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*
Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010;
Transplantation Immunology Unit,
Department of Community Medicine, and
Central Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland; and
¶ Department of Medical Biochemistry, University Medical Center, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first clinical trial demonstrating that synthetic peptides could elicit functional anti-sporozoite Abs in humans used a peptide-protein conjugate, consisting of the Plasmodium falciparum CS repeat B cell epitope, (NANP)3, conjugated to tetanus toxoid as a carrier (2). Second generation peptide vaccines, termed multiple Ag peptides (MAP), have been developed in which the relevant T and B cell epitopes are synthesized on a branched lysine core, eliminating the requirement for a foreign protein carrier (3). MAPs containing CS repeat epitopes induced high levels of anti-sporozoite Abs and protective immunity in the murine malarias, Plasmodium berghei and Plasmodium yoelii (4, 5), and in a Saimiri monkey/Plasmodium vivax primate malaria model (6).
We have recently completed phase I clinical trials to assess the safety
and immunogenicity of a P. falciparum MAP vaccine in
volunteers of defined class II genotypes (7). This
vaccine, termed (T1B)4 MAP, contains the
(NANP)3 B cell epitope synthesized in tandem with
a repeat region Th cell epitope, T1, that was originally defined using
CD4+ T cells of a P. falciparum
sporozoite-immunized volunteer (8). Immunization of
volunteers with the (T1B)4 MAP vaccine elicited
high anti-sporozoite Ab titers, comparable to those obtained in
volunteers immunized by multiple exposures to the bites of hundreds of
irradiated P. falciparum-infected mosquitoes (7, 9). However, this high responder phenotype was genetically
restricted to three HLA class II genotypes (DRB1*0401, *1101, and
DQB1*0603), which are found in 
25–35% of the population, depending
on ethnic background (7).
Effective immunoprophylaxis against malaria requires a vaccine that can elicit parasite-specific humoral and cell-mediated immunity in individuals of diverse HLA types. The ideal Th epitope would be "universal," that is capable of interacting with a large number, if not all, class II molecules. To identify a parasite-derived universal T cell epitope, we used CD4+ T cell clones derived from several volunteers immunized with P. falciparum sporozoites who were protected against sporozoite challenge (9, 10, 11). These human T cell clones recognized an epitope located in the C terminus of the CS protein that was restricted by a broad range of class II molecules (11). Peptides containing this epitope sequence, designated T*, also bound with high affinity to multiple DR and DQ molecules in vitro and thus was predicted to be a potential universal T cell epitope (12). Consistent with this prediction, MAPs containing the T* peptide were found to elicit Th cells in mice of diverse genetic backgrounds (12).
The ability to construct a triepitope MAP, containing this universal T*
epitope in combination with the T1B repeats, was limited by the
step-wise solid-phase synthesis used to construct MAPs. This limitation
was overcome by chemoselective oxime ligation, in which reciprocally
modified peptide modules, consisting of the epitopic peptide and a
branched core, are first synthesized and purified before linkage via
oxime bonds (13). A malaria (T1BT*)4
polyoxime vaccine was constructed using a branched core modified
to contain the lipopeptide palmitoyl-S-glyceryl cysteine
(P3C), to function as an endogenous adjuvant (Refs. 14 and
15 ; Fig. 1
). This totally
synthetic vaccine formulation was of sufficient homogeneity for
chemical characterization by mass spectrometry (16).
Polyoxime constructs containing malaria or flu epitopes were found to
be highly immunogenic in mice (16, 17), but the
immunogenicity of these polyoxime synthetic peptides in humans had not
yet been determined. An open-label phase 1 trial was designed to assay
the safety and immunogenicity of the triepitope
(T1BT*)4-P3C polyoxime vaccine in 10 volunteers
of diverse HLA types.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten healthy male and female volunteers (19–29 years old) were
recruited at the University of Geneva in Geneva, Switzerland. Medical
history, physical examination, and routine laboratory tests (complete
blood count, serum chemistries, electrocardiogram, and urinalysis) were
obtained to exclude individuals with cardiovascular, hepatic, or renal
functional abnormalities or past malaria infection. Serology for
hepatitis B surface Ag and HIV-1 and -2 Abs was used to rule out
volunteers with hepatitis or HIV infection. Women were tested for
pregnancy by measuring urinary 
-hCG. Informed consent was obtained
from all volunteers before admission into the study, which was approved
by the ethics Committee of the Geneva University Hospital.
The 10 volunteers were typed for class I and II HLA haplotypes (Table I). The class II genotypes were
determined by PCR/sequence-specific oligonucleotide probes and
PCR/sequence-specific primers (PCR-SSP) methodologies. DNA extracted
from Ficoll-purified PBL from each volunteer was amplified by PCR using
primer pairs specific for exon 2 of DRB1/B3/B4/B5 and DQB1 class II
loci, respectively. The PCR products were analyzed in a
microtiter-plate oligotyping assay (18) using HLA-DRB and
DQB-oligodetection kits that were generously supplied by Dr. B. Mougin
(BioMerieux, Lyon, France). HLA-DRB1 allelic subtypes of DR4, DR8,
DR11, DR13-DR16 were determined by PCR-SSP using commercial kits
(Dynal). HLA-A and -B class I Ags were first typed by serology using
Ficoll-purified PBL in a standard microlymphocytotoxicity assay, and
then by low resolution PCR-SSP (12th International Histocompatibility
Workshop, Dynal or Biotest kits). HLA-A2 subtypes were determined by
PCR/sequence-specific oligonucleotide probes as described
(19).
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The polyoxime construct (T1BT*)4-P3C
contained the repeat T and B cell epitopes, T1B, in combination with a
universal T cell epitope, T*, of the P. falciparum (NF54
strain) CS protein (Fig. 1
). The T* epitope, representing the
C-terminal aa 326–345 (EYLNKIQNSLSTEWSPCSVT), was originally
identified using CD4+ T cells derived from
sporozoite-immunized volunteers (10, 11). These human T
cell clones recognized the peptide in the context of DR 1,4, 7, and 9
class II molecules (11) and the T* peptide bound to the
corresponding soluble DR 1, 4, and 7 class II molecules in vitro
(12). In addition, the T* peptide also bound to DR 2, 3,
8, 11, and 13 and to DQ 7 and DQ 9 soluble class II molecules, as well
as to cells expressing DRB1*1501 (12), suggesting the
potential of T* to function as a universal T cell epitope in
vivo.
The (T1BT*)4-P3C polyoxime vaccine contains a
48-mer malaria sequence
[(DPNANPNV)2(NANP)3
EYLNKIQNSLSTEWSPCSVT] in each of the four branches (Fig. 1). The core
was modified to contain the synthetic lipopeptide
N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl)
(P3C), to function as an endogenous adjuvant (14, 15).
Each of the functional peptide components (i.e., the 48-mer malaria
epitopic module and the lipopeptide-modified tetrabranched core) were
purified and fully characterized by HPLC and mass spectrometry before
assembly by oxime bond ligation, thus ensuring a synthetic product of
high purity and defined composition (16).
Briefly, for construction of the polyoxime, the terminal lysines on each branch of the lipopeptide core module were modified with aminooxyacetyl groups (NH2OCH2CO). The 48-mer epitopic module was synthesized to contain Ser on a C-terminal Lys side chain, which was oxidized with periodate to create the reciprocal aldehyde group (O=CH-CO)-NH-. Spontaneous oxime bond formation (-COCH=NOCH2CO) was initiated in aqueous solution at pH 4 by mixing the aldehydic peptide derivatives in a 4-fold excess over the aminooxyacetyl-modified core. After 16 h at room temperature, the tetra-oxime was isolated as a single peak by HPLC. Matrix-assisted laser desorption- time of flight mass spectrometry gave an experimental molecular mass of 23,936 (±60), in agreement with the calculated mass of 23,973, as previously found in the small-scale batches of (T1BT*)4-P3C polyoximes produced for preclinical studies (16). The polyoxime migrated as a single major band on tricine SDS-PAGE, and this band stained with a mAb specific for P. falciparum CS repeats (mAb 2A10) in a Western blot (data not shown). The vaccine preparation was sterilized by ultrafiltration and was negative when tested in endotoxin and bacteriological assays.
Study design
The study was an open-label trial to assess the safety and immunogenicity of (T1BT*)4-P3C in volunteers of known HLA class I and class II haplotypes. Vaccinees received 1 mg of (T1BT*)4-P3C administered s.c. in the deltoid area on days 0, 28, and 84. After each injection of vaccine, volunteers were observed for 60 min and examined for local and systemic side effects by a physician. An interval history of axillary temperature and systemic and local reactions following each vaccination were recorded by each volunteer.
CBC, serum chemistries and urinalysis were determined immediately
before each immunization and at 2 wk after each vaccine dose. Blood
samples for serology and T cell assays were drawn at the same time
points. Additional samples were obtained 1, 2, 5, and 10 mo after
the third and final dose of vaccine to measure persistence of the Ab
and cellular immune responses.
Serological assays
Peptide-specific Ab titers were measured by ELISA using peroxidase-labeled Abs specific for IgM or IgG (Cappel, West Chester, PA) and IgE (Hybridoma Research Lab, Baltimore, MD). Qualitative determination of IgG subgroups, IgG1 through IgG4, was obtained by ELISA using sera diluted 1:100 and enzyme-labeled murine mAb specific for each human IgG subgroup (Southern Biotechnology Associates, Birmingham, AL).
ELISA were conducted using 96-well plates coated with the
(T1BT*)4 polyoxime or the corresponding
triepitope MAP construct. The fine specificity of the Ab response was
determined using the T1B repeats or universal T* peptide as Ag in the
ELISA. Following incubation for 1 h with 2-fold dilutions of sera
in PBS/0.05% Tween/2.5% BSA, and washing, the presence of bound Ig
was detected by incubation with peroxidase-labeled anti-IgG or IgM
Abs followed by the addition of
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD). The end point
titer was defined as the dilution of sera giving a 
OD (OD
peptide-coated wells - BSA-coated wells) that was greater than
the mean + 2 SD obtained with day 0 sera. IgE titers were determined
using a murine mAb specific for human IgE, followed by sequential
incubations with biotinylated anti-murine IgG (Vector Laboratories,
Burlingame, CA), HRP-labeled strepavidin (Calbiochem, La Jolla, CA),
and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) substrate.
An indirect immunofluorescence assay (IFA) was conducted using P. falciparum (NF54 isolate) sporozoites, dissected from the salivary glands of infected Anopheles mosquitoes. The slides containing air-dried sporozoites were incubated with 2-fold dilutions of sera, followed by FITC-labeled anti-human IgG or IgM (Kierkegaard & Perry Laboratories) diluted in PBS/0.4% Evans blue.
Reactivity with viable P. falciparum sporozoites was assayed using the CS precipitin (CSP) assay (20, 21). Twofold dilutions of sera, obtained 20 days after the final immunization, were mixed with viable P. falciparum sporozoites and incubated for 45 min at 37°C. The endpoint titer was the last serum dilution that could elicit a positive terminal precipitate visible by phase microscopy, the CSP reaction, in 2/20 sporozoites.
Cellular assays
Peptide-specific T cell responses were measured by proliferation
and cytokine assays using PBL obtained at various time points during
immunization. Ficoll-purified PBL were incubated in triplicate wells
with medium, or with 10-fold dilutions of polyoximes in RPMI 1640/10%
human serum starting at 1 µM concentration. The triepitope
(T1BT*)4 immunogen, as well as polyoximes
representing the individual epitopes, (T1)4,
(B)4, or (T*)4, were tested
in each assay. Positive controls included T cell mitogens PHA (Difco,
Detroit, MI) or PWM (Life Technologies, Grand Island, NY) and a control
recall Ag, tetanus toxoid (kindly provided by Wyeth-Ayerst
Laboratories, Marietta, PA). For the proliferation assays, cultures
received 10 U/ml IL-2 on day 7 and were pulsed on day 9 with 1 µCi
[3H]Tdr for 18 h before harvesting and
scintillation counting. Significant responses were taken as >1100 
cpm (cpm in cultures stimulated with peptide - cpm in cultures
without peptide), representing the mean + 2 SD of responses obtained
with preimmune PBL from the 10 volunteers.
T cell lines (TCL) were established using PBL depleted of
CD8+ T cells by magnetic cell sorting (Miltenyi
Biotec, Auburn, CA). The purified CD4+ T cells
were expanded in vitro by stimulation with 4 µM T1BT* linear peptide
for 7 days followed by the addition of fresh medium containing 100 U/ml
IL-2 at 3- to 4-day intervals. After 3 wk in vitro, the TCL were
assayed for proliferation and lymphokine production in response to
stimulation with 4 µM T1BT*, or equimolar concentrations of the
monoepitope peptides containing T1, B, or T*, using irradiated
autologous EBV-immortalized B cells as APCs.
IL-2 assays were conducted using PBL culture supernatants collected at
72 h, or TCL culture supernatants obtained 24 h after peptide
stimulation. The amount of IL-2 was measured by bioassay using an
IL-2-dependent T cell line (10, 11). Stimulation indices
(SI) were calculated as follows: cpm induced by supernatants from
peptide stimulated cultures/cpm induced by supernatants from medium
only cultures. SI > 3.2 were considered positive for the
triepitope peptide and SI > 2.5 positive for the mono-epitope
peptides (corresponding to 35 mU/ml rIL-2 standard). The IFN-
levels
were measured in TCL supernatants collected at 48 h using a
commercial ELISA (R&D Systems, Minneapolis, MN).
CD8+ T cells were assayed by IFN- ELISPOT
using target cells pulsed with an HLA-A*0201-restricted CTL epitope of
the CS protein (22, 23). Purified
CD8+ T cells were isolated by magnetic cell
sorting (Miltenyi Biotec) of cryopreserved PBL from five HLA-A*0201
volunteers, obtained 2 wk after the second or third dose of vaccine.
The cells were expanded by in vitro culture with a 9-mer
HLA-A*0201-restricted CD8+ CTL epitope, which is
present within the universal T* peptide sequence (23). As
a positive control, parallel cultures were stimulated with a
HLA-A*0201-restricted 9-mer peptide derived from influenza
hemagglutinin (24). After 7–10 days of in vitro
expansion, varying concentrations of CD8+
effector cells were cocultured with 104
HLA-A*0201-positive T2 target cells, pulsed with or without the CS or
flu peptides. After overnight incubation of the cells in
anti-IFN- mAb-coated nitrocellulose microtiter wells, lymphokine
secretion was detected by staining the washed wells with
peroxidase-labeled anti-IFN-, followed by
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazoluim substrate
(Kirkegaard & Perry). The number of spots in triplicate wells was
counted and the results were expressed as mean number of
CD8+ IFN--secreting
cells/105 cells. Responses were considered
significant if this number was at least 2-fold greater than that
obtained with the negative control at the cell concentration
used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The (T1BT*)4-P3C vaccine was well tolerated
and elicited minimal local and systemic reactions in the majority of
volunteers (Table II). None of the
vaccinees developed fever (defined as axillary temperature 
37.5°C) and laboratory safety parameters remained within normal
ranges. Systemic side effects included mild to moderate fatigue, mild
arthralgia, headache, and nausea, which resolved spontaneously without
treatment. One individual (volunteer no. 7) had an increase of
C-reactive protein with intercurrent streptococcal angina on day 7,
unrelated to the vaccine.
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Humoral Immune Response
Polyoxime elicits Ab responses in individuals of diverse HLA.
Following three immunizations with (T1BT*)4-P3C, all of the
volunteers developed detectable levels of IgG Abs reactive with the
triepitope immunogen (Fig. 2). The Ab
titers ranged from 160 to 20,240, with peak anti-peptide titers
exceeding 103–104 in the majority (8/10) of
the vaccinees.
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, 
), which had been shown to function in T1- or T*- specific
CD4+ T cell responses in P. falciparum
sporozoite-immunized volunteers (11, 12). The IgG
geometric mean titer (GMT) in the five volunteers who expressed these
known class II responder alleles was not significantly different from
the titers of five vaccinees expressing HLA molecules of unknown in
vivo function (p = 0.8; Fig. 2, 
). Consistent
with the broad spectrum of T* binding to soluble HLA class II
molecules, all of the volunteers in this latter group expressed at
least one class II molecule that was predicted to function as a
restriction element based on peptide binding studies in vitro
(12).
Isotypes and kinetics of anti-polyoxime response.
The majority of the volunteers seroconverted following a single dose of
the (T1BT*)4-P3C vaccine. Volunteers (8/10) had
peptide-specific IgM Abs (GMT 320) and 9/10 volunteers had IgG (GMT
296) 1 mo after the first immunization. Following a booster
inoculation, all of the volunteers were seropositive with IgM GMT 529
and IgG GMT 1670 (Fig. 3). Ab titers
declined 2- to 4-fold over a 2-mo interval. Administration of a third
dose of vaccine at this time point (day 84), lead to a rapid increase
in Ab titers, which reached IgM GMT of 802 and IgG GMT 2605. The
peptide-specific Ab levels decreased over the 10-mo period following
the final immunization, falling to an IgG GMT 137 on day 373 when the
study was terminated.
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).
Volunteers with the highest IgG titers also had increased levels of
IgG2 and IgG4 peptide-specific Abs. The two volunteers with the lowest
Ab titer, volunteers no. 03 and no. 06, had only IgG1 subtype Abs
detectable.
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Fine specificity of Ab response.
Peptides representing the individual B or T cell epitopes contained in
the vaccine (i.e., the T1B repeats or the T* universal T cell epitope)
were used to determine the fine specificity of the anti-peptide Ab
response (Table III). For all sera, the
titers to the (T1B) repeats were within a 4-fold dilution of the Ab
titer against the entire triepitope (T1BT*)4 immunogen,
indicating the repeats contained the immunodominant B cell epitope. In
contrast to the anti-repeat response, little or no reactivity was
detected against the T* epitope, with low titers measured in only five
volunteers (GMT 320). The polyoxime core sequence also was not
immunogenic as comparable ELISA titers were obtained using a
(T1BT*)4 MAP, which differed in the structure and amino
acid composition of the branched core (data not shown). Moreover, no
positive reactions were observed when the lipidated Pam3Cys-modified
core was used as Ag in the ELISA (data not shown).
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). 8/10 volunteers
seropositive for anti-repeat Abs (GMT 1114) were also positive by
IFA conducted using air-dried P. falciparum sporozoites
(GMT 830). However, volunteer no. 07, despite good titers to the
peptide immunogen, reacted less well with the repeat peptide and poorly
with P. falciparum sporozoites. Volunteers no. 03 and
no. 06, the two individuals with the lowest anti-repeat ELISA
titers 160–320(160–320), did not have detectable IFA reactions (<80).
The biological relevance of the peptide-induced Abs was also
demonstrated using viable P. falciparum sporozoites.
When tested in the CSP assay, 7/10 volunteers with highest
anti-repeat ELISA titers gave positive CSP reactions, at sera
dilutions of 1:5–1:10 (Table III). Consistent with the requirement for
high levels of Ab to induce the terminal precipitin reaction on the
viable parasite, the sera of volunteers with low or negative IFA titers
were CSP negative (volunteers no. 03, no. 06, and no. 07).
Cellular immunity
PBL proliferation and IL-2 production.
Proliferative responses to the (T1BT*)4 peptide were
detectable after multiple inoculations of the vaccine. None of the PBL
obtained after the first immunization (day 14), and 1/10 after the
second dose of vaccine (day 42), proliferated in response to peptide
stimulation (Fig. 5). At 2–8 wk after
the third and final dose of vaccine (days 98, 112, and 150), the PBL of
the majority of volunteers (7/10) proliferated in response to
(T1BT*)4 peptide. When retested 10 mo after the third dose
(day 373), PBL of 57% (4/7) of these volunteers still had
peptide-specific T cell responses detectable in the proliferation
assay.
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). The fine specificity of the cellular
response was determined using supernatants from cultures stimulated
with monoepitope peptides, (T)4, (B)4, or
(T*)4. PBL of the majority of volunteers produced
lymphokine only in response to the T* peptide (SI range, 4.5–21.5).
IL-2 was not detected in supernatants from cultures stimulated with the
T1 or B cell peptides (mean SI for T1 peptide 1.3 ± 0.4; for B
peptide 1.0 ± 0.2). Volunteers no. 03 and no. 07 did not produce
significant IL-2 in response to any peptide, consistent with the lack
of proliferative response. The PBL of these volunteers gave normal
responses to PHA and PWM in all of the assays.
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CD4+ TCL.
To further analyze the T cell responses, CD4+ TCL were
established from five volunteers by expanding PBL using a single cycle
of (T1BT*) peptide stimulation in vitro. TCL that specifically
recognized the (T1BT*) peptide were obtained from 4/5 volunteers,
including individuals with low PBL responses, such as volunteer no. 15
(Table IV). Consistent with the fine
specificity noted with PBL, the CD4+ TCL proliferated
and/or produced IL-2 when stimulated with the T* peptide, but not the
repeat, T1, or B peptides (data not shown). TCL from volunteer no. 07
did not proliferate or produce lymphokine in response to any of the
peptides, consistent with the lack of response noted with bulk PBL
cultures.
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, a potent inhibitor of intracellular hepatic
stages of the parasite (25). All of the CD4+
TCL, with the exception of volunteer no. 07, produced IFN- in
response to T1BT* and/or the T* peptide, suggesting a Th1- or Th0-type
cellular response. Further analysis of the cytokines and fine
specificity of cellular responses of the polyoxime-immunized volunteers
is in progress using peptide-specific T cell clones (J.M.C.-C.,
manuscript in preparation). CD8+ T cells. The T* epitope contains, in addition to a Th cell epitope, an HLA-A*0201-restricted CD8+ CTL epitope that is recognized by cells of peptide-immunized mice and naturally infected individuals (23). The P3C lipopeptide adjuvant has been shown to enhance CD8+ T cell responses in peptide-immunized murine and primate hosts (26, 27).
To determine whether malaria-specific CD8+ T cells were
elicited by immunization with (T1BT*)4-P3C,
CD8+ T cells were isolated from PBL of five
HLA-A*0201-positive vaccinees. The purified CD8+ T cells
were expanded in vitro by coculture with a 9-mer peptide containing the
CD8+ CTL epitope, or a control peptide from influenza
hemmagglutinin, and tested in a IFN- ELISPOT assay. In the control
cultures expanded with the flu peptide, CD8+ T cells of all
five HLA-A*0201 volunteers produced IFN- when assayed using
HLA-A*0201 target cells pulsed with flu peptide (mean 2472
spots/105 cells). An additional cycle of in vitro peptide
expansion further increased the levels of flu-specific CD8+
T cells (> 4000 spots/105 cells). In contrast, despite
multiple cycles of in vitro expansion with the malaria CS peptide,
CD8+ T cells of the five HLA-A*0201 volunteers failed to
produce positive ELISPOT when tested against malaria peptide-pulsed
target cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All 10 volunteers, of diverse HLA haplotypes, seroconverted following
two doses of the (T1BT*)4-P3C polyoxime vaccine
(Fig. 2). The majority of Abs elicited in the vaccinees specifically
recognized the CS repeats (Table III). The immunodominance of the CS
repeat B cell epitope, and absence of significant responses to the
nonrepeat T* helper epitope, is consistent with the fine specificity of
the Ab responses elicited by immunization with P. falciparum
sporozoites in human volunteers and murine hosts (1). Of
particular importance was the ability of the peptide-induced Abs to
react with P. falciparum sporozoites. Sera of the seven
volunteers with high anti-repeat Ab titers all had positive IFA
titers and CSP reactions. The CSP reaction effectively immobilizes the
sporozoite (28), thereby inhibiting invasion of the
hepatocytes, which is dependent on parasite motility (29).
High levels of Abs reactive with the sporozoite surface may also
function by sterically blocking host cell receptor interactions
mediated by the CS ligand (30).
The 100% seroconversion of the polyoxime vaccinees is most likely due to inclusion of the parasite-derived T* epitope. The T* peptide functions as a Th cell epitope in 8/8 inbred strains of mice and can bind to a broad range of HLA class II molecules in vitro (12). These murine and in vitro studies suggested that the T* epitope could function as a universal T cell epitope in humans. These predictions were confirmed in the present study, as high Ab titers were detectable in the majority of volunteers (7/10) of diverse HLA types following immunization with the (T1BT*)4-P3C vaccine. In contrast, a (T1B)4 MAP, which contains the same CS repeat epitopes but lacks the universal T* epitope, could elicit high Ab titers in only limited class II genotypes (DRB1*0401, *1101, and DQB1*0603; Ref. 7).
The suggestion that the T * epitope was functioning as a universal Th
epitope in the polyoxime-immunized volunteers is also supported by the
correlation of Ab and cellular responses. T*-specific proliferation,
IL-2 and IFN- production were observed in PBL and/or TCL derived
from all volunteers who developed high levels of anti-parasite Abs.
The inability to detect cells of this specificity before immunization
indicate that these T*-specific cellular responses were induced by the
polyoxime vaccine.
In contrast to the T* epitope, significant cellular responses to T1, a second malaria Th epitope contained in the vaccine, were not detected using either bulk PBL or TCL. This was despite the fact that two volunteers, no. 05 and no. 09, were of the high responder genotype for the T1 epitope (DQB1*0603 and DR *0401, respectively). In contrast, CD4+ T cells specific for the T1 helper epitope were detected in volunteers of these genotypes following immunization with (T1B)4 MAP (Ref. 7 ; G.A.O., manuscript in preparation).
These findings indicate that T* was the immunodominant T cell epitope within the (T1BT*)4 polyoxime vaccine. Although the mechanisms controlling immunodominance remain to be defined (31), it is noteworthy that the HLA binding affinity of the T* epitope was significantly higher than that of the T1 peptide, as measured by peptide binding to soluble class II molecules (12). The absence of detectable T1-specific T cells in the polyoxime-immunized volunteers expressing T1 responder genotypes (DRB1*0401 and DQ603) suggest that peptide competition may occur in vivo, as has been found in vitro (J.M.C.-C., unpublished observations). The correlation of broad spectrum T* peptide HLA binding in vitro and high levels of immunogenicity of the (T1BT*)4 polyoxime in vivo support the use of computer-driven algorithms that predict T cell epitopes with high affinity for multiple class II molecules to identify potential T cell epitopes for inclusion in peptide vaccines (32).
Although peptide vaccines can be engineered to include universal epitopes from bacterial or viral proteins or artificial universal epitopes (5, 6), these foreign epitopes are not functionally equivalent to a parasite-derived universal T cell epitope. The inclusion of T* in a malaria vaccine provides the potential to elicit anamnestic responses in individuals living in endemic areas, and to maintain vaccine induced responses following exposure to the bites of infected mosquitoes. The (T1BT*)4 polyoxime induced strong anamnestic responses (250-fold increase in titer) when injected in mice primed with P. falciparum sporozoites (J.M.C.-C., unpublished observations).
In addition to the requirement for universal Th epitopes, other factors
may play a role in determining the magnitude of the Ab response to CS
peptide vaccines. It is noteworthy that volunteers no. 03 and no. 07,
who had low anti-sporozoite Ab titers and no detectable
peptide-specific PBL or TCL responses, were heterozygous for the
DR*0701 allele. Similarly, in our recent phase I MAP trial, individuals
of high responder genotypes who were heterozygous for DR7 also failed
to develop the expected high levels of Ab and T cell responses
(7). Poor humoral responses following immunization with
hepatitis and measles vaccines have also been noted in DR7
heterozygotes (33, 34). However, one of the three
DR7-positive polyoxime vaccinees, volunteer no. 10, developed high
levels of Ab (Fig. 2), as well as strong cellular responses to the
immunogen and the T* epitope (Figs. 5 and 6). The comparative analysis
of DR7-linked genes expressed in low vs high responder haplotypes may
help identify additional genetic mechanisms that function in
immunoregulation.
The identification of regulatory cell populations would facilitate the rationale design of vaccines by ensuring that only functional T and B cell epitopes are included in peptide vaccines. However, such minimal peptide epitopes lack the nonspecific immunostimulatory properties of more complex recombinant protein or viral subunit vaccines and thus peptide vaccines require potent adjuvants to elicit optimal immune responses. In the current trial, the lipophilic adjuvant P3C, which can enhance peptide-induced Ab as well as CD4+ and CD8+ T cell responses (14, 17), was tested as a synthetic endogenous adjuvant.
The inclusion of the P3C adjuvant may have helped to broaden the range
of IgG subgroups elicited by the vaccine. The majority of the
volunteers developed IgG1 and IgG3 anti-peptide Abs, and IgG4 Abs
were obtained in 50% of these vaccinees (Fig. 4), suggesting T cell
help was provided by both Th1 and Th2 subsets. In contrast, the
(T1B)4 MAP formulated with alum and QS21 as
coadjuvants, elicited IgG1 and IgG3 anti-repeat Abs with little or
no IgG4, suggesting a predominantly Th1 response (7). To
directly determine the T cell subset(s) induced by the lipidated
polyoxime, the cytokine profiles of peptide-specific T cell clones are
being analyzed.
The inclusion of the P3C adjuvant also provided, in principle, the
potential to elicit class I-restricted T cells. However,
malaria-specific CD8+ T cells were not detectable
in the five HLA-A*0201-positive vaccinees when assayed by IFN-
ELISPOT. Whether CD8+ T cells are required for
protection of man against P. falciparum sporozoite-induced
infection is currently unknown. Although there is ample evidence that
CD8+ T cells are protective in the experimental
murine model, additional studies have shown that sterile immunity
against sporozoites can also be obtained in the absence of
CD8+ T cells (35). High levels of
anti-repeat Abs alone can protect against sporozoite challenge in
MAP-immunized mice and monkeys (5, 6). In the murine
model, peptide-induced protective immunity against sporozoite challenge
can also be mediated by IFN- produced by CD4+
T cells (36).
Additional support for the role of class II-restricted responses in
protection has also been provided by recent phase I/II studies of a
recombinant CS/hepatitis B surface Ag protein, termed RTS,S
(37). High levels of CS-specific Abs and IFN- producing
CD4+ T cells could be detected in volunteers
protected against challenge with P. falciparum sporozoites,
in the absence of malaria-specific CD8+ T cells
(37, 38). Of particular relevance to vaccine design,
protection in that study was dependent on a combination of strong
adjuvants, consisting of a mixture of MPL and QS21 in an oil-in-water
emulsion (37). Although encouraging results were obtained
with the P3C adjuvant in the current trial, it is expected that peptide
vaccines will also require more potent adjuvants to elicit optimal
immune responses.
The use of multiepitope polyoximes as immunogens can overcome the limitations of chemical synthesis and genetic restrictions that have hindered the development of synthetic peptide malaria vaccines. The results of this phase I trial demonstrate the immunogenic potential of polyoximes in humans and support further studies to develop precisely defined, broadly applicable synthetic peptide vaccines for malaria immunoprophylaxis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Elizabeth Nardin, New York University School of Medicine, Department of Medical and Molecular Parasitology, 341 East 25th Street, New York, NY 10010.
3 Abbreviations used in this paper: CS, circumsporozoite; CSP, CS precipitin; SI, stimulation indices; MAP, multiple Ag peptides; PCR-SSP, PCR/sequence-specific primers; IFA, immunofluorescence assay; TCL, T cell lines; GMT, geometric mean titer.
Received for publication March 22, 2000. Accepted for publication September 29, 2000.
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) and IgG (
) Ab response to
(T1BT*)4 measured in sera of the polyoxime-immunized
volunteers. ELISA results are expressed as GMT for the 10 volunteers.
The arrows indicate days of immunization.
