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Immunization of
Aotus
monkeys with a functional
domain of the
Plasmodium falciparum
variant
antigen induces protection against a lethal
parasite line
Dror I. Baruch*
†
, Benoit Gamain*, John W. Barnwell
‡
, JoAnn S. Sullivan
‡
, Anthony Stowers
§
, G. Gale Galland
¶
,
Louis H. Miller*, and William E. Collins
‡
*Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
‡
Division of
Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341;
§
Malaria Vaccine
Development Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Rockville, MD 20852; and
¶
Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and
Prevention, Atlanta, GA 30333
Contributed by Louis H. Miller, January 11, 2002
Immunity to Plasmodium falciparum in African children has been
correlated with antibodies to the P. falciparum erythrocyte mem-
brane protein 1 (PfEMP1) variant gene family expressed on the
surface of infected red cells. We immunized Aotus monkeys with
a subregion of the Malayan Camp variant antigen (MCvar1) that
mediates adhesion to the host receptor CD36 on the endothelial
surface and present data that PfEMP1 is an important target for
vaccine development. The immunization induced a high level of
protection against the homologous strain. Protection correlated
with the titer of agglutinating antibodies and occurred despite the
expression of variant copies of the gene during recurrent waves of
parasitemia. A second challenge with a different P. falciparum
strain, to which there was no agglutinating activity, showed no
protection but boosted the immune response to this region during
the infection. The level of protection and the evidence of boosting
during infection encourage further exploration of this concept for
malaria vaccine development.
C
linical immunity to malaria requires numerous and repeated
exposure to the pathogen and can take years to develop (1).
Ample evidence indicates that antibodies to the variant antigen,
Plasmodium falciparum erythrocyte membrane protein 1
(PfEMP1) (2–4), are a major component of protective immu-
nity, particularly during early childhood (5–8). Variant antigens,
however, are used by organisms to evade immunity and are not
considered as good vaccine targets to control infection (9). Vast
diversity, multiple copies, and clonal antigenic variation are the
hallmark of these variant antigens leading to variant specific
immune response (10, 11). Immunity to variants of PfEMP1 also
results from multiple antibodies specific for each variant and not
from cross-reactive epitopes (12). Why then should PfEMP1 be
considered an appropriate candidate for a malaria vaccine?
Besides its role in evasion of antibody-dependent immunity,
PfEMP1 mediates the attachment of mature parasitized eryth-
rocytes (PEs) to the host endothelium, a process that prevents
clearance of mature parasites by the spleen (13; for review, see
refs. 14 and 15). The immunodominant epitopes of PfEMP1 are
likely to be the least cross-reactive, but the diversity of functional
domains of PfEMP1 may be restricted to maintain function.
Although PEs express antigenically distinct PfEMP1s, almost all
bind to CD36 (16), a vital receptor for P. falciparum sequestra-
tion in the microvasculature (17, 18). This interaction is medi-
ated by a 179-amino acid variant fragment (179 region) of the
cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 (19).
Therefore, we immunized monkeys against this region from one
PfEMP1 to determine whether it would lead to protection from
parasite challenge, despite antigenic variation and the extensive
diversity of PfEMP1s.
Materials and Methods
Recombinant Proteins. The recombinant proteins y179 and the
Plasmodium yoelii circumsporozoite protein (yPyCSP) were
cloned into the YEpRPEU3 plasmid that supplied a C-terminal
six-histidine tag. The proteins were expressed in Saccharomyces
cerevisiae VK1 cells and purified from the supernatant by
Ni-NTA chromatography (Qiagen, Chatsworth, CA) as de-
scribed (20). Protein concentrations were determined by BCA
protein assay (Pierce). Endotoxin levels were determined by
Limulus amebocyte lysate assay (Charles River Endosafe,
Charleston, SC). The final y179 product had ⬍11 endotoxin
units兾mg compared with ⬍1.2 units兾mg for the yPyCSP.
Animals. Twenty-four spleen-intact male Aotus nancymai mon-
keys, negative for evidence of Plasmodium infection and reac-
tivity with Malayan Camp (MC) R⫹ parasites, were used. The
protocol was approved by the Institutional Animal Care and Use
Committee of the Centers for Disease Control and Prevention in
accordance with U.S. Public Health Service Policy, 1986 (pro-
tocol 1056-COL-MON-B). Starting 1 month before the vaccine
trial, animals were observed daily by trained personnel, weighed
weekly, and bled biweekly for complete blood count and serum
collection, and as needed for clinical chemistry. All observations
and laboratory values were recorded on a daily basis. Animals
were under the supervision of a resident clinical veterinarian.
Immunization. The monkeys were assigned to four trial groups of
six monkeys each taking into consideration weight through the
use of a table of random numbers for distribution to a group.
Each animal was injected with 200
g per injection of yeast
recombinant protein in an adjuvant formulation. Groups I and
II received three injections of yPyCSP and y179, respectively, on
days 0, 28, and 56, with Freund’s complete on day 0 and
incomplete adjuvant on days 28 and 56. These monkeys were
injected s.c. at four sites in the back. Groups III and IV received
four intramuscular injections in the thighs of yPyCSP or y179,
respectively, on days 0, 28, 56, and 84 with the MF59 adjuvant
(Chiron).
Challenge Infections. On day 114, each monkey was challenged
with 50,000 MC R⫹ ring-stage PEs from a donor monkey.
Abbreviations: CIDR, cysteine-rich interdomain region; CSP, circumsporozoite protein; FA,
Freund’s adjuvant; FVO, Vietnam Oak Knoll; MC, Malayan Camp; PE, parasitized erythro-
cyte; PfEMP1, P. falciparum erythrocyte membrane protein 1.
†
To whom reprint requests should be addressed. E-mail: dbaruch@niaid.nih.gov.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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Parasitemia was followed daily by quantitative Giemsa-stained
thick films according to the methodology of Earle and Perez
(21). Parasite counts were recorded as PEs兾
l of blood. Fifty-six
days after the first challenge, all monkeys were drug treated and
allowed to rest for 70 days. The monkeys were then challenged
with 25,000 Vietnam Oak Knoll (FVO) ring-stage PEs and
followed as above. Monkeys developing parasite counts higher
than 200,000 PE兾
l or developing a hematocrit lower than 20%
were cured with mefloquine (20 mg) and quinine (50 mg). Blood
was checked for subpatent parasites by nested PCR-based am-
plification (22). Samples from some of the primary and recru-
descent peaks of parasitemia were inoculated into additional
monkeys, and the resulting infected erythrocytes tested for
antigenic phenotype by agglutination as described below. In
some cases, immune sera were also collected from these
monkeys.
Measurements of Immune Responses. ELISA, agglutination, and
flow cytometry assays were performed as described (23). ELISA
was performed with glutathione S-transferase-179, y179, or
yPyCSP at 1
g兾ml by using 1:5,000 dilution alkaline phos-
phatase-conjugated goat anti-human IgG (Kirkegaard & Perry
Laboratories). Agglutination scores (0–5) were determined ac-
cording to size and number of agglutinates as described (23).
Flow cytometry was performed as described (23) with monkey
sera diluted 1:250 followed by fluorescein-labeled goat anti-
human IgG (Kirkegaard & Perry Laboratories) at 1:100 dilution.
Results are given as median fluorescence intensity.
Results
Efficacy of the Vaccines Against the
P. falciparum
MC Challenge. We
vaccinated four groups of monkeys to determine the efficacy of
the CIDR1 subdomain produced in S. cerevisiae (y179) in
protecting A. nancymai monkeys from usually lethal P. falcipa-
rum challenge. Two groups were vaccinated with y179 or a
control antigen, yPyCSP, in Freund’s adjuvant (FA), and two
were vaccinated with y179 or the control antigen in MF59, an
adjuvant used for influenza vaccination (24). We challenged the
monkeys with 50,000 Malayan Camp rosetting positive (MC R⫹)
PEs and compared the efficacy of each formulation of y179 to
its control group.
The monkeys vaccinated with y179 in FA demonstrated a very
high level of protection. None of them developed parasitemia
that required drug treatment compared with four of the six
monkeys in the FA control group that received drug treatment
(Fig. 1 and Table 1). Two monkeys of the y179兾FA group never
developed detectable parasitemia, and the peak parasitemia for
the other four monkeys were between 1,400 and 6,700 PEs兾
l,
compared with an average of 211,939 PEs兾
l (11,635–416,000)
in the control monkeys. All y179兾FA vaccinated monkeys had a
delayed onset of parasitemia except for one monkey (T794) that
rapidly suppressed the infection followed by a subsequent re-
crudescence on day 22 reaching 150 PEs兾
l. This recrudescence
as well as parasites from at least some of the monkeys having a
significant delay in primary infection expressed a variant antigen
phenotype that was antigenically distinct from the MCvar1 type
expressed by parasites in the primary peak (Fig. 2). The highly
significant delays in the prepatent period indicate that control of
parasitemia was achieved by preexisting antibodies to PfEMP1
from the vaccination. This is supported by the presence of high
PE agglutination titers in these monkeys before challenge (Table
1; see also Fig. 3). The four y179-vaccinated monkeys that had
parasitemia during the study were positive by PCR at the end of
the study, indicating persistent low grade parasitemia (Table 1).
This suggests that PfEMP1-based vaccination leads to low-grade
chronicity of the infection, and the monkeys harbored parasites
without developing detectable parasitemia.
Monkeys immunized with the y179兾MF59 formulation
showed variable degrees of protection, having higher overall
peak parasitemia and shorter prepatent periods than monkeys
Fig. 1. Parasitemia in control and immunized monkeys challenged with MC R⫹ P. falciparum parasites. (A) yPyCSP兾FA formulation (control); (B) y179兾FA
formulation; (C) yPyCSP兾MF59 formulation (control); (D) y179兾MF59 formulation. Parasitemia are given as PEs兾
l on a log scale. Drug treatments for high
parasitemia are indicated by down arrows, and appearances of secondary peak recrudescence are marked by arrowheads. ‡, Died during trial.
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immunized with y179兾FA. Five of the six monkeys controlled
their infection without treatment. The sixth monkey (T502)
unexpectedly died after rapidly reaching parasitemia of 112,000
PEs兾
l and was classified as a vaccine failure (Fig. 1 and Table
1). Another monkey, T-690, died on day 36 because of excessive
internal bleeding from splenic rupture. The onset of parasitemia
was delayed in three monkeys (T-796, T-690, and T-829) that had
the lowest primary peaks in this group; in two of them, the
primary infection was of recrudescences or a mixture of MCvar1
and recrudescent type (Fig. 2). One monkey in the control group,
T-735, had a long delay in the onset of parasitemia. This animal
developed agglutinating antibodies before challenge, which
might explain the apparent protection, but was negative for
antibodies to y179 (Fig. 1 and Table 1). We found recrudescence
in all five monkeys in the y179兾MF59 group that controlled their
initial acute parasitemia (Fig. 1 and Table 1). Interestingly, some
of the recrudescents developed into more substantial infections
than in the primary peaks. Yet, none of these recrudescences
required treatment.
Immunization Protects from Recrudescent Parasites Expressing Anti-
genically Distinct PfEMP1s.
One of our important goals was to show
that the immunization protects from recrudescent parasites
expressing variant PfEMP1. To establish this, we collected
parasites from some of the primary and secondary peaks and
tested a panel of sera for agglutination with these PEs at dilutions
ranging from 1:10 to 1:3,125 (Fig. 2A and data not shown). To
obtain sufficient amounts, parasites collected from vaccinated
animals were inoculated into naive monkeys. The rapid progres-
sion to fulminant infection that required drug treatment (data
not shown) indicated that these (recrudescent) parasites are
highly virulent, yet fully controlled by the immunized monkeys.
Parasites from control animals expressed a MCvar1 type
PfEMP1 (Fig. 2A). Contrary to that, all of the isolates from
vaccinated monkeys expressed PfEMP1s different from MCvar1
except for T-690 from day 22, which had a mixture of MCvar1
and variant PfEMP1 (Fig. 2A). These PEs were not agglutinated
by mouse anti-MC-179 sera or the monkey prechallenge sera but
were agglutinated by Aotus anti-MC hyperimmune sera. Inter-
estingly, PEs taken from peaks delayed only by 2–4 days also
expressed PfEMP1 differently from the inoculums. Our results
clearly show that immunization with y179 generated variant
transcending protection within MC strain parasites.
Antibody Response to the 179 Region Is Boosted by Exposure to MC
Rⴙ Parasites.
We analyzed the antibody response among the
various monkeys before and after challenge. We measured the
specific response to the 179 region by ELISA against recombi-
Table 1. Homologous challenge of Aotus monkeys with MC R ⴙ P. falciparum parasites
Monkey Day of patency
Peak parasitemia (day)*
Drug treatment PCR
†
(d56)
ELISA titer
‡
Agglut. titer
§
Primary Secondary Pre Post Pre Post
yPyCSP兾FA
T-193 6 416,000 (15) ⫹ ND ⬍100 ⬍100 0 298
T-487 9 212,000 (17) ⫹ ND ⬍100 ⬍100 0 269
T-505 5 312,000 (11) ⫹ ND ⬍100 ⬍100 0 12
T-515 6 11,635 (14) ⫺⫺⬍100 ⬍225 0 ⬎625
T-784 5 236,000 (14) ⫹ ND ⬍100 ⬍100 0 309
AI-2133 5 84,000 (15) ⫺⫺⬍100 ⬍100 0 625
Average 6 211,939 (14) 4兾60兾2 ⬍100 ⬍100 0 490
y179兾FA
T-221 ⬎56 0 ⫺⫺11,776 23,988 ⬎5,625 ⬎5,625
T-532 ⬎56 0 ⫺⫺11,324 12,134 ⬎5,625 ⬎5,625
T-590 22 6,660 (30) ⫺⫹18,535 19,861 ⬎5,625 ⬎5,625
T-756 16 5,580 (24) ⫺⫹45,709 36,559 ⬎5,625 ⬎5,625
T-794 7 1,350 (13) 150 (25) ⫺⫹10,914 18,493 1,537 ⬎5,625
T-1008 32 6,600 (44) ⫺⫹5,395 2,606 ⬎5,625 ⬎5,625
Average 31.5 3,365 (37) 0兾64兾6 19,346 18,880 ⬎5,625 ⬎5,625
yPyCSP兾MF59
T-450 5 272,000 (15) ⫹ ND ⬍100 ⬍100 0 ⬎625
T-682 5 112,000 (11) ⫺⫹⬍100 ⬍100 0 ⱖ625
T-735
¶
18 72,000 (31) ⫺⫺⬍100 ⬍100 ⬎125 214
T-789 6 212,000 (11) ⫹ ND ⬍100 ⬍100 0 ⱖ625
T-817 6 256,000 (10) ⫹ ND ⬍100 ⬍100 0 126
Average 8 184,800 (12) 3兾51兾2 ⬍100 ⬍100 0 533
y179兾MF59
T-488 5 74,000 (11) 150 (50) ⫺⫹117 579 625 ⬎5,625
T-502 5 112,000 (16) D
17
ND 294 298 0 25
T-690 14 1,080 (18) 20,000 (30) ⫺(D
36
) ND 671 2,089 625 ⬎5,625
T-751 4 122,170 (12) 16,920 (53) ⫺⫹670 1,303 0 3,515
T-796 17 20 (18) 16,560 (25) ⫺⫺774 565 3,858 ⬎5,625
T-829 9 667 (14) 41,632 (26) ⫺⫹1,607 8,054 1,875 ⬎5,625
Average 9 51,656 (15) 19,052 (37) 0兾53兾4 614 1,910 1,057 ⬎5,625
*Peak parasitemia of primary and secondary (recrudescence) are given as PEs兾
l. The day of peak is in parentheses.
†
Blood collected from monkeys on last day of the trial (day 56) was tested for presence of P. falciparum parasites by PCR reaction.
‡
Sera dilution that gave an OD of 0.5 by standard ELISA assay with glutathione S-transferase-179 (MC).
§
Lowest serum dilution that gave agglutination of 1⫹.
¶
Monkey T-735 spontaneously developed antibodies that agglutinated MC R ⫹ PEs but did not react with y179 about 60 days before challenge. ND, not
determined; D, died; day of death is superscript.
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nant glutathione S-transferase-179 (19) and to PfEMP1 by PEs
agglutination. The antibody response to glutathione S-
transferase-179 was positive by ELISA in all y179 immunized
animals. Agglutinating antibodies were found in 10 of the 12 y179
immunized monkeys before challenge and in all monkeys (im-
munized and control) after challenge (Table 1). The challenge
with MC R⫹ parasites boosted the antibody titer in immunized
monkeys, particularly in the y179兾 MF59 group that had initially
lower antibody titers (Table 1). Of particular note was the
absence of antibodies to the 179 region in the control groups
after infection (except for T515), although they developed
agglutinating antibodies after challenge (Table 1). This indicates
that immunity to PfEMP1 induced by infection is directed
against parts of the molecule outside the 179 region. In the
y179兾MF59 immunized group, the rise in agglutination titers was
also associated in many monkeys with increased reactivity to the
glutathione S-transferase-179 recombinant protein as measured
by ELISA (Table 1). These results indicate that the 179 region
is not only immunogenic when presented as recombinant protein
but also that vaccination makes it a target for the immune system
during the infection.
Protection Is Correlated with Antibody Response. A number of
malaria vaccine trials have shown measurable antiparasitic pro-
tection but failed to demonstrate correlation between the mea-
sured immune responses and the degree of protection (25–27).
We measured immune responses to the y179 region and PfEMP1
to determine whether they correlate with protection. In general,
we found association between antibody titers and protection.
Monkeys in the y179兾FA group had a higher degree of protec-
tion than those in the y179兾MF59 group in line with the higher
ELISA and agglutination responses of the former group (Table
1). Overall, although higher ELISA titers were not always
associated with greater protection, they positively correlated
(P ⬍ 0.01 by Spearman rank correlation) with lower parasitemia
and extended prepatent period. We found even higher correla-
tion between protection and agglutination titers (P ⬍ 0.0001),
and agglutination titers of individual monkeys were associated
with the degree of protection (Table 1 and Fig. 3). The one
monkey (T-794) in the y179兾FA group having parasites on day
7 had the lowest agglutination titer (Fig. 3A), but not ELISA
response, in the group (Table 1). The two monkeys with the
highest peak parasitemia in the y179兾MF59 group, T-502 and
T-751, had no agglutinating antibodies before challenge, al-
though the ELISA titer of T-751 was similar to other monkeys
in the group. In contrast, those that had higher agglutination
titers, particularly T-796 and T-829, had the lowest peak para-
sitemia (Table 1). Thus, agglutination titers may serve as pre-
challenge indicators for the degree of protection.
Vaccination with y179 Does Not Protect from Challenge with Heter-
ologous FVO Strain Parasites.
After demonstrating protection from
homologous MC R⫹ challenge, we tested whether the immu-
nization could protect from a highly virulent heterologous strain
expressing a variant PfEMP1 (FVOvar1). We did not observe
significant protection in any of the y179 immunized groups, and
there were no significant differences in day of patency or
Fig. 2. Parasitemia and agglutination results with PEs taken from various peaks during the MC R⫹ challenge. (A) Agglutination scores of various isolates
depicted at 1:25 sera dilution. Agglutination was scored as described (23). (B) Parasitemia courses in monkeys from the Freund’s adjuvant groups (immunized
and control) tested in A. Arrows indicate the day of challenge PEs were taken. (C) Parasitemia courses in monkeys from the MF59 adjuvant groups (immunized
and control) tested in A. Arrows indicate the day of challenge PEs were taken.
Fig. 3. Agglutination titers of y179 immunized monkeys with MC R⫹
parasites on day of MC R⫹ challenge. (A) y179兾FA immunized monkeys; (B)
y179兾MF59 immunized monkeys.
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reduction in peak parasitemia (Table 2). We attribute the
apparent protection in the control FA group to the combined
effect of the Freund’s adjuvant and the high parasite burden
experienced during the previous challenge. The difference in the
number of animals requiring treatment may indicate some
protection among y179兾MF59 immunized monkeys but also
could arise from the previous exposure to recrudescent parasites
during the first challenge. We did not find any agglutination of
FVO parasites with sera taken before the FVO challenge, and
the monkeys developed FVO-specific agglutinating antibodies
only after the challenge (Table 2). The lack of detectable
agglutinating antibodies and the rapid increase in parasitemia
can account for the lack of significant protection to this heter-
ologous challenge. An unanticipated result of this challenge was
the much higher antibody response to the FVO CIDR1 and the
FVO-179 region developed in the y179 immunized monkeys
(P ⬍ 0.018), although their agglutinating antibody titers were
lower than in control monkeys (Table 2). Thus, despite the fact
that immunization with y179 did not elicit strain transcending
protective immunity, it effectively diverted the response to this
normally silent region of PfEMP1 in a homologous and heter-
ologous challenge. These findings provide evidence that addi-
tional exposure to parasites could induce variant and strain
transcending protective immunity directed against the minimal
CD36-binding region of PfEMP1.
Discussion
Developing vaccines based on PfEMP1 may seem counterintui-
tive as these proteins evolved to evade host immunity. PfEMP1
is encoded by the large and diverse var gene family and is clonally
expressed from a set of multiple copies (50 genes) of PfEMP1
that are highly variant (2–4). Switching between var genes
(antigenic variation) can be very rapid (up to 2% per generation)
(28), and the parasite expresses new PfEMP1s that are not
recognized by antibodies raised against variants from previous
exposures (13, 29). These new variants can escape anti-PfEMP1
immunity and rapidly develop into virulent infection.
Our strategy was to choose a functional region of PfEMP1,
CIDR1, that may be conserved in structure for binding of CD36
(19) on endothelium. After immunization, the monkeys con-
trolled the primary infection and subsequently the equally
virulent recrudescent parasites that expressed antigenically dif-
ferent copies of PfEMP1. This indicates that immunization with
this region could protect against severe infection and a diverse
range of variants.
The lower immune responses and variable outcomes in ani-
mals immunized with y179兾MF59 formulation are consistent
with previous studies using MF59 as an adjuvant in Aotus
monkeys (30). Nevertheless, most of the y179兾MF59 monkeys
were protected from high parasitemia during primary infection
and recrudescence, although less than monkeys immunized with
Freund’s adjuvant. Our findings indicate that this immunogen
may be effective even when eliciting moderate antibody re-
sponses. This suggests that other adjuvants suitable for use in
humans that are less toxic than Freund’s adjuvant but more
effective than MF59 may provide high protective efficacy.
Antibodies may function to this region to induce blocking of
adhesion, agglutination of infected erythrocytes, or opsonization
of infected erythrocytes. Whatever the mechanism, immuniza-
tion with this vaccine target led to highly effective immunity.
Low grade parasitemia is unlikely to induce immunity or elicit
antibodies to other proteins such as those related to parasite
invasion. Higher initial parasitemia is usually required to protect
against a second challenge. This was evident from the higher
resistance to the FVO challenge among the Freund’s adjuvant
control animals compared with the susceptible y179 immunized
monkeys that had only low grade parasitemia (maximum of 8,700
PEs兾
l) during the MC challenge. We found a highly significant
correlation between the degree of protection and anti-PfEMP1
antibodies that agglutinated infected erythrocytes. This corre-
lation provides an important tool in developing PfEMP1-based
vaccines, as it provides a link between in vitro assays and
protection not found with other vaccine candidates (26, 27).
Thus, other constructs of PfEMP1 can be tested for inducing
broadly reactive antibodies against field isolates without the
need to challenge during the development phases of the vaccine.
Children in Africa eventually develop immunity that protects
against severe and clinical disease (1, 6, 8, 31). In the course of
developing this immunity, they experienced many infections,
including some that can be life threatening, and continue to be
routinely exposed to parasites for years (1, 31, 32). These
chronic, recurrent infections boost immunity that may recognize
new variants and is associated with antibodies that agglutinate
infected erythrocytes (5, 6, 8). The correlation between protec-
tion and agglutinating antibodies in this trial provide additional
support that such protection is associated with PfEMP1 (5).
An ideal vaccine against P. falciparum blood-stage infections
will rapidly accelerate the development of immunity and will
prevent disease without compromising the natural immunity
acquired by residents of endemic areas (1, 5, 31). Our study
indicates that var gene vaccines may do just that in that they boost
immunity without eliminating exposure to the parasite. We did
not observe sterile immunity in most monkeys. The monkeys
Table 2. Heterologous challenge of Aotus monkeys with FVO P.
falciparum parasites
Monkey
Day of
patency
Peak*
parasitemia
Drug
treatment
FACS
(median)
†
FVO-179
Agglut.
titer
‡
Pre Post Pre Post
yPyCSP兾FA
T-193 6 259,200 (14) ⫹ 0.9 18.2 0 ⬎625
T-487 7 10,908 (11) ⫺ 1.1 21.1 0 ⬎625
T-505 6 96,660 (12) ⫺ 1.2 29.9 0 ⬍125
T-515 7 515 (12) ⫺ 0.9 11.7 0 ⬍125
T-784 7 69,328 (14) ⫺ 0.7 38.1 0 ⬎625
Average 7 87,322 (13) 1兾5 1.0 23.8 0 490
y179兾FA
T-221 7 236,000 (18) ⫹ 6.9 59.6 0 ⬎625
T-532 6 48,268 (13) D
13
7.5 NA 0 NA
T-590 6 47,208 (16) D
16
8.1 53.7 0 NA
T-756 6 255,000 (16) ⫹ 63.6 159.9 0 ⬎625
T-794 6 376,000 (14) ⫹ 4.2 97.1 0 517
T-1008 8 252,000 (16) ⫹ 15.3 102.2 0 296
Average 7 202,413 (16) 4兾4 17.6 94.5 0 ⬍625
yPyCSP兾MF59
T-450 7 132,000 (16) ⫺ 28.3 55.5 0 ⬎625
T-682 7 1,091 (14) ⫺ 0.9 6.2 0 0
T-735 8 244,000 (17) ⫹ 0.8 19.4 0 ⬎625
T-789 6 376,000 (15) ⫹ 1.0 61.1 0 ⬎625
T-817 7 244,000 (14) ⫹ 0.8 42.6 0 ⬎625
Average 7 199,418 (15) 3兾5 6.4 36.9 0 ⬎625
y179兾MF59
T-488 5 384,000 (14) ⫹ 0.9 93.3 0 488
T-751 7 136,000 (15) ⫺ 3.1 85.4 0 ⬎625
T-796 7 2,727 (10) ⫺ 0.8 25.6 0 561
T-829 7 14,908 (12) ⫺ 0.9 21.4 0 ⱕ625
Average 7 134,409 (13) 1兾4 1.4 56.4 0 ⬍625
*Peak parasitemia are given as PEs兾
l. The day of peak is in parentheses.
†
Median fluorescence intensity of sera reactivity with Chinese hamster ovary
cells expressing the 179 region of the FVO CIDR1 measured by flow cytometry.
‡
Serum dilution that gave agglutination of 1⫹. NA, not applicable; D, died;
day of death is superscript.
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continued to harbor infected erythrocytes, detectable only by
PCR, but suppressed development of significant infection, par-
ticularly in those animals with higher antibody titers. This type
of immunity is typical of immune adults in regions of heavy
malaria transmission who carry the blood infection at low levels
of parasitemia without developing disease (1, 31, 32). Thus, a
PfEMP1-based vaccine apparently accelerates immunity to
other copies of PfEMP1 and may induce a young child to develop
immunity similar to that in an older child without paying the
price of frequent illness and sometimes death.
We found that although immunization with the MC R⫹ y179
region protected against variant PfEMP1s of MC parasites, it did
not elicit agglutinating antibodies against FVO parasites and
failed to protect against this strain. Overcoming this limitation
is a major concern for this type of vaccine. It is conceivable that
the rapid progression of the FVO infection, the most virulent P.
falciparum strain in Aotus monkeys, did not allow sufficient time
for effective immune responses to be mounted before the
animals succumb to the infection. Thus, protection may be
evident in a less virulent challenge or whether the proliferation
of the parasite is attenuated by combining y179 with other
vaccine candidates. Another possibility is that after immuniza-
tion, further exposure to a limited number of infections will
induce and facilitate the development of clinical protection. This
is suggested by the boost in the antibody response to the
respective 179 region by exposure to PEs expressing homologous
and heterologous CIDR1 and was not observed in monkeys
without y179 immunization (19). It is possible that this critical
region is cryptic because of immunodominant variant epitopes in
other regions of PfEMP1 (19, 23, 33). We provide data that
immunization with y179 overcomes its cryptic nature and can
lead to ‘‘determinant spreading’’ with subsequent P. falciparum
infections (34).
Vaccines against blood stages of P. falciparum are in the early
stages of development and field testing. Our approach to attack
a functionally critical region of the variant antigen is a first step
in directing immunity toward a molecule that is exposed to
antibody for much of the parasite cycle and plays an important
role in clinical immunity. Importantly, we have shown that
antibodies to CIDR1 will protect against challenge and that this
immunity spreads to other var genes. The objective of this
approach is not to eliminate the parasite but instead to lead to
low grade, asymptomatic parasitemia while boosting broad
protection. We face many difficulties and challenges along the
way, but whether we are able to develop immunogens that induce
immunity to multiple CIDR1s and accelerate spreading of the
immunity to that domain during natural infection, it will become
an important component in future malaria vaccines.
We thank Carter Diggs, U.S. Agency for International Development
(USAID) Malaria Vaccine Development Program, for his support and
Chiron for providing us the MF59 adjuvant. This work was supported in
part by USAID Interagency Agreement No. 936-6001 between the
Division of Parasitic Diseases, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, and the U.S. Agency for
International Development.
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MEDICAL SCIENCES
