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Divisions of
*
Viral Products and
Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research/Food and Drug Administration, Bethesda, MD 20892; and
Vaccine Research Center, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD 20892
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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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Due to evolutionary divergence in CpG recognition between species, ODN
that are highly active in rodents are poorly immunostimulatory in
primates, and vice versa (15, 16, 17). Extensive studies
involving human PBMC identified two distinct classes of
immunostimulatory CpG ODN (17, 18). "K" type ODN have
phosphorothioate backbones, encode multiple TCGTT and/or
TCGTA motifs (CpG motif is underlined), trigger the
maturation of plasmacytoid DC, and stimulate the production of IgM and
IL-6 (17, 19). "D" ODN have mixed
phosphodiester/phosphorothioate backbones and contain a single
hexameric purine/pyrimidine/CG/purine/pyrimidine
motif flanked by self-complementary bases that form a stem-loop
structure capped at the 3' end by a poly G tail (17). D
ODN trigger the maturation of APC and preferentially induce the
secretion of IFN-
and IFN-
(Refs. 17 and
18 and M. Gursel, unpublished observations).
There is considerable interest in evaluating the safety and activity of CpG ODN planned for human use in a relevant animal model. Although Davis and colleagues (20, 21, 22) showed that K ODN increased the seroconversion rate and Ab response of orangutans and aotus monkeys immunized with hepatitis B vaccine and malaria proteins, that group was unable to document improved protection against infection by challenge studies. Moreover, no studies have compared the activity of K ODN with the recently discovered D class of ODN in primates.
This work examines whether rhesus macaques provide a useful model for assessing the activity of CpG ODN in vivo. In vitro studies established that PBMC from rhesus macaques responded to the same panel of K and D ODN that were highly active on human PBMC. Building on results from murine studies (23, 24), CpG ODN were coadministered with a mixture of OVA plus alum. The ODN significantly boosted the Ag-specific IgG response of macaques, with D being superior to K ODN. A cutaneous Leishmania infection model was used to examine whether CpG ODN could boost protective immunity in primates. The nature, severity, duration, and histopathology of the cutaneous disease caused by Leishmania major in macaques is quite similar to that in humans (25, 26). Results indicate that D ODN significantly improve the protection conferred by coadministered heat-killed Leishmania vaccine (HKLV).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy 3-year-old female rhesus macaques (Macaca mulatta) were obtained from the Food and Drug Administration colony in Morgan Island, SC. All studies were approved by the Center for Biologics Evaluation and Research Animal Care and Use Committee, and were conducted in an American Association for the Accreditation of Laboratory Animal Care-accredited facility. Animals were monitored daily by veterinarians. No systemic or local adverse reactions to CpG ODN, OVA, or HKLV immunizations were observed. Treatments were administered and peripheral blood samples obtained from ketamine-anesthetized animals (10 mg/kg, Ketaject; Phoenix Pharmaceuticals, St. Joseph, MD).
Vaccination groups and protocol
Two in vivo studies were conducted: 1) three monkeys per group were immunized s.c. and boosted 12 wk later with a mixture of 4 µg of OVA, 250 µg of ODN, and 125 µg of alum (Rehydragel HPA; Reheis, Berkeley Heights, NJ); 2) five to six monkeys per group were immunized s.c., and boosted 4 wk later with 250 µg of GMP-grade HKLV (Biobras, Montes Claros, Brazil) plus 125 µg of alum, as previously described (27). The HKLV was administered alone, or combined with 500 µg of ODN. Preliminary studies established that this dose of ODN was active in vivo and well-tolerated. Animals were exposed to nonviable Leishmania amazonensis metacycle promastigotes on wk 8, a treatment that induced no disease and no change in Ab titer or proliferative response to Leishmania Ags when compared with control animals. Animals were challenged on the forehead on wk 14 with 107 viable L. major (WHOM/IR/-/173) metacyclic promastigotes intradermally. The monkeys developed a typical self-limited in situ lesion characterized by erythema, induration, and ulceration. Lesion size, which reflects the severity of infection (25, 26), was measured weekly.
Oligodeoxynucleotides
ODN (Table I
) were synthesized by
the Center for Biologics Evaluation and Research Core Facility. All ODN
had <0.1 EU of endotoxin per milligram of ODN as assessed by a
Limulus amebocyte lysate assay (QCL-1000; BioWhittaker,
Gaithersburg, MD).
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Human and monkey mononuclear cells were isolated by density gradient centrifugation of PBMC over Ficoll-Hypaque as described (17). Cells were washed three times and cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 1.5 mM L-glutamine, and 100 U/ml penicillin/streptomycin at 5 x 105 cells/well in the presence of 3 µM ODN. Supernatants were collected after 72 h and tested by ELISA for cytokine and Ab levels.
ELISA
Ninety-six-well microtiter plates (Millipore, Bedford, MA) were
coated with Abs that cross-reactively recognized human and macaque IL-6
(R&D Systems, Minneapolis, MN), IFN- (PBL Biomedical Laboratories,
New Brunswick, NJ), and IgG (Boehringer Mannheim, Mannheim,
Germany). The plates were blocked with PBS-5% BSA (17).
Culture supernatants from PBMC cultures were added, and their cytokine
content quantitated by the addition of biotin-labeled anti-cytokine
Ab followed by phosphatase-conjugated avidin and phosphatase-specific
colorimetric substrate. Standard curves were generated using known
amounts of recombinant human cytokine or purified Ig. All assays were
performed in triplicate. Titers of Abs to OVA in sera were assayed on
OVA-coated plates.
ELISPOT
The number of PBMC secreting IFN- in response to soluble
Leishmania Ag (SLA) was determined by ELISPOT as described
(28). Briefly, Millipore 96-well filtration plates
(Millipore, Bedford, MA) were coated overnight at 4°C with 1 µg/ml
of anti-human IFN- Abs (clone GZ4; Alexis, San Diego, CA) in PBS
and then blocked with PBS-5% BSA for 2 h. The plates were
overlaid with 5 x 105 cells/well (1–2
series per monkey) and incubated at 37°C in a humidified 5%
CO2 in air incubator for 18 h in the
presence of 25 µg of SLA. The plates were then washed with
water-0.025% Tween and overlaid with biotin-conjugated anti-human
IFN- (clone 76-B-1; Mabtech, Nacka, Sweden). After 2 h, the
plates were washed again and then overlaid with alkaline
phosphatase-conjugated streptavidin. Spots were visualized by the
addition of 5-bromo-4-chloro-3-indolyl phosphate (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) and counted using the KS ELISPOT
Imagine System (Carl Zeiss, Thornwood, NY).
Cell proliferation assay
A total of 105 PBMC/well were incubated with 3 µM of ODN for 68 h, pulsed with 1 µCi of [3H]thymidine and harvested 4 h later. All assays were performed in triplicate.
Statistical analysis
Statistically significant differences were determined using a two-tailed nonparametric ANOVA with Dunnett’s post test analysis. Differences in lesion sizes were tested by repeated measures ANOVA using the Proc Mixed procedure from the statistical analysis system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies established that human PBMC respond to two
structurally distinct classes of CpG ODN (17). D-type ODN
triggered the secretion of IFN- and IFN- (17),
whereas K ODN induced human PBMC to proliferate and secrete IL-6 and
IgM (Fig. 1, Ref. 17 , and
data not shown). Analysis of several hundred CpG ODN identified several
D and K ODN that strongly activated human PBMC (17). These
ODN were tested for their ability to stimulate PBMC from rhesus
macaques.
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production. Results show that macaque PBMC are
activated by the same D ODN that stimulate human PBMC
(p < 0.002, Fig. 1
). In contrast, neither K
ODN, nor control ODN that are structurally similar to D but lack the
critical CpG dinucleotide have this effect.
Proliferation and IL-6 secretion were used to compare the response of
macaque and human PBMC to K ODN (Fig. 1). PBMC from both species were
stimulated by K ODN to proliferate (p < 0.002)
and secrete IL-6 (p < 0.01), whereas controls
of the same structure as K ODN, but lacking the critical CpG motif,
failed to trigger immune stimulation. These findings demonstrate that
the pattern of reactivity of PBMC from rhesus macaques
(n = 20) and humans (n = 8–20) to K
and D ODN is quite similar.
Ongoing studies in our lab indicate that individual humans and monkeys
vary in their response to specific K and D sequences. Indeed, no single
D or K motif is optimally stimulatory in all donors (Ref.
29 and C. Leifer, unpublished observations).
However, mixtures of ODN were identified that strongly stimulated PBMC
from all human donors. These mixtures were tested on PBMC from macaques
and found to be uniformly active (Fig. 2). Subsequent in vivo studies were
conducted with these ODN mixtures.
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Previous studies in mice showed that CpG ODN could boost the
immune response to a coadministered protein Ag (such as OVA). This
effect was amplified by adding alum to the mixture of CpG ODN plus Ag
(23, 30, 31). Building on these results, macaques were
immunized and boosted with a mixture of OVA, alum, and ODN. Animals
immunized with mixtures containing D ODN increased their IgG
anti-OVA response 470-fold after primary (p
< 0.05) and 600-fold after secondary (p <
0.01) immunization (Fig. 3). By
comparison, K ODN boosted the IgG Ab response 7-fold after primary, and
35-fold after secondary immunization when compared with pretreatment
values (p < 0.05). Macaques immunized with OVA
plus control ODN generated only a 4-fold increase in anti-OVA
titer. These findings indicate that D ODN are particularly effective at
boosting the Ag-specific humoral response to a coadministered
Ag.
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Previous human clinical trials showed that HKLV was safe, but
poorly immunogenic (26). Building on evidence that HKLV
combined with alum and IL-12 induces short-term protective immunity in
rhesus macaques (27), and that CpG ODN plus alum increased
the immune response to the hepatitis B vaccine in cyalomongus monkeys
(20), we immunized and boosted macaques with a mixture of
HKLV, alum, and CpG ODN. PBMC from these animals were isolated 10 days
postboost and restimulated in vitro with Leishmania Ag for
18 h. As seen in Fig. 4, both K and
D ODN significantly increased the number of PBMC triggered to secrete
IFN- (p < 0.05). In contrast, animals
immunized with alum-adsorbed HKLV alone showed no increased IFN-
production when compared with unimmunized controls.
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). Monkeys vaccinated with
HKLV-alum plus K ODN developed lesions of similar size, although the
peak lesion formation was slightly delayed. Animals immunized with
HKLV-alum plus D ODN had significantly smaller lesions (maximal size
80 ± 13 mm2, p < 0.05),
consistent with a reduced parasite burden (32).
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All animals treated with CpG ODN, either alone or with Ag, remained healthy and active throughout the study. No hematologic or serologic abnormalities were observed 3 days or 9 mo after injection, and no weight loss or change in behavior was detected following administration of CpG ODN at therapeutic doses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in animals
vaccinated with HKLV. Of greater importance, D ODN significantly
increased the protective response elicited by a coadministered HKLV
vaccine.
Several previous reports examined whether K ODN could act as immune
adjuvants in nonhuman primates (20, 21, 22). Studies by Davis
and colleagues (20, 21, 22) demonstrated that K ODN boosted
the Ag-specific serum IgG response to alum-adjuvanted hepatitis B
vaccine, and to a peptide from the circumsporozoite protein of malaria
in orangutans and aotus monkeys. This was consistent with results from
earlier studies in mice showing that CpG ODN plus alum synergize to
boost immunity to Ag (23, 30, 31, 33). Yet these
experiments did not establish whether the resulting immune response
conferred protection against infection. The current experiments confirm
that K ODN boost the Ab response to a coadministered protein (OVA).
They further document that D ODN are significantly more effective in
this role, boosting Ab production by >500-fold over pretreatment
levels and >100-fold over OVA plus alum (Fig. 3).
Cutaneous infection of macaques with L. major provides a
means for testing the protective efficacy of CpG ODN vaccine
combinations. The nature, severity, and duration of the cutaneous
disease caused by L. major in macaques is quite similar to
that in humans (25). The leading Leishmania
vaccine candidate (HKLV) has proven safe but poorly immunogenic in
clinical trials (26). Coadministration of both
D and K ODN with this alum-adjuvanted HKLV vaccine significantly
increased the number of PBMC triggered to secrete IFN- when
stimulated with Leishmania Ag in vitro. However, the
critical test of any vaccine/adjuvant combination is its ability to
induce protective immunity. Results show that the cutaneous lesions of
macaques vaccinated with HKLV plus D ODN were significantly reduced
when compared with HKLV-alum alone. Previous studies established that a
reduction in lesion size correlates with a reduced parasite load (Ref.
32 and R. A. Seder, unpublished observations).
These findings suggest that the ability of D ODN to stimulate IFN-
and IFN- production while promoting the maturation of APC may be
particularly useful for the induction of a protective response against
Leishmania (17, 18).
K and D ODN have unique structural properties. Optimally active K ODN
have a phosphorothioate backbone and express multiple TCGTT
and/or TCGTA motifs. D ODN have a mixed
phosphodiester/phosphorothioate backbone, express a single
self-complementary purine/pyrimidine/CpG/purine/pyrimidine motif, and
are capped by a 3' poly G tail. These two types of ODN trigger human
and rhesus PBMC to mount distinct responses. K ODN stimulate B cells to
proliferate and secrete IgM, plasmacytoid DC precursors to mature and
secrete IL-8, and monocytes to produce IL-6 (Fig. 1 and Refs. 17, 18, 34 and 35). By comparison, D ODN trigger
plasmacytoid DC to produce large amounts of IFN-, and directly or
indirectly trigger NK cells to secrete IFN-, and myeloid DC to
mature (Fig. 1, Refs. 17, 18, 19 , and M. Gursel, unpublished
observations). We postulate that D ODN may be superior vaccine
adjuvants when a Th1-dependent immune response is required, whereas K
ODN may excel at the induction of proinflammatory responses.
It is likely that differences in the recognition, uptake, and/or processing of K and D ODN underlie their distinct functional properties. It was recently established that Toll-like receptor 9 plays a critical role in CpG ODN-mediated activation of human and murine immune cells (35, 36). Using HEK 293 cells transfected with human Toll-like receptor 9, our lab confirmed that the recognition of K-type ODN was mediated by this receptor (37). However, our ongoing studies indicate that these transfected cells do not respond to D ODN, suggesting that a second type of receptor may be involved in D ODN-mediated immune activation.
Clinical trials exploring the utility of CpG ODN as vaccine adjuvants, immunotherapeutic agents, and anti-allergens have been initiated (38). Current results suggest that rhesus macaques may be a useful model for evaluating the safety and activity of these agents in vivo. In this context, neither local nor systemic adverse reactions to K or D CpG ODN were detected in any of the animals studied. Moreover, although K ODN similar to those currently in human clinical trials were found to be active in vivo, our results indicate that D may be superior vaccine adjuvants, improving the humoral response and protective efficacy to certain coadministered vaccines.
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Acknowledgments |
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Footnotes |
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2 Current address: Iomai Corporation, 20 Firstfield Road, Suite 250, Gaithersburg, MD 20878.
3 Address correspondence and reprint requests to: Dr. Dennis Klinman, Center for Biologics Evalutaion and Research/Food and Drug Administration, Building 29 A, Room 3 D 10, Bethesda, MD 20892-4555. E-mail address: Klinman{at}CBER.FDA.GOV
4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; SLA, soluble Leishmania Ag; DC, dendritic cell; HKLV, heat-killed Leishmania vaccine.
Received for publication July 19, 2001. Accepted for publication December 4, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Note that macaques immunized with HKLV plus D
ODN had significantly smaller lesions (p <
0.01).