Gowda, K., Gwadz, R., Kumar, S., Gold, M. et al.
(2001). Multistage multiantigen heterologous prime boost vaccine
for Plasmodium knowlesi malaria provides partial protection in rhesus
macaques. Infect. Immun.
69,5565
-5572.
Sabchareon, A., Burnouf, T., Ouattara, D., Attanath, P., Bouharoun-Tayoun, H., Chantavanich, P., Foucault, C., Chongsuphajaisiddhi, T. and Druilhe, P. (1991). Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am. J. Trop. Med. Hyg. 45,297 -308.[Medline]
Sacci, J. B., Jr, Aguiar, J. C., Lau, A. O. and Hoffman, S. L. (2002). Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites. Mol. Biochem. Parasitol. 119,285 -289.[CrossRef][Medline]
Sanni, L. A., Fonseca, L. F. and Langhorne, J. (2002). Mouse models for erythrocytic-stage malaria. Methods Mol. Med. 72,57 -76.[Medline]
Schneider, J., Gilbert, S. C., Hannan, C. M., Degano, P., Prieur, E., Sheu, E. G., Plebanski, M. and Hill, A. V. (1999). Induction of CD8+ T cells using heterologous prime-boost immunisation strategies. Immunol. Rev. 170, 29-38.[Medline]
Schofield, L., Hewitt, M. C., Evans, K., Siomos, M. A. and Seeberger, P. H. (2002). Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature 418,785 -789.[CrossRef][Medline]
Sedegah, M., Hedstrom, R. C., Hobart, P. and Hoffman, S. L.
(1994). Protection against malaria by immunization with plasmid
DNA encoding circumsporozoite protein. Proc. Natl. Acad. Sci.
USA 91,9866
-9870.
Sedegah, M., Charoenvit, Y., Minh, L., Belmonte, M., Fallarme, V., Abbot, S., Ganeshan, H., Sacci, J., Kumar, S., Meek, J. et al. (in press). Reduced immunogenicity of P. falciparum DNA vaccine plasmids in a nine-plasmid mixture. J. Immunol.
Sedegah, M., Tosta, C. E., Henderson, D. C. and Wedderburn, N. (1982). Cross-reactivity and cross-protection in murine malaria. Ann. Trop. Med. Parasitol. 76,219 -221.[Medline]
Sette, A. and Sidney, J. (1998). HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr. Opin. Immunol. 10,478 -482.[CrossRef][Medline]
Sette, A. and Sidney, J. (1999). Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50,201 -212.[CrossRef][Medline]
Sette, A., Livingston, B., McKinney, D., Appella, E., Fikes, J., Sidney, J., Newman, M. and Chesnut, R. (2001). The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation. Biologicals 29,271 -276.[CrossRef][Medline]
Sette, A., Keogh, E., Ishioka, G., Sidney, J., Tangri, S., Livingston, B., McKinney, D., Newman, M., Chesnut, R. and Fikes, J. (2002). Epitope identification and vaccine design for cancer immunotherapy. Curr. Opin. Investig. Drugs 3, 132-139.[Medline]
Sim, B. K., Narum, D. L., Liang, H., Fuhrmann, S. R., Obaldia, N., 3rd, Gramzinski, R., Aguiar, J., Haynes, J. D., Moch, J. K. and Hoffman, S. L. (2001). Induction of biologically active antibodies in mice, rabbits, and monkeys by Plasmodium falciparum EBA-175 region II DNA vaccine. Mol. Med. 7, 247-254.[Medline]
Sinnis, P. and Nussenzweig, V. (1996). Preventing sporozoite invasion of hepatocytes. In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L Hoffman), pp. 15-34. Washington, DC: ASM Press.
Snow, R. W., Nahlen, B., Palmer, A., Donnelly, C. A., Gupta, S. and Marsh, K. (1998). Risk of severe malaria among African infants: direct evidence of clinical protection during early infancy. J. Infect. Dis. 177,819 -822.[Medline]
Southwood, S., Sidney, J., Kondo, A., del Guercio, M. F.,
Appella, E., Hoffman, S., Kubo, R. T., Chesnut, R. W., Grey, H. M. and Sette,
A. (1998). Several common HLA-DR types share largely
overlapping peptide binding repertoires. J. Immunol.
160,3363
-3373.
Sykes, K. F. and Johnston, S. A. (1999). Linear expression elements: a rapid, in vivo, method to screen for gene functions. Nat. Biotech. 17,355 -359.[CrossRef][Medline]
Thompson, J., Janse, C. J. and Waters, A. P. (2001). Comparative genomics in Plasmodium: a tool for the identification of genes and functional analysis. Mol. Biochem. Parasitol. 118,147 -154.[CrossRef][Medline]
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R.
J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A. et
al. (2001). The sequence of the human genome.
Science 291,1304
-1351.
Vounatsou, P., Smith, T., Kitua, A. Y., Alonso, P. L. and Tanner, M. (2000). Apparent tolerance of Plasmodium falciparum in infants in a highly endemic area. Parasitology 120,1 -9.[Medline]
Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M. and Yates, J. R., III (2002). Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal. Chem. 74,1650 -1657.[CrossRef][Medline]
Waters, A. P. (2002). Orthology between the genomes of Plasmodium falciparum and rodent malaria parasites: possible practical applications. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 357,55 -63.[CrossRef][Medline]
Wickham, M. E., Thompson, J. K. and Cowman, A. F. (2003). Characterisation of the merozoite surface protein-2 promoter using stable and transient transfection in Plasmodium falciparum. Mol. Biochem. Parasitol. 129,147 -156.[CrossRef][Medline]
Wirth, D. F. (2002). Biological revelations. Nature 419,495 -496.[CrossRef][Medline]
Witney, A. A., Doolan, D. L., Anthony, R. M., Weiss, W. R., Hoffman, S. L. and Carucci, D. J. (2001). Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy. Mol. Biochem. Parasitol. 118,233 -245.[CrossRef][Medline]
World Health Organization (2002). WHO Report. http://www.who.int/infections-disease-report/2002/index.html.
Zapata, J. C., Perlaza, B. L., Hurtado, S., Quintero, G. E., Jurado, D., Gonzalez, I., Druilhe, P., Arevalo-Herrera, M. and Herrera, S. (2002). Reproducible infection of intact Aotus lemurinus griseimembra monkeys by Plasmodium falciparum sporozoite inoculation. J. Parasitol. 88,723 -729.[Medline]
Related articles in JEB: