Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe, elicits high titer antibody and induces IFN-gamma/IL-2 CD4+ T cells but does not protect against experimental Plasmodium falciparum infection

Article (PDF Available)inVaccine 28(31):5135-44 · October 2009with171 Reads
DOI: 10.1016/j.vaccine.2009.08.046 · Source: PubMed
Abstract
Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region termed "LSA-NRC." We describe the first Phase I/II study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system. We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10microg LSA-NRC/0.5ml AS01 (n=5), high dose (HD) LSA-NRC/AS01: 50microg LSA-NRC/0.5ml AS01 (n=13); LD LSA-NRC/AS02: 10microg LSA-NRC/0.5ml AS02 (n=5) and HD LSA-NRC/AS02: 50microg LSA-NRC/0.5ml AS02 (n=13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity controls underwent experimental malaria sporozoite challenge. The vaccines showed a reassuring safety profile but were moderately reactogenic. There were no serious adverse events. All subjects seroconverted after the first immunization. Following the second immunization, LSA-1-specific CD4+ T cells producing two cytokines (IL-2 and IFN-gamma) were found by intra-cellular staining in all subjects in the LD LSA-NRC/AS01B group and in 3 of 5 subjects in the LD LSA-NRC/AS02 group. In contrast, the HD LSA-NRC/AS01 and HD LSA-NRC/AS02 group subjects had fewer LSA-1-specific CD4+ T cells, and minimal to no IFN-gamma responses. There was no increase in LSA-1-specific CD8+ T cells found in any group. Per protocol, 22 high dose vaccinees, but no low dose vaccinees, underwent P. falciparum homologous malaria challenge (3D7 clone). All vaccinees became parasitemic and there was no delay in their pre-patent period versus controls (p=0.95). LSA-NRC/AS01 and LSA-NRC/AS02 elicited antigen-specific antibody and CD4+ T cell responses, but elicited no protective immunity. Although the optimal antigen dose of LSA-NRC may not have been selected for the challenge portion of the protocol, further vaccine development based upon LSA-1 should not be excluded and should include alternative vaccine platforms able to elicit additional effector mechanisms such as CD8+ T cells.
Vaccine 28 (2010) 5135–5144
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Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Recombinant Liver Stage Antigen-1 (LSA-1) formulated with AS01 or AS02 is safe,
elicits high titer antibody and induces IFN-/IL-2 CD4+ T cells but does not
protect against experimental Plasmodium falciparum infection
James F. Cummings
a,
, Michele D. Spring
a
, Robert J. Schwenk
a
, Christian F. Ockenhouse
a
,
Kent E. Kester
a
, Mark E. Polhemus
a
, Douglas S. Walsh
a
, In-Kyu Yoon
a,6
, Christine Prosperi
a
,
Laure Y. Juompan
a
, David E. Lanar
a
, Urszula Krzych
a
, B. Ted Hall
a
, Lisa A. Ware
a
, V. Ann Stewart
a,5
,
Jack Williams
a
, Megan Dowler
a
, Robin K. Nielsen
a
, Collette J. Hillier
a,4
, Birgitte K. Giersing
b,2
,
Filip Dubovsky
b,3
, Elissa Malkin
b,3
, Kathryn Tucker
c
, Marie-Claude Dubois
d
, Joe D. Cohen
d
,
W. Ripley Ballou
d,1
, D. Gray Heppner Jr
a,7
a
Walter Reed Army Institute of Research, Silver Spring, MD, USA
b
PATH Malaria Vaccine Initiative (MVI), Bethesda, MD, USA
c
Statistics Collaborative Incorporated, Washington, DC, USA
d
GlaxoSmithKline Biologicals, Rixensart, Belgium
article info
Article history:
Received 30 June 2008
Received in revised form 12 August 2009
Accepted 14 August 2009
Available online 6 September 2009
Keywords:
Malaria vaccine
Plasmodium falciparum
Liver Stage Antigen
abstract
Plasmodium falciparum Liver Stage Antigen 1 (LSA-1) is a pre-erythrocytic stage antigen. Our LSA-1 vaccine
candidate is a recombinant protein with full-length C- and N-terminal flanking domains and two of the
17 amino acid repeats from the central repeat region termed “LSA-NRC.” We describe the first Phase I/II
study of this recombinant LSA-NRC protein formulated with either the AS01 or AS02 adjuvant system.
We conducted an open-label Phase I/II study. Thirty-six healthy malaria-naïve adults received one of four
formulations by intra-deltoid injection on a 0 and 1 month schedule; low dose (LD) LSA-NRC/AS01:10 g
LSA-NRC/0.5 ml AS01 (n = 5), high dose (HD) LSA-NRC/AS01: 50 g LSA-NRC/0.5 ml AS01 (n = 13); LD
LSA-NRC/AS02: 10 g LSA-NRC/0.5 ml AS02 (n = 5) and HD LSA-NRC/AS02: 50 g LSA-NRC/0.5 ml AS02
(n = 13). Two weeks post-second immunization, the high dose vaccinees and 6 non-immunized infectivity
controls underwent experimental malaria sporozoite challenge.
The vaccines showed a reassuring safety profile butweremoderately reactogenic. There were no serious
adverse events. All subjects seroconverted after the first immunization. Following the second immuniza-
tion, LSA-1-specific CD4+ T cells producing two cytokines (IL-2 and IFN-) were found by intra-cellular
staining in all subjects in the LD LSA-NRC/AS01B group and in 3 of 5 subjects in the LD LSA-NRC/AS02
group. In contrast, the HD LSA-NRC/AS01 and HD LSA-NRC/AS02 group subjects had fewer LSA-1-specific
CD4+ T cells, and minimal to no IFN- responses. There was no increase in LSA-1-specific CD8+ T cells
found in any group. Per protocol, 22 high dose vaccinees, but no low dose vaccinees, underwent P. falci-
parum homologous malaria challenge (3D7 clone). All vaccinees became parasitemic and there was no
delay in their pre-patent period versus controls (p = 0.95).
Disclaimer: The opinions expressed herein are private and do not represent official positions of the Departments of the Army or of Defense.
Corresponding author at: Department of Clinical Trials, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA.
Tel.: +1 301 319 9312; fax: +1 301 319 9585.
E-mail addresses: james.cummings@us.army.mil (J.F. Cummings), dwalsh@wrp-ksm.org (D.S. Walsh), InkyuYoon@afrims.org (I.-K. Yoon), astewart@wrp-ksm.org
(V.A. Stewart), Chillier@arana.com (C.J. Hillier), giersingb@ebsi.com (B.K. Giersing), dubovskyf@medimmune.com (F. Dubovsky), malkine@medimmune.com
(E. Malkin), rip.ballou@gatesfoundation.org (W.R. Ballou), donald.heppner@us.army.mil (D.G.H. Jr).
1
Current address: Bill & Melinda Gates Foundation, PO Box 23350, Seattle, WA 98102, USA.
2
Current address: Emergent Product Development UK Ltd., 540-545 Eskdale Road, Winnersh Triangle, Wokingham, Berkshire RG41 5TU, United Kingdom.
3
Current address: MedImmune Inc., One MedImmune Way, Gaithersburg, MD 20878, USA.
4
Current address: Arana Therapeutics Ltd., Level 2, 37 Epping Road, Macquarie Park, NSW 2113, Australia.
5
Current address: United States Army Medical Unit-Kenya, Unit 64109, APO 09831-4109, LTC, USA.
6
Current address: Armed Forces Research Institute of Medical Sciences, APO AP 96546, USA.
7
Current address: Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA.
0264-410X/$ see front matter © 2009 Published by Elsevier Ltd.
doi:10.1016/j.vaccine.2009.08.046
5136 J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144
LSA-NRC/AS01 and LSA-NRC/AS02 elicited antigen-specific antibody and CD4+ T cell responses, but
elicited no protective immunity. Although the optimal antigen dose of LSA-NRC may not have been
selected for the challenge portion of the protocol, further vaccine development based upon LSA-1 should
not be excluded and should include alternative vaccine platforms able to elicit additional effector mech-
anisms such as CD8+ T cells.
© 2009 Published by Elsevier Ltd.
1. Introduction
Liver Stage Antigen-1 (LSA-1), expressed during Plasmodium fal-
ciparum hepatic schizogony, is a candidate vaccine antigen based
upon data correlating the acquisition of LSA-1 specific B cell and
T cell responses in humans with immunity to clinical malaria
[1–6] and experimental infection [8]. Three additional reasons also
support its development as a vaccine antigen. First, it is highly
conserved suggesting that a candidate vaccine based on the 3D7
strain might elicit immune responses that cross-react with all
other strains of P. falciparum [9,10]. Secondly, LSA-1 is abundantly
expressed from early through late schizogony, presumably allow-
ing time for both circulating and memory-recalled effector cells to
infiltrate the liver and exert their effector function. Third, it is pos-
sible that high titer antibody could act upon the cloud of flocculent
liver stage antigen enveloping hepatic merozoites to impede the
latter’s emergence and subsequent invasion of erythrocytes [11].
However, because LSA-1 appears to be unique to P. falciparum, there
are no data from animal models of malaria to predict its suitability
as a potential human vaccine antigen.
LSA-1 is a 230 kDa protein, with a large central repeat region
(over 80 repeats of 17 amino acids each) flanked by two highly con-
served N- and C-terminal regions, known to contain B cell and CD4+
and CD8+ T cell epitopes. We expressed LSA-NRC in Escherichia coli
based upon the 3D7 clone [12] and demonstrated that formula-
tions of this recombinant protein with either AS01 or AS02 elicited
potent B and T cell responses in mice [13]. Subsequent evaluation
of LSA-NRC/AS01 in Asian Macaca mulatta showed that immuniza-
tion elicited LSA-1-specific antibody and CD4+ T cells producing
both IFN- and IL-2 [14]. The present study was conducted in 2006
to determine for the first time the safety, reactogenicity, immuno-
genicity and preliminary efficacy against homologous sporozoite
challenge of LSA-NRC/AS01 and LSA-NRC/AS02 in healthy, malaria
naïve adults.
2. Materials and methods
2.1. Ethics
The study was conducted according to Good Clinical Practices
under a protocol approved by the Human Use Review Com-
mittee of the Walter Reed Army Institute of Research (WRAIR),
The Western Institutional Review Board, Olympia, Washington,
and by the US Army Medical Research and Materiel Command’s
Human Subjects Research Review Board, Fort Detrick, Mary-
land under an Investigational New Drug application filed at the
US Food and Drug Administration. This study was registered at
www.ClinicalTrials.gov under reference identifiers NCT00312702
and NCT00312663.
2.2. Vaccines
The expression, purification, and biochemical and immuno-
logical characterization of E. coli produced, GMP manufactured
LSA-NRC antigen, with conserved T cell epitopes in the N- and
C-terminal region and 2 of the 17 amino acid repeats, has
been described previously [12]. The antigen was produced and
lyophilized into single-dose vials at the WRAIR Pilot Bioproduc-
tion Facility at Forest Glen, Maryland, and adjuvanted at the time
of administration by mixing with either AS01 or AS02 Adjuvant
Systems manufactured by GlaxoSmithKline Biologicals, Rixensart,
Belgium. AS01 is a liposome-based formulation of 3-deacylated
monophosphoryl lipid A (3-D-MPL) and the saponin derivative
Quillaja saponaria (QS) 21. AS02 is a proprietary oil-in-water emul-
sion with 3-D-MPL and the QS21.
2.3. Screening and enrolment
Written informed consent was obtained from all volunteers
prior to screening and enrollment into the study. Volunteers
provided a medical history and underwent a physical examina-
tion and laboratory testing consisting of a complete blood count
(CBC), serum biochemistries (creatinine, alanine aminotransferase,
aspartate aminotransferase and bilirubin), and serologic tests to
characterize hepatitis B, hepatitis C and HIV infection status. Vol-
unteers were excluded from participation if they had had malaria or
recent travel to a malaria endemic country, had previously received
an experimental malaria vaccine, had recently taken or planned to
take any other experimental vaccine, were asplenic, had a positive
-HCG test, were immunodeficient, or had any acute or chronic pul-
monary, cardiovascular, hepatic, neurologic or renal disease that
might confound evaluation of the vaccines.
2.4. Allocation and immunization
Two groups of five volunteers each were recruited and ran-
domly assigned to participate in the low dose vaccination schedule,
and two groups of thirteen volunteers each were recruited and
randomly assigned to participate in the high dose vaccination
schedule. Subjects in the low dose groups were immunized with
10 g LSA-NRC protein in a final volume of 0.5 ml AS01 or AS02
(LD LSA-NRC/AS01 or LD LSA-NRC/AS02, respectively) and subjects
in the high dose groups were immunized with 50 g LSA-NRC in a
final volume of 0.5 ml of AS01 or AS02 (HD LSA-NRC/AS01 or HD
LSA-NRC/AS02, respectively). Immunizations were administered in
the deltoid muscle of the same non-dominant arm at 0 and 1 month.
The low dose and high dose groups were staggered for immuniza-
tions at 14-day intervals to assess adverse events before proceeding
to the higher doses (Fig. 1).
2.5. Safety assessments
The primary objective was to assess the safety and reac-
togenicity of the LSA-NRC/AS01 and LSA-NRC/AS02 vaccines in
malaria-naïve adults. The primary endpoints were the occurrence
of solicited signs and symptoms during 14-day periods after each
vaccination, i.e., on day of vaccination (day 0) and post-vaccination
Days 1, 2, 3, 7, and 14 days, and the occurrence of serious
adverse events (SAEs) during the study period. Clinical safety lab-
oratory tests were performed at baseline and 7 days after each
immunization. Volunteers were observed for 30 min after each
immunization for evidence of anaphylaxis or other acute reactions.
The presence of solicited local and general signs and symptoms,
including measurement of oral temperature, were assessed after
J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144 5137
Fig. 1. Flow of participants from screening to challenge and study completion.
each immunization and 1, 2, 3 and 7 days post-immunization.
The solicited injection site adverse events were pain, redness
and swelling. Solicited general adverse events were fever, nausea,
fatigue, headache, malaise, myalgia and joint pain. In addition to
the solicited signs and symptoms, investigators recorded any other
adverse events occurring within a 28-day follow-up period (day
of immunization and 27 subsequent days) as unsolicited adverse
events. Adverse events were assessed for intensity. Injection site
pain was graded as 0 = absent, 1 = mild pain not interfering with
function, 2 = moderate pain; pain not interfering with activities of
daily living (ADL), and 3 = severe pain; pain that interferes with
ADL. Solicited symptoms were graded as 0 = normal, 1 = easily tol-
erated, 2 = interferes with normal activity, and 3 = prevents normal
daily activity. Additional grading scales were applied to visible
swelling or redness at the injection site; 0 = none, 1 = 0–20 mm,
2 > 20–50 mm, and 3 50 mm, and to oral temperature; 0 37.5
C,
1 = 37.5–38
C, 2 > 38–39
C, and 3 39
C. Serious adverse events
(SAEs) were reported from enrollment until study completion 3
months after final immunization. A serious adverse event was
defined as any untoward medical occurrence that resulted in death,
was life threatening, resulted in persistent or significant disabil-
ity/incapacity, required hospitalization or prolongation of existing
hospitalization, or was associated with a congenital anomaly/birth
defect in the offspring of a study subject.
2.6. Humoral immunity
Serum was obtained at baseline and 4 weeks after the first
immunization (Days 0 and 28) for both low dose and high dose
vaccine groups, and at day of challenge (DOC) (Day 42) and study
conclusion on Day 84 for the high dose groups. Analysis of antibody-
mediated immunity to the vaccine was assessed by ELISA. Briefly,
plates were coated at 50 ng/well with purified bulk LSA-NRC anti-
gen [14] and incubated overnight at 4
C. Plates were washed
four times with PBS between all subsequent steps. After overnight
incubation, plates were blocked and all subsequent steps until
substrate addition performed in a 0.5% casein in PBS solution.
Test sera were serially diluted in triplicate on the plates from
1:500 to 1:64,000 and incubated for 2 h, followed by horseradish
peroxidase-conjugated goat antihuman IgG (KPL, Gaithersburg,
MD) for 1 h. After addition of ABTS peroxidase substrate (Kirkegaard
& Perry Laboratories Inc., Gaithersburg, MD), plates were incu-
bated for 1 h, and the reaction stopped with 10 l of 20% SDS
(Sigma, St. Louis, MO). Plates were read at 414 nm (Vmax Molecu-
lar DevicesTM) and the serial dilutions used to fit a four-parameter
curve using SoftMax Pro v4.1 (Molecular Devices). Results were
expressed in titer values, the titer endpoint being defined as the
serum dilution yielding an optical density of 1.0. All plates included
two positive and two negative controls, with stringent quality
control limits to ensure precision of readout. Criteria for sero-
conversion to vaccine antigen required that the magnitude of the
antibody response (midpoint titers) differed significantly (p < 0.05)
from pre-immune serum as assessed using the paired Student’s
t-test.
2.7. Intra-cellular cytokine staining (ICS)
PBMCs were isolated by centrifugation on Ficoll-Hypaque (ICN
Biomedicals Inc., Aurora, OH) and stored in liquid nitrogen until
used. PBMC were collected before the first immunization and 2–3
weeks after the second dose of vaccine.
The LSA-NRC antigen used in the in vitro cultures was the
same as that used to immunize the donors (vide supra). LSA-NRC
peptides: 108 overlapping 15-mer peptides corresponding to the
entire LSA-NRC protein were each dissolved at 11 mg/ml in DMSO.
Two pools of overlapping peptides (Pool 1 = peptides 1–54; Pool
2 = peptides 55–108) were prepared at 2 g/peptide/ml in com-
plete medium (CM) consisting of RPMI 1640 (Gibco BRL, Grand
Island, NY) supplemented with 8 mM Glutamax (Life Technologies,
Grand Island, NY), 50 units/ml penicillin–50 units/ml streptomycin
(Life Technologies), 0.1 mM MEM Non-Essential Amino Acids (Life
Technologies), 0.042 mM 2-mercaptoethanol (Sigma–Aldrich, St.
5138 J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144
Louis, MO), 1 mM MEM sodium pyruvate (Life Technologies).
Although PBMC from two of the 24 subjects receiving two doses
of HD LSA-NRC (one receiving HD LSA-NRC/AS01 and one receiv-
ing LSA-NRC/AS02) were stimulated with a slightly different pool
of peptides (Pool 1 = peptides 1–38 and Pool 2 = peptides 35–108),
all HD LSA-NRC ICS data are reported by their respective vaccine
group.
All antibodies, with the exception of Pacific Orange-anti-human
CD8+ (Invitrogen Corporation, Carlsbad, CA), were obtained from
BD Pharmingen (San Jose, CA). Anti-human FcR Block was from
Miltenyi Biotech (Auburn, CA) and the Fixable Blue Dead Cell Stain
was from Invitrogen Corporation. 100 l of thawed and washed
PBMC (2 × 10
6
cells/ml in CM supplemented to 20% with heat inac-
tivated Gem Cell human AB serum (Gemini Bioproducts, Woodland,
CA) or for some of the CD8+ T cell assays, heat inactivated FBS
(Hyclone, Logan, UT)) were added to eight wells of a 96-well U-
bottom plate (Corning, Corning, NY) along with 100 l/well of CM
containing anti-CD28 and anti-CD49d co-stimulants (2 g/ml) plus
either no antigen, LSA-NRC at 20 g/ml or LSA-NRC peptide pool at
2 g/peptide/ml. Each assay also included at least one sample stim-
ulated with SEB (Sigma–Aldrich) at 2 g/ml as a positive control.
For 66% of the subjects a second positive control consisting of PBMC
stimulated with a pool of class I-restricted CEF peptides was also
included in the assays for CD8+ T cells. After 2 (peptides) or 3 (LSA-
NRC) hours at 37
, brefeldin A (BD Biosciences; San Jose, CA) was
added at 1/1000 final dilution and cultures were continued for an
additional 15–16 h.
Cells from each antigen group were harvested into a FACS tube,
washed, re-suspended in 100–200 l of PBS/BSA and stained with
a pool of antibodies containing 3 l PerCP-anti-human CD3, 1.0 l
Pacific Blue-anti-human CD4, 2 l Pacific Orange-anti-human CD8
and 0.5 l UV viability dye for 1 h on ice. After washing (PBS), the
cells were cytofixed/cytopermed (BD Biosciences) on ice for 30 min,
washed twice with cold Permwash buffer (BD Biosciences) and then
stained with a pool of antibodies containing 1.0 l anti-FcR Block,
0.3 l PerCP-anti-human CD3, 1.0 l FITC-anti-human IFN- and
0.5 l APC-anti-human IL-2. After 30 min on ice, cells were washed
twice with Permwash re-suspended in PBS, and placed at 4
Cin
the dark until acquired on a LSR II (BD Biosciences) flow cytometer.
The data were analyzed using Flow Jo (TreeStar Inc., Ashland, OR)
and Prism Graph (Graphpad Software Inc., San Diego, CA) software.
2.8. Malaria sporozoite challenge
Two weeks after second immunization, the volunteers who
had received two immunizations with the high dose formula-
tions and six concurrent infectivity controls underwent standard
malaria challenge as previously described [15,16]. On the DOC,
five mosquitoes were allowed to feed on each volunteer for 5 min,
after which they were dissected to confirm how many infected
mosquitoes had fed, and the salivary glands were scored for sporo-
zoite burden. If required, additional mosquitoes were allowed to
feed until a total of five infected mosquitoes with a minimum
2+ salivary gland score had fed. Blood for malaria smear was
collected from Day 5 post-DOC up to 30 days, depending on devel-
opment of parasitemia or until resolution of parasitemia. Thick film
slides were stained with Giemsa for each blood sample. If a sub-
ject was found to be parasitemic, antimalarial treatment with oral
chloroquine was initiated immediately and completed under direct
observation of a physician.
2.9. Statistical analysis
Demographic, safety and adverse event data were summarized
and presented in tabular format (Statistics Collaborative, Inc.). For
analyses of antibody responses, data were log-transformed to nor-
malize it prior to statistical analysis. Comparisons among groups
were made by one-way ANOVA with Tukey’s post-test analysis.
When no differences were observed among groups within a time-
point, comparisons across time were made with pooled data. Day
42 of the study (DOC) data for the six challenge controls was con-
sidered with Day 0 data for the other volunteers, as these samples
were taken prior to any experimental manipulation. For analyses
of cellular responses, the Mann–Whitney test was used to compare
pre-immune to post-second dose immunologic responses, and to
compare post-second dose intergroup responses (Figs. 5 and 6).
Vaccine efficacy was estimated by performing Kaplan–Meier anal-
yses on the time to onset of parasitemia (Statistics Collaborative,
Inc.). No adjustment of p-values was made for multiple analyses.
3. Results
3.1. Study population
Thirty-six subjects were enrolled in the immunization phase of
the study, with five each assigned to the LD LSA-NRC/AS01 and
LD LSA-NRC/AS02 groups, and 13 each assigned to the HD LSA-
NRC/AS01 and HD LSA-NRC/AS02 groups. Seventy of 72 scheduled
vaccine doses were administered. Twenty-two of 24 eligible high
dose vaccinees elected to undergo malaria challenge along with
six subjects enrolled to serve as infectivity controls for the efficacy
phase of the study (Fig. 1). Volunteer demographics were similar
for all groups (Table 1).
3.2. Safety and reactogenicity
Vaccine-related adverse events are depicted in Fig. 2a and b.
The most common local side effect was pain at the injection site,
with no increase in frequency associated with the second immu-
Fig. 2. Local and general vaccine-related adverse events after first and second
immunization.
J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144 5139
Table 1
Volunteer demographics.
LSA-NRC/AS02 LSA-NRC/AS01 Infectivity controls
Low dose, n = 5 High dose, n = 13 Total, n = 18 Low dose, n = 5 High dose, n = 13 Total, n = 18 Total, n =6
Male (%) 3 (60%) 6 (46%) 9 (50%) 4 (80%) 5 (38%) 9 (50%) 5 (83%)
Mean age (SD) 25 (6) 33 (8) 31 (8) 31 (8) 33 (8) 32 (8) 24 (6)
Caucasian (%) 5 (100%) 11 (85%) 16 (89%) 4 (80%) 6 (46%) 10 (56%) 5 (83%)
African (%) 0 0 0 0 3 (23%) 3 (17%) 1 (17%)
Other (%) 0 2 (15%) 2 (11%) 1 (20%) 4 (31%) 5 (28%) 0
nization in any of the groups. There were no instances of grade
3 pain after either immunization. Vaccine-related general adverse
events were uncommon after the first immunization, but common
after the second immunization. There were no statistically signif-
icant differences in local side effects or general adverse events
between vaccines formulated with AS01 or AS02. There were no
instances of grade 3 vaccine related systemic adverse events. No
severe adverse events or significant abnormal clinical laboratory
test results occurred.
3.3. ELISA
All four groups experienced an approximate 10-fold increase in
antibody titer after the first dose as measured at 28 days. Of note,
all four groups were not statistically different from one another
at baseline (p = 0.50 by ANOVA) or at 28 days (p = 0.98 by ANOVA)
(Fig. 3). In the high dose formulation groups, a second dose resulted
in a greater than 10-fold increase in antibody titer and those two
groups remained equivalent (p = 0.75 by unpaired t-test) at DOC. At
Day 84, antibody titers in vaccinees remained elevated and were
quite different from the challenge controls (p < 0.0001 by ANOVA)
but not distinguishable between themselves (p > 0.05 by Tukey’s).
However, the titers had fallen significantly from 2 weeks after
the last vaccine (p < 0.0001 by paired t-test between Days 42 and
84 using pooled data from both high dose groups). Exposure to
challenge with live parasites in infectivity controls resulted in no
significant increase in antibody titers for the infectivity control
Fig. 3. Antibody to LSA-NRC by treatment group. Titers are optical density units, the
dilution of serum which would yield an absorbance of 1.0 in a standard ELISA. Bars
represent mean ± 95% C.I. of mean of log-transformed data. Day 0 is prior to first
immunization; Day 28 is just prior to second immunization; Day 42 is just prior to
malaria mosquito challenge and Day 84 is 42 days post-malaria mosquito challenge.
group, although one individual did evince a 4-fold rise in titer from
base line.
3.4. Cellular immunity
IFN- released from antigen-specific T cells is one of the key
mediators of protective immunity against pre-erythrocytic stage
Plasmodia parasites [17]. In addition, for human CD4+ T cells there
is some consensus that a shift from predominantly IL-2 produc-
tion to predominantly IFN-g-production reflects a transition of
T cells from a central memory to an effector phenotype [18].T
cells producing both an effector (IFN-) and a growth promoting
(IL-2) cytokine have also been shown to be the most effective in
controlling infection, presumably because IL-2 helps expand the
frequency of the effector cells [19]. To determine whether vacci-
nation with LSA-NRC induced IL-2 and/or IFN- responses, PBMC’s
obtained from the vaccinees at pre-immunity and after the second
immunization just prior to infectious challenge were stimulated
overnight with LSA-NRC, LSA-1 peptide pools or medium alone and
the responding cells were examined by intra-cellular staining for
antigen-specific IL-2 and IFN- recall responses. A typical gating
scheme and LSA-1-recalled CD 4+ T cell responses from a volun-
teer immunized with LD LSA-NRC/AS01 are shown in Fig. 4A and
B, respectively. As can be seen for this subject, among the LSA-1
recalled CD4+ T cells, some produced only IL-2 (0.46%), others only
IFN- (0.04%) and yet another population of CD4+ T cells produced
both cytokines (0.12%). Upon complete analyses of the responses
from each subject in the entire cohort, we observed a large varia-
tion in the percentage as well as in absolute cell numbers among
the single cytokine or double cytokine producing CD4+ T cells.
Therefore, we chose to present individual CD4+ T cells responses
at baseline and post-second immunization for each subject. The
responses show both IL-2 producing CD4+ T cells (Fig. 5) and IFN-
producing CD4+ T cells (Fig. 6) (as a sum of both the single plus
the double producers for each cytokine) minus the culture medium
background. As can be seen, CD4+ T cell IL-2 responses, recalled
with either LSA-NRC or LSA-1 peptide pools from both the low
dose and the high dose vaccines administered with either AS01
or AS02, were significantly higher than those obtained at baseline
in all four vaccine groups. Subjects receiving LD LSA-NRC/AS01 or
HD LSA-NRC/AS01 also showed significantly higher CD4+ T cell IFN-
responses recalled with either LSA-NRC or LSA-1 peptide pools
than their baseline reactivity. By contrast, CD4+ T cells from only
3 of 5 subjects in the LD LSA-NRC/AS02 group exhibited IFN-
responses recalled with either LSA-NRC or LSA-1 peptide pools that
were above baseline; however neither the LD LSA-NRC/AS02 group
nor the HD LSA-NRC/AS02 group exhibited any significant group
differences in CD4+ T cell IFN- responses from those obtained
at baseline. Overall, the IL-2 and IFN- responses induced with
the LD LSA-NRC/AS01 were also significantly greater than those
induced with HD LSA-NRC/AS01. With one exception, CD4+ T cell
IL-2 and IFN- responses induced in the presence of the AS01B
adjuvant were not significantly different from those induced in
the presence of the AS02A adjuvant. The CD4+ T cells responses
5140 J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144
Fig. 4. (A) Gating scheme for identification of CD4+ and CD8+ T cells. PBMC were stimulated in vitro with recall antigen, surface and intra-cellularly stained with the indicated
antibodies (Section 2) and then acquired on an LSR II flow cytometer and analyzed with FlowJo software. Cells were displayed as forward scatter (FSC) versus viability dye,
live cells were gated and displayed as FSC versus side scatter (SSC) and SSC lo cells were selected and gated for CD3+ T cells and then CD4+ and CD8+ T cells. (B) IL-2 and
IFN- production by CD4+ T cells. PBMC from a single donor immunized with LD LSA-NRC/AS01 analyzed by intra-cellular staining for cytokine production. The plots show
IL-2 (y-axis) and IFN- (x-axis) production by CD4+ T cells. Left panel: preimmune PBMC cultured with LSA-NRC; Central panel: post-second immunization PBMC cultured
with media, and Right panel; post-second immunization PBMC cultured with LSA-NRC.
induced with HD LSA-NRC in AS01B and recalled with the LSA
antigen, the IFN- responses were significantly higher (p < 0.014)
than those induced with HD LSA-NRC in AS02A. In addition, for the
CD4+ T cells induced with LD LSA-NRC in AS01B and recalled with
the LSA peptides, the IFN- responses were significantly higher
(p < 0.016) than those induced with LD LSA-NRC in AS02A, when
the one outlier in the latter group was excluded from the analy-
sis. It is also worth mentioning that CD4+ T cells induced with LD
LSA-NRC in AS01B and recalled with LSA showed a trend (p < 0.1)
towards higher IFN- responses than those induced with LD LSA-
Fig. 5. Pre-immunization and post-second immunization CD4+ T cell IL-2 responses. Pre-immunization and post-second immunization PBMC from donors immunized with
either a low dose or a high dose of LSA-NRC in either the AS01 or the AS02 adjuvant were stimulated in vitro with either (A) LSA-NRC or (B) LSA-1 peptide pools. CD4+ T
cells were then analyzed for IL-2 production as described in Fig. 4. Culture medium only backgrounds were subtracted from values obtained in the presence of antigen. For
70% of the pre-immune PBMC, culture medium backgrounds for the assay involving recall with intact LSA-NRC were not available and therefore pre-immune PBMC culture
medium backgrounds from the assay involving recall with LSA-1 peptides were also used for the LSA-NRC assay. Values shown in (B) represent the response to peptide pool
1 plus the response to peptide pool 2. Data from 3 of 31 subjects were excluded from the analyses due to very high backgrounds. The post-second immunization responses
for all groups were significantly different from the corresponding pre-immune responses. Significant differences between other appropriate groups are indicated. Horizontal
black bars depict geometric mean values for each group and timepoint. p-Values appear with brackets to indicate intergroup comparisons.
J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144 5141
Fig. 6. Pre-immunization and post-second immunization CD4+ T cell IFN- responses. Pre-immunization and post-second immunization PBMC from donors immunized
with either a low dose or a high dose of LSA-NRC in either the AS01 or the AS02 adjuvant were stimulated in vitro with either (A) LSA-NRC or (B) LSA-1 peptide pools. CD4+
T cells were then analyzed for IFN- production as described in Fig. 4. Culture medium only; refer to legend for Fig. 5 backgrounds were subtracted from values obtained in
the presence of antigen. Data from 3 of 31 subjects were excluded from the analyses due to very high backgrounds. Values shown in (B) represent the response to peptide
pool 1 plus the response to peptide pool 2. Statistical differences between appropriate groups are as indicated. Horizontal black bars depict geometric mean values for each
group and timepoint. p-Values appear with brackets to indicate intergroup comparisons.
NRC in AS02A. The CD4+ T cells induced with LD LSA-NRC in AS01B
and recalled with LSA peptides showed a trend (p < 0.1) towards
higher IL-2 responses than those induced with LD LSA-NRC in
AS02A.
We attempted numerous times to measure CD8+ T cell IFN-
responses using both LSA-NRC and the LSA-1 peptide pools as the
in vitro recall antigen. Although it is conceivable that immunization
with LSA-NRC may have induced a low frequency of antigen-
specific CD8+ T cells secreting a low level of cytokine, the CD8+ T
cell cytokine responses remained undetectable above the baseline
responses (Fig. 7).
Fig. 7. Pre-immunization and post-second immunization CD8+ T Cell IFN-
responses. PBMC from donors immunized with either a low dose or a high dose
of LSA-NRC in either the AS01 or the AS02 adjuvant were stimulated in vitro
with LSA-1 peptide pool 1 or LSA-1 peptide pool 2. CD8+ T cells were then ana-
lyzed for IFN- production as described in Fig. 4. Values shown represent the
response to peptide pool 1 plus the response to peptide pool 2. Culture medium
only backgrounds were subtracted from values obtained in the presence of anti-
gen. Mean ± SD pre-immune medium background (IFN-+ CD8+ T cells\10
6
CD8+
T cells) = 470 ± 250 (AS01); 400 ± 190 (AS02); Mean ± SD post-second-immune
medium background = 490 ± 230 (AS01); 390 ± 190 (AS02). Horizontal black bars
depict geometric mean values for each group and timepoint.
3.5. Efficacy
Twenty-two vaccinees (10 HD LSA-NRC/AS01 and 12 HD
LSA-NRC/AS02) and six infectivity controls underwent standard
challenge with the bites of five malaria-infected mosquitoes. All
volunteers developed parasitemia between Days 9 and 13 and were
treated. There was no difference in the time to parasitemia between
vaccinees and controls (p = 0.99) (Fig. 8).
4. Discussion
We report that administration of two doses of recombinant LSA-
NRC protein with either the AS01 or AS02 adjuvant, has a favorable
safety profile, elicits potent antigen-specific antibody and CD4+ T
cell responses, but does not protect against experimental P. fal-
ciparum malaria challenge. These findings have implications for
the use of recombinant LSA-NRC formulated with potent adjuvant
systems as a pre-erythrocytic vaccine.
4.1. Antibody to LSA-NRC
Total IgG responses to this vaccine were brisk; peak titers in the
high dose groups were greater than the titers (range: 900–1200
(i.e., log 2.95–3.07)) (Ann Stewart-unpublished data)) observed in
a random sample of serum collected from 12 blood bank samples in
a hyper-endemic area of Western Kenya. There was no difference
between the four different formulations in their capacity to induce
antibody 28 days after the first immunization, and a second immu-
nization with either high dose formulation boosted the response
in excess of 10-fold. Although a causal relation between LSA-1-
specific antibodies and clinical immunity to malaria has previously
been postulated, LSA-1 is contained within the parasitophorous
vacuole until the rupture of the hepatocyte, and thus it is difficult
to hypothesize a direct role for anti-LSA-1 antibodies in interfer-
ing with hepatic schizogony. Post-rupture agglutination of hepatic
merozoites ensconced in a cloud of LSA-1 would require large
amounts of high affinity antibody in order to exert a measurable
physiologic effect against short-lived merozoites. We did not mea-
sure the isotype of LSA-1 antibodies in this study, since antibody
dependent cell mediated immunity was not a postulated mecha-
nism of LSA-NRC mediated protection. Of note, a robust serologic
5142 J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144
Fig. 8. Kaplan–Meier curves for time to Plasmodium falciparum Parasitemia. Kaplan–Meier curve depicting time to parasitemia after experimental malaria challenge for
subjects immunized with HD LSA-NRC/AS01 or HD LSA-NRC/AS02, and for the un-immunized infectivity controls.
response to LSA-NRC was not detected in the challenge controls,
suggesting anti-LSA-1 antibody in natural exposure may be the
sequel of multiple prior malaria episodes, a pre-requisite for devel-
oping partial immunologic protection [20].
4.2. LSA-NRC-specific T cells
The capacity of malaria-specific CD4+ T cells to confer pro-
tection has been demonstrated previously. For example, murine
CD4+ T cells specific for an epitope in the P. yoelii circumsporo-
zoite protein can both eliminate infected hepatocytes in vitro and
adoptively transfer protection in vivo [21,22]. In clinical trials, the
adjuvanted RTS,S vaccine induces the activation of CD4+ T cells
that recognize multiple Pf CS protein epitopes. These T cells can
be recalled in vitro with CS protein-based peptides to produce IFN-
which is known to inhibit intra-cellular stages of the parasite.
Moreover, the production of IFN- by CD4+ T cells has been shown
to significantly associated with the protection of RTS,S-immunized
volunteer against experimental malaria challenge [23,24].Inthe
present study, it was demonstrated that immunization with LSA-
NRC in either the AS01 or the AS02 adjuvants primed CD4+ T cells
that were able to develop LSA-specific IL-2 recall responses in vitro;
in addition both the low and high dose formulations of LSA-NRC
with AS01 also primed CD4+ T cells that were able to develop LSA-
specific IFN- recall responses. PBMC from volunteers receiving the
LD LSA-NRC/AS01 adjuvant elaborated significantly higher CD4+
T cell IL-2 and IFN- responses than PBMC from subjects immu-
nized with the HD LSA-NRC/AS01. PBMC from volunteers receiving
HD LSA-NRC/AS02 developed IFN- recall responses that were too
weak to be detected by intra-cellular staining. Although it is unclear
why the low dose formulations of LSA-NRC induced higher cytokine
responses than the high dose formulations, several mechanisms
can be considered. First, it is conceivable that the CD4+ T cells
responding to the high dose formulations produced such a high
level of IFN- that they underwent IFN- auto-induced cell death
[25]. Secondly, the high dose formulations of LSA-NRC may have
provoked a more rapid response than did the low dose formula-
tions and by 14 days post-second immunization this response may
have already declined from its peak level. The low IFN- responses
observed in the volunteers immunized with HD LSA-NRC/AS01
and the undetectable IFN- responses observed in the volunteers
immunized with HD LSA-NRC/AS02 may explain, at least in part,
why these individuals were not protected against infectious chal-
lenge.
By means of the ELISPOT assay, we have demonstrated
recently that PBMC from the LSA-NRC immunized volunteers also
make intact LSA-NRC-induced IL-10 recall responses (prelimi-
nary analyses revealed 653 +/- 342 (mean ± STD) IL-10 SFC/10
6
PBMC for LD LSA-NRC/AS01; 411 ± 449 IL-10 SFC/10
6
PBMC
for LD LSA-NRC/AS02; 223 ± 112 IL-10 SFC/10
6
PBMC for HD
LSA-NRC/AS01and 209 ± 156 IL-10 SFC/10
6
PBMC for HD LSA-
NRC/AS02). Although the nature of the cells producing the IL-10
has not been identified, the requirement for antigen to recall the
response suggests that they might be TH1 cells that produce both
IFN- + IL-10 [26] or possibly IL-10 producing regulatory T cells.
In any case, IL-10 is known to minimize the effects of inflamma-
tory cytokines such as IFN- and it is conceivable that this may
also account for the lack of protection observed in the present vac-
cine trial. In two other studies it was reported that LSA-1- induced
IL-10 responses correlated with increased resistance to infection
with P. falciparum [5,7]. However, these trials dealt with individ-
uals who had experienced recent blood stage infections and the
IL-10 responses were measured by ELISA; consequently, the results
cannot be readily compared with those of the present study.
4.3. Is adjuvanted recombinant LSA-NRC a vaccine candidate?
Sporozoite challenge models of P. falciparum are not reliably
established in non-human primates [27] and there are no known
homologous LSA-1 antigens in other plasmodia species, hence
there are no animal models of malaria to address the feasibility
of LSA-1 as a potential vaccine antigen [28]. In humans, there have
been two Phase IIa trials of virally vectored multi-antigen vaccines
expressing portions of the LSA-1 antigen. Both of these vaccines
demonstrated a significant physiologic effect against malaria, and
one was able to elicit LSA-1-specific T cell responses; however attri-
bution of these effects to the LSA-NRC component was confounded
by the potential protective effect of other pre-erythrocytic antigens
in the vaccine constructs [29,30].
The negative efficacy of the present study challenges the con-
cept of using adjuvanted recombinant LSA-NRC protein to direct
cellular immune responses against hepatic-stage falciparum par-
J.F. Cummings et al. / Vaccine 28 (2010) 5135–5144 5143
asites. However, this trial did not rule out that LSA-NRC-specific
CD4+ T cells elaborating IFN- plus a second cytokine (IL-2, TNF-)
might exert a physiologic effect. Such an effect might be achieved
using a lower dose of the vaccines tested, or even an alternative
vaccine platform, such as a viral vector with some trophism for
hepatic antigen presenting cells to present LSA-NRC [31]. Also, this
truncated test antigen (LSA-NRC) may have inadvertently omitted
repeat region B cell and T cell epitopes that might have elicited a
protective immune response. Although in the present study, the LD
LSA-NRC/AS01 formulation elicited bifunctional LSA-NRC-specific
CD4+ T cell responses, by protocol, the group receiving this formu-
lation unfortunately was not subjected to a malaria challenge.
As was seen in three previous clinical trials involving proteins
adjuvanted with either AS01 or AS02 [24,32], Dr. Michel Spring-
unpublished), in the present study antigen-specific ex vivo CD8+ T
cells were not detected in any of the four vaccine groups. Although
CD8+ T cells targeting the CS antigen have been shown to medi-
ate sterile immunity against sporozoite challenge in mice [33],
CS antigen-specific CD8+ T cells elicited by a DNA-based vaccine
expressing the protective CS antigen failed to exert a detectable
protective effect against malaria challenge in humans [34]. The use
of a prime-boost regimen including a recombinant adenovirus com-
ponent might also envoke greater numbers of effector T cells [35],
or additional populations of effector T cells, such as antigen-specific
CD8+ T cells [36].
We conclude that the feasibility of LSA-1 as a vaccine can-
didate requires additional investigation. Future development of
adjuvanted recombinant LSA-NRC will focus on vaccine regimens
that significantly augment the breadth and magnitude of T cell
responses to this antigen.
Acknowledgements
Funding: The cGMP manufacture of LSA-NRC and this trial were
funded by the PATH Malaria Vaccine Initiative, Bethesda, Mary-
land and by the Military Infectious Disease Research Program, Fort
Detrick, Maryland. Conflict of interest: J.D. Cohen, W.R. Ballou, M.C.
Dubois were employees of and owned stock in GSK Biologicals, the
manufacturer of the described Adjuvant Systems when this study
was planned and conducted. D.E. Lanar and L.A. Ware have applied
for patents involving the described vaccine antigen, LSA-NRC. J.D.
Cohen, W.R. Ballou, V.A. Stewart, C.F. Ockenhouse and D.G. Heppner
are listed as inventors on patents or have patent applications cov-
ering various malaria vaccine candidates. Disclosure: Presented in
part at the 55th annual meeting of the American Society of Tropical
Medicine and Hygiene held in Atlanta, Georgia, 12–16 November
2006.
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    • "Adjuvanted RTS,S (RTS,S/AS), a candidate malaria vaccine consisting of the recombinant protein RTS,S, which is comprised of sequences of the circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg), is uniquely able to protect malaria-naïve adult subjects after experimental malaria challenge against infection [1] [2] [3] [4] [5], and African adults and children exposed to diverse strains against clinical and severe disease [6] [7] [8] [9] [10] [11]. Other strategies have been concurrently explored to improve the efficacy of adjuvanted RTS,S, including formulation with more potent adjuvants [12–14], prime-boost regimens with alternative vaccine platforms expressing the CSP [15] [16] [17] [18] and evaluation of other adjuvanted Plasmodium falciparum antigens [19] [20] [21] individually or in combination with RTS,S [22] [23]. We report two clinical evaluations which aimed at improving adjuvanted RTS,S by combining it with the recombinant thrombospondin related anonymous protein (TRAP) of P. falciparum, PfTRAP [24]. "
    [Show abstract] [Hide abstract] ABSTRACT: In an attempt to improve the efficacy of the candidate malaria vaccine RTS,S/AS02, two studies were conducted in 1999 in healthy volunteers of RTS,S/AS02 in combination with recombinant Plasmodium falciparum thrombospondin-related anonymous protein (TRAP). In a Phase 1 safety and immunogenicity study, volunteers were randomized to receive TRAP/AS02 (N = 10), RTS,S/AS02 (N = 10), or RTS,S + TRAP/AS02 (N = 20) at 0, 1 and 6-months. In a Phase 2 challenge study, subjects were randomized to receive either RTS,S + TRAP/AS02 (N = 25) or TRAP/AS02 (N = 10) at 0 and 1-month, or to a challenge control group (N = 8). In both studies, the combination vaccine had an acceptable safety profile and was acceptably tolerated. Antigen-specific antibodies, lymphoproliferative responses, and IFN-γ production by ELISPOT assay elicited with the combination vaccine were qualitatively similar to those generated by the single component vaccines. However, post-dose 2 anti-CS antibodies in the RTS,S + TRAP/AS02 vaccine recipients were lower than in the RTS,S/AS02 vaccine recipients. After challenge, 10 of 11 RTS,S + TRAP/AS02 vaccinees, 5 of 5 TRAP/AS02 vaccinees, and 8 of 8 infectivity controls developed parasitemia, with median pre-patent periods of 13.0, 11.0, and 12.0 days, respectively. The absence of any prevention or delay of parasitemia by TRAP/AS02 suggests no apparent added value of TRAP/AS02 as a candidate vaccine. The absence of significant protection or delay of parasitemia in the 11 RTS,S + TRAP/AS02 vaccine recipients contrasts with previous 2 dose studies of RTS,S/AS02. The small sample size did not permit identifying statistically significant differences between the study arms. However, we speculate, within the constraints of the challenge study, that the presence of the TRAP antigen may have interfered with the vaccine efficacy previously observed with this regimen of RTS,S/AS02, and that any future TRAP-based vaccines should consider employing alternative vaccine platforms.
    Full-text · Article · Nov 2014
    • "It has been shown that the gene products of liver-stage sporozoites are accessible to the host MHC class I-dependent antigen-processing machinery that is required for CD8 + T cell recognition and are thus considered potential vaccine targets against the disease (Birkett et al., 2013). Among these genes, the immunogenic properties of Plasmodium liver-stage antigen-1 have been investigated, and this antigen has been currently evaluated in vaccine protocols aimed at inducing protection from malaria liver-stage parasites (Hill et al., 1992; Pichyangkul et al., 2008; Rodríguez et al., 2009; Cummings et al., 2010). The choice of liver-stage development genes as vaccine targets seems to be of relevance because the antigens can be expressed early or late during parasite development in the liver, thus varying the efficacy of the immunity to the infected hepatocytes. "
    [Show abstract] [Hide abstract] ABSTRACT: Plasmodium sporozoites and liver stages express antigens that are targeted to the MHC-Class I antigen-processing pathway. After the introduction of Plasmodium sporozoites by Anopheles mosquitoes, bone marrow-derived dendritic cells in skin-draining lymph nodes are the first cells to cross-present parasite antigens and elicit specific CD8(+) T cells. One of these antigens is the immunodominant circumsporozoite protein (CSP). The CD8(+) T cell-mediated protective immune response against CSP is dependent on the interleukin loop involving IL-4 receptor expression on CD8(+) cells and IL-4 secretion by CD4(+) T cell helpers. In a few days, these CD8(+) T cells re-circulate to secondary lymphoid organs and the liver. In the liver, the hepatic sinusoids are enriched with cells, such as dendritic, sinusoidal endothelial and Kupffer cells, that are able to cross-present MHC class I antigens to intrahepatic CD8(+) T cells. Specific CD8(+) T cells actively find infected hepatocytes and target intra-cellular parasites through mechanisms that are both interferon-γ-dependent and -independent. Immunity is mediated by CD8(+) T effector or effector-memory cells and, when present in high numbers, these cells can provide sterilizing immunity. Human vaccination trials with recombinant formulations or attenuated sporozoites have yet to achieve the high numbers of specific effector T cells that are required for sterilizing immunity. In spite of the limited number of specific CD8(+) T cells, attenuated sporozoites provided multiple times by the endovenous route provided a high degree of protective immunity. These observations highlight that CD8(+) T cells may be useful for improving antibody-mediated protective immunity to pre-erythrocytic stages of malaria parasites.
    Full-text · Article · Aug 2014
    • "However, Phase I/IIa clinical trials in which humans were immunized with RTS, S/TRAP failed to provide protection in the majority of volunteers (unpublished data, as written by [30]). Moreover, immunization of humans with a recombinant liverstage antigen-1 (LSA-1)-based vaccine elicited high antibody titers, but did also not protect against P. falciparum infection [31]. While the induction of sterile immunity with subunit vaccines has been shown to be difficult, sterile immunity against malaria can be achieved experimentally by whole-parasite immunization with attenuated sporozoites in animal models and human volunteers, targeting the sporozoite/liver-stage parasites (pre-erythrocytic stages). "
    [Show abstract] [Hide abstract] ABSTRACT: Long-lasting and sterile protective immunity against Plasmodium falciparum can be achieved by immunization of malaria-naive human volunteers under chloroquine prophylaxis with sporozoites delivered by mosquito bites (CPS-immunization). Protection is mediated by sporozoite/liver-stage immunity. In this study, the capacity of CPS-induced antibodies to interfere with sporozoite functionality and development was explored. IgG was purified from plasma samples obtained before and after CPS-immunization from two separate clinical trials. The functionality of these antibodies was assessed in vitro in gliding and human hepatocyte traversal assays, and in vivo in a human liver-chimeric mouse model. Whereas pre-treatment of sporozoites with 2 mg/ml IgG in the majority of the volunteers did not have an effect on in vitro sporozoite gliding motility, CPS-induced IgG showed a distinct inhibitory effect in the sporozoite in vitro traversal assay. Pre-treatment of P. falciparum sporozoites with post-immunization IgG significantly inhibited sporozoite traversal through hepatocytes in 9/9 samples when using 10 and 1 mg/ml IgG, and was dose-dependent, resulting in an average 16% and 37% reduction with 1 mg/ml IgG (p = 0.003) and 10 mg/ml IgG (p = 0.002), respectively. In vivo, CPS-induced IgG reduced liver-stage infection and/or development after a mosquito infection in the human liver-chimeric mouse model by 91.05% when comparing 11 mice receiving post-immunization IgG to 11 mice receiving pre-immunization IgG (p = 0.0008). It is demonstrated for the first time that CPS-immunization induces functional antibodies against P. falciparum sporozoites, which are able to reduce parasite-host cell interaction by inhibiting parasite traversal and liver-stage infection. These data highlight the functional contribution of antibody responses to pre-erythrocytic immunity after whole-parasite immunization against P. falciparum malaria.
    Full-text · Article · Apr 2014
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