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Cooperative Research Center for Vaccine Technology, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
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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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Use of LHRH-based vaccines in humans as an immunocontraceptive, although theoretically possible, is unlikely, because profound behavioral modification can occur as a consequence of inhibiting the production of testosterone in males. However, a very active area of research is the use of LHRH-based vaccines in the control of hormone-dependent cancers, including breast (4) and prostate cancers (5, 6). Because the sequence of LHRH is conserved in all mammals, LHRH-based immunocontraceptives are candidates for use in the companion animal and livestock arenas. Here the concerns of behavioral modification are not of such overriding importance and the use of immunocontraception as an alternative to surgical castration has attracted a great deal of attention (5, 7, 8, 9, 10).
Although the peptide nature of the hormone makes it an obvious candidate for epitope-based vaccine design, the major problem of poor immunogenicity in the absence of an adjuvant confronts this as it does all peptide-based vaccine candidates. Of the many adjuvants currently available, many are too toxic for use in humans and animals or are ineffective. However, lipids and lipopeptides have been shown to be capable of adjuvanting otherwise weak immunogens (11, 12, 13, 14, 15) and show none of the harmful side effects of other adjuvant formulations. In lipopeptide constructs, a lipid moiety with known adjuvanticity can be covalently coupled to peptide to generate a fully synthetic self-adjuvanting and potentially safe vaccine (16, 17, 18, 19). There have been a number of successful applications of lipopeptides as immunogens to generate Ab and cellular responses (12, 17, 20), and several different lipids have been used for these purposes. Tripalmitoyl-S-glyceryl cysteine (Pam3Cys) is a synthetic version of the N-terminal moiety of Braun’s lipoprotein that spans the inner and outer membranes of Gram-negative bacteria and has been shown to be capable of stimulating virus-specific CTL responses against influenza virus-infected cells (21) and to elicit protective Abs against foot-and-mouth disease (17) when coupled to the appropriate synthetic epitopes. Recently dipalmitoyl-S-glyceryl cysteine (Pam2Cys), an analog of Pam3Cys, has been synthesized (22) and been shown to correspond to the lipid moiety of macrophage-activating lipopeptide 2 isolated from myoplasma that lack cell walls (23, 24, 25). In contrast to Pam3Cys, Pam2Cys has only two ester-bound palmitic acids and a free amino group, which leads to improved water solubility of Pam2Cys-peptide constructs compared with Pam3Cys-peptide constructs. It has also been reported that Pam2Cys is a more potent stimulator of splenocytes (22) and macrophages (24, 25) than is Pam3Cys. The structural similarity of Pam2Cys to Pam3Cys and its enhanced macrophage-activating properties, as well as its less hydrophobic nature, indicate that Pam2Cys could also be used as a self-adjuvanting moiety.
In this study, we describe the synthesis of Pam3Cys- and Pam2Cys-peptide constructs based on LHRH and report on their immunogenicity and ability to sterilize female mice when inoculated in the absence of additional adjuvants. These lipopeptides consist of an epitope for CD4+ T cells and the LHRH sequence, which contains one or more epitopes for B cells. Because the LHRH sequence contains no T cell epitopes, the CD4 + T cell epitope GALNNRFQIKGVELKS from the L chain of influenza virus hemagglutinin (26) was incorporated in the construct to provide T cell help. In a previous publication from this laboratory, it was shown that a peptide composed of these two epitopes assembled as a colinear tandem construct was a potent immunogen and able to induce reproductive sterility in female mice when inoculated in CFA (27). In the present study, either Pam3Cys or Pam2Cys was attached to the peptide-based vaccine to allow delivery in the absence of additional adjuvant. The lipid-containing groups were attached at two different positions within the peptide to produce linear and branched configurations of lipopeptide. The immunogenic properties of these vaccines were determined by examining the titer and isotype of anti-LHRH Ab, and the biological effects were determined by measuring any influence on the reproductive capabilities of vaccinated mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unless otherwise stated, chemicals were of analytical grade or its equivalent. N,N'-dimethylformamide, piperidine, trifluoroacetic acid (TFA), O'benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate, 1-hydroxybenzotriazole, and diisopropylethylamine and diisopropylcarbodiimide were obtained from Auspep (Melbourne, Australia) and Sigma-Aldrich (Castle Hill, New South Wales, Australia). O'benzotriazole-N,N,N',N'-tetramethyl-uronium-tetrafluoroborate was obtained from Bachem (Bubendorf, Switzerland). Dichloromethane (DCM) and diethylether were obtained from Merck (Kilsyth, Australia). Phenol and triisopropylsilane were obtained from Sigma-Aldrich; trinitrobenzylsulfonic acid and diaminopyridine (DMAP) were obtained from Fluka (Buchs, Switzerland); 1,8-diazabicyclo-[5.4.0]undec-7-ene was obtained from Sigma-Ald-rich; and palmitic acid was obtained from Fluka.
Synthesis of Pam3Cys and Pam2Cys
Pam3Cys was prepared according to the method described by Wiesmuller et al. (28) and modified by Zeng et al. (29). The synthesis of Pam2Cys was adapted from previously described methods (30, 31); specifically, 3-bromo-propan-1,2-diol was used instead of 3-chloro-propan-1,2-diol, and centrifugation, not filtration, was used to recover the product.
Peptide synthesis
The general procedure used in this laboratory for the synthesis of peptides has been described previously (32). To enable lipid attachment between the CD4+ T cell epitope and LHRH, F-moc-lysine(Mtt)-OH was inserted at a point between the two epitopes in the approximate center of the resin-bound peptide. Following completion of peptide synthesis, the Mtt group was removed by continual flow washing with 1% TFA in DCM over a period of 30–45 min.
Pam3Cys or Pam2Cys was then coupled to the exposed 
-amino group
according to the procedure described previously (29).
Briefly, a 2-fold excess of Pam3Cys or Pam2Cys,
O'benzotriazole-N,N,N',N'-tetramethyl-uronium-tetrafluoroborate,
and 1-hydroxybenzotriazole was dissolved in DCM, and a 3-fold excess of
diisopropylethylamine was added. This solution was then added to the
resin-bound peptide to generate lipopeptide. Monitoring the reaction by
analytical reversed phase (RP)-HPLC, we found that quantitative
palmitoylation was achieved after 16 h.
All resin-bound peptide constructs were cleaved from the solid-phase support with reagent B (88% TFA, 5% phenol, 2% triisopropylsilane, and 5% water) for 2 h, and purified by RP chromatography as described previously (33). Analytical RP-HPLC was conducted using a Vydac C4 column (4.6 x 300 mm; Edwards Instruments, New South Wales, Australia) installed in a Waters (Box Hill, Victoria, Australia) HPLC system and developed at a flow rate of 1 ml/min using 0.1% TFA in H2O and 0.1% TFA in CH3CN as the limit solvent. All products presented as a single major peak on analytical RP-HPLC and had the expected mass when analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry on a Bruker BIFLEX instrument (Bruker Daltonics, Bremen, Germany) equipped with delayed ion extraction. The final quantitation of the immunogens was done by measuring the absorption at 280 nm, exploiting the presence of a tryptophan and a tyrosine residue in the peptide constructs (molar extinction coefficient of 6.6 x 103).
Two serine residues are present between the protein and lipid portion of Braun’s lipoprotein from which Pam3Cys is derived (28). Furthermore, the presence of serine between the lipid and peptide moieties of Pam3Cys-containing lipopeptides has been said to improve immunogenicity (34). Therefore, we also investigated the effect of serine by incorporating two residues between the peptide and lipid moieties of the Pam2Cys-containing peptide constructs. This was simply done by sequential addition of two serine residues to the peptide before covalent attachment of the lipid moiety.
A schematic diagram of the peptides and lipopeptides used in this study
is shown in Fig. 1
and a summary of their
characteristics, conducted by analytical RP-HPLC and mass spectrometry,
is presented in Table I.
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Groups of five female BALB/c mice, 6–8 wk old, were inoculated at day 0 and again on day 28. For s.c. inoculations (100 µl volume per dose), lipopeptide constructs were prepared in saline and nonlipidated peptides formulated as an emulsion in an equal volume of CFA for the primary injection or IFA for the secondary inoculation. For intranasal (i.n.) inoculations, 50 µl of peptide in saline were applied to the nares of mice anesthetized with penthrane for inhalation. Sera were prepared from blood taken at 4 wk after the primary inoculation and 2 wk after the secondary inoculation.
ELISAs
ELISAs were conducted on serum samples as described previously (27) using LHRH as the coating Ag. The titers of Ab are expressed as the reciprocal of the highest dilution of serum to achieve an OD of 0.2, which represents approximately five times the background binding in the absence of Ab. The isotype of Abs specific for LHRH was determined using rabbit antisera directed against mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA (ICN Pharmaceuticals, Costa Mesa, CA) as previously described (27).
Fertility studies
After being inoculated with peptide immunogen and following exposure to male mice, female mice were tested for their ability to drop litters. A group of mice immunized with saline in CFA was used as a control. A male mouse was introduced into a cage in which two or three female mice were kept, and male mice were rotated between each cage to expose each group of female mice to every male. Males and females were kept together for a total of 3 wk, at the end of which the males were removed and the females kept under observation.
Dendritic cell (DC) culture
DC were cultured in medium based on complete IMDM. This
consisted of IMDM containing 25 mM HEPES and without 
-thioglycerol
or L-glutamine (JRH Bioscience, Lenexa, KS), supplemented
with 10% (v/v) heat-inactivated (56°C, 30 min) FCS (CSL, Victoria,
Australia), gentamicin (24 µg/ml), glutamine (2 mM), sodium pyruvate
(2 mM), penicillin (100 IU/ml), streptomycin (180 µg/ml), and 2-ME
(0.1 mM). For DC generation, complete IMDM was further supplemented
with 30% supernatant from cultured NIH/3T3 cells and 5% GM-CSF in the
form of a supernatant from Ag8653 cells transfected with the GM-CSF
gene (DC medium).
The culture method for immature DC was adapted from Winzler et al. (35). Spleen cells from a BALB/c mouse were seeded at 1.5 x 106 cells per 55-mm dish (Techno-Plas, South Australia, Australia) in 3 ml of DC medium and incubated at 37°C with 5% CO2. All the equipment used for culturing was pyrogen free. The medium was changed every 4 days, and all cells were returned to the dish. On day 12, both suspended and weakly adherent cells were collected by forcefully pipetting and then aspirating the medium. The procedure was repeated with 2 ml of PBS. The remaining strongly adherent cells were discarded. The collected cells were pelleted by centrifugation and reseeded into a new dish. Cells were subsequently maintained on a 4-day alternating cycle of medium change and passage. After 1 mo of continuous culturing, the floating and semiadherent cells took on the appearance and staining characteristics of immature DC and are referred to as D1 cells. Under these passage conditions, the majority of cultured D1 cells maintain an immature phenotype characterized by an intermediate expression level of cell surface MHC class II molecules.
Flow cytometric analysis of D1 cells
D1 cells (8 x 104 cells per sample)
were seeded in a new petri dish with 1 ml of DC medium and incubated
with 0.0045 nmol lipopeptide, dissolved in complete IMDM. LPS purified
from Escherichia coli serotype O111:B4 (Difco, Detroit, MI;
a kind gift from Dr. E. M. Anders, Department of Microbiology and
Immunology, University of Melbourne) was used at 5 µg/ml as a
positive control for DC maturation. After overnight incubation, the
cells were harvested and washed once with PBS with 1% FCS. To prevent
nonspecific binding to Fc
RII/III, the cells were preincubated with
20 µl of normal mouse serum for 5 min at room temperature. The cells
were then exposed to FITC-conjugated mAb 14-4-4S (IgG2a,
anti-I-Ek,d) (36) for 30 min on
ice. mAb 36/1 (37) specific for the hemagglutinin of
influenza virus was used as an isotype control. All Abs were used at
2.5 µg/ml. The samples were washed once with PBS containing 1% FCS
and fixed with PBS containing 4% paraformaldehyde on ice for 15 min.
Flow cytometry analysis was performed using a FACSort (BD Biosciences,
San Jose, CA), and the data were analyzed using FlowJo software (Tree
Star, San Carlos, CA)
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Visual inspection (Fig. 1
) of the different lipopeptide
preparations showed that they differed markedly in their solubilities.
Better solubility was most evident in those cases where lipid was
attached between the two epitopes at the approximate center of the
molecule; [T]-Lys(Pam2Cys)-[B] and
[T]-Lys(Pam3Cys)-[B] were soluble in saline at
concentrations of at least 8 mg/ml (no higher concentrations were
examined), whereas constructs in which lipid was attached to the N
terminus of the sequence formed opalescent solutions at concentrations
as low as 0.25 mg/ml. Efforts to increase the solubility of peptides
with N-terminally linked lipid by the incorporation of two hydrophilic
serine residues between the lipid and peptide moieties, i.e.,
Pam2Cys-Ser-Ser-[T]-[B] and Pam3Cys-Ser-Ser-[T]-[B], proved
unsuccessful. In fact, the lipopeptide Pam3Cys-Ser-Ser-[T]-[B] was
so insoluble that it could not be purified by RP-HPLC under conditions
used for the other lipopeptides.
Immunogenicity of lipopeptides
The three lipopeptides, Pam2Cys-Ser-Ser-[T]-[B],
[T]-Lys(Pam3Cys)-[B], and
[T]-Lys(Pam2Cys)-[B], when administered s.c. in saline,
induced high levels of anti-LHRH Ab. In fact, Ab titers induced
after two doses of these lipopeptides were similar to those obtained
with [T]-[B] or [T]-Lys-[B] when they were administered in CFA
(Fig. 2). The titers of anti-LHRH Abs
in sera of mice that had received Pam2Cys-[T]-[B] or
Pam3Cys-Ser-Ser-[T]-[B] may be slightly lower. The two soluble
branched lipopeptides [T]-Lys(Pam3Cys)-[B] and
[T]-Lys(Pam2Cys)-[B] induced 10- to 100-fold higher
levels of anti-LHRH Ab following the primary inoculation than did
the other less soluble lipopeptide constructs. Two groups of five mice
receiving [T]-[B], which was simply admixed with
Pam3Cys-Ser-(Lys)4 in the ratio 1:1 or 1:5, did
not elicit significant levels of anti-LHRH Ab, a finding that
contrasts with other results reported using
Pam3Cys-Ser-(Lys)4 (38) as an
externally added adjuvant.
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) indicate that none of the mice
that received either of the two branched soluble lipopeptide constructs
[T]-Lys(Pam3Cys)-[B] or
[T]-Lys(Pam2Cys)-[B], which were administered in saline,
or the two nonlipidated constructs, [T]-[B] or [T]-Lys-[B],
which were administered in CFA, became pregnant. Two animals from the
groups that received Pam2Cys-[T]-[B] or Pam3Cys-Ser-Ser-[T]-[B]
did drop litters 2 wk after the second dose of vaccine and one mouse
from the group that received Pam2Cys-Ser-Ser-[T]-[B] also delivered
a litter. All members of control groups, i.e., mice that received
saline in CFA or the peptide [T]-[B] coadmixed with
Pam3Cys-S-(Lys)4, dropped litters.
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Pam2Cys is a more potent adjuvant than Pam3Cys
The results presented in Fig. 2 and Table II indicate that
the two branched lipopeptides [T]-Lys(Pam3Cys)-[B] and
[T]-Lys(Pam2Cys)-[B] not only were more soluble but also
elicited higher Ab titers, particularly in the primary Ab response,
than the linear immunogens Pam2Cys-[T]-[B],
Pam3Cys-Ser-Ser-[T]-[B], and Pam2Cys-Ser-Ser-[T]-[B]. The
results presented in Fig. 2 and Table II also indicate that
Pam2Cys-containing peptides may be better immunogens than
Pam3Cys-containing peptides. To examine this further, we investigated
the effect of decreasing the dose on the immunogenicity of
[T]-Lys(Pam3Cys)-[B] and
[T]-Lys(Pam2Cys)-[B]. The results of the Ab titer
determinations (Table III) is suggestive
of a trend toward somewhat higher Ab titers elicited by
[T]-Lys(Pam2Cys)-[B], but a striking difference is
observed in the mating trial; 1 of 5 and 0 of 5 mice receiving 10 and 1
nmol [T]-Lys(Pam2Cys)-[B], respectively, dropped
litters, whereas 3 of 5 and 5 of 5 mice receiving
[T]-Lys(Pam3Cys)-[B] at these doses dropped litters. The
effect of including two additional serine residues into the
Pam2Cys-containing immunogens had little effect on the fertility status
of animals, although there was some improvement in the Ab titers that
were generated following the second dose (1 and 10 nmol) of
[T]-Lys(Pam2Cys-Ser-Ser)-[B].
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It has been reported previously that immunization with Pam3Cys-containing peptides by the mucosal (oral or i.n.) route induces strong mucosal and/or systemic immune responses (33, 39, 40). In the present series of experiments, we compared the immunogenicity of the linear Pam2Cys-Ser-Ser-[T]-[B] and branched [T]-Lys(Pam2Cys-Ser-Ser)-[B] peptides following inoculation in saline by the i.n. route. The same vaccines were also inoculated by the s.c. route, and the systemic anti-LHRH Ab responses were measured. The solution used for inoculation of [T]-Lys(Pam2Cys-Ser-Ser)-[B] was clear and the one for Pam2Cys-Ser-Ser-[T]-[B] was opalescent, indicating solubility differences between the two preparations.
Following two i.n. inoculations, each of the vaccines induced
similar titers of serum anti-LHRH Abs, which were slightly lower
than those induced following s.c. inoculation (Table IV). The more soluble
[T]-Lys(Pam2Cys-Ser-Ser)-[B] induced significantly
higher levels of anti-LHRH Ab 4 wk after a single dose than did the
less soluble Pam2Cys-Ser-Ser-[T]-[B] (p =
0.00007); in fact, this was similar to the result obtained following
s.c. inoculation. The results of the fertility trial showed that two
i.n. inoculations of [T]-Lys(Pam2Cys-Ser-Ser)-[B]
prevented all mice from becoming pregnant in contrast to those animals
receiving Pam2Cys-Ser-Ser-[T]-[B] i.n., in which 3 of 5 mice
dropped litters.
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. Twenty-six weeks following the second dose of vaccine, the
levels of Ab in all mice had dropped below those observed 2 wk after
the second dose. However, the decrease in anti-LHRH Ab in the group
that received [T]-Lys(Pam2Cys-Ser-Ser)-[B] s.c. was much
less apparent, again indicating the immunological superiority of the
configuration in which Pam2Cys-Ser-Ser is attached at the center of the
molecule.
We also determined the titers of individual Ab isotypes that were
directed toward LHRH and obtained from animals following two s.c. or
i.n. doses of the soluble lipopeptide
[T]-Lys(Pam2Cys-Ser-Ser)-[B] (Fig. 3). i.n. inoculation appeared to induce
higher levels of IgG3, IgG2b, and possibly IgM than did s.c.
inoculation, even though the amount of total Ig induced by i.n.
inoculation was less.
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The priming of naive CD4+ T cells in
secondary lymphoid organs by DC is preceded by maturation of DC upon
exposure to Ag. This maturation is characterized by up-regulation of
MHC products and costimulatory molecules on the DC surface.
Therefore, we determined whether the various peptides and lipopeptides
could differentially activate DC in an attempt to explain the different
immunogenic properties of these vaccine candidates. The results
of experiments (Fig. 4) in which a line
of immature DC, D1 cells, were exposed to peptides, stained for surface
expression of MHC class II molecules, and then analyzed by flow
cytometry demonstrated that [T]-Lys(Pam2Cys-Ser-Ser)-[B]
was the most effective and Pam2Cys-[T]-[B] was the least effective
in causing maturation of DC. The ability of
[T]-Lys(Pam2CysS-er-Ser)-[B] to up-regulate class II
expression approached that of bacterial LPS, and
Pam2Cys-Ser-Ser[T]-[B] and [T]-Lys(Pam2Cys)-[B]
displayed intermediate levels of activation. The nonlipidated peptide
was unable to induce maturation of D1 cells at a rate greater than
26%, which is the rate that occurs spontaneously in culture. The
ability of the lipopeptides to induce the maturation of D1 cells was
concentration dependent (data not shown). The relative abilities of
these lipopeptides to induce maturation of D1 cells directly reflected
their ability to induce Ab, providing a possible mechanism for
differences in immunogenicity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the solubility of the resulting vaccine was greatly improved by placing lipids in the approximate center of the peptide immunogen between the T cell epitope and LHRH instead of at the more usual position at the N terminus. A clear solution in saline at the concentration required for inoculation could easily be obtained with these branched structures. In contrast, the immunogens in which the lipid was coupled at the N terminus were less soluble, giving a cloudy or opalescent solution in saline. Investigation of the Ab responses and subsequent fertility trials indicated that the water-soluble lipopeptides induced higher Ab titers 4 wk after the primary inoculation and were also more efficient in preventing pregnancy than were the less soluble lipopeptides where lipid was attached to the N terminus. A water-soluble self-adjuvanting vaccine has clear advantages over partially soluble or insoluble material, allowing for simplification of the manufacturing process and more accurate metering of dose.
Investigations into the effects of varying the lipopeptide dose
indicated that Pam2Cys-containing lipopeptides are better immunogens
than are Pam3Cys-containing peptides. This may correlate with the
finding that Pam2Cys is a more potent macrophage and splenocyte
stimulator than Pam3Cys (22, 24, 25). In the present
study, we found that insertion of two serine residues between the lipid
moiety and the peptide sequence increased the potency of the resulting
Pam2Cys-containing immunogens. Two serine residues are present in the
original Braun’s lipoprotein sequence found in bacterial cell walls
from which Pam3Cys is derived (28) and were reported in
early publications (17, 34) to be important for activity
of Pam3Cys-containing peptide constructs. Later studies also
demonstrated that insertion of Ser-D-Ser instead of Ser-Ser
markedly decreased immunogenicity (41). However, serine
residues are absent in naturally occurring Pam2Cys-containing
structures including macrophage-activating lipopeptide 2
(25) and the splenocyte-stimulating lipopeptides initially
investigated (22). In this study, where lipid is attached
to the N terminus, the two serine residues could be acting either as an
inert spacer between the lipid and the peptide sequence or as an
extension of the T helper cell epitope and perhaps modulating
immunological activity. In those cases where lipid is coupled to the
-amino group of a lysine residue at the center of the molecule, the
two serine residues are probably acting as a spacer. Experiments where
the two serine residues are replaced by an inert spacer such as
6-aminohexanoic acid may resolve the question of whether these serine
residues per se are important.
To determine a mechanism for the enhanced immunogenicity of particular lipid-containing constructs, we considered the known ability of Pam3Cys and Pam2Cys to activate macrophages. It is now understood that macrophages can be stimulated by microbial products that bind to cell surface receptors; the signal resulting from this binding event is transmitted via Toll-like receptors and results in the production of pro-inflammatory cytokines and chemokines. These receptors are also present in populations of DC (for review, see Ref. 42) and, when engaged, transmit signals for cellular maturation and migration as well as for the production of molecules required for efficient Ag presentation.
The various synthetic lipopeptide vaccines used in this study were found to induce the up-regulation of class II MHC molecules, a marker used to assess DC maturation, on the surface of immature DC. In contrast, the nonlipidated peptide construct was unable to cause maturation of DC, indicating that the lipid moiety is responsible for the effect. Microbial lipopeptides have been similarly shown to activate DC of human origin via an interaction involving Toll-like receptor 2 (43). In this study, we demonstrate that the hierarchy of lipopeptide-induced maturation of DC reflects the hierarchy of immunogenicity exhibited by the peptide constructs, implying that the ability of the vaccine to interact with and induce maturation of DC leads to a better immune response, possibly by increasing the efficiency of CD4+ T cell priming by DC that have been signaled to mature and migrate to the draining lymph node.
The lipopeptides can trigger an immune response in the absence of additional adjuvant and can therefore be delivered by nonparenteral routes. Therefore, we investigated the Ab response following i.n. inoculation of Pam2Cys-containing peptides. Previous reports have shown that oral or i.n. inoculation of Pam3Cys-peptides induces strong mucosal and/or systemic responses (33, 39, 40). The results obtained in this study showed that i.n. inoculation of [T]-Lys(Pam2Cys-Ser-Ser)-[B] or Pam2Cys-Ser-Ser-[T]-[B] induced lower titers of systemic anti-LHRH Ab than those induced by inoculation by the s.c. route and also that the isotype profiles of immunoglobulins were different. i.n. inoculation of the soluble lipopeptide [T]-Lys(Pam2Cys-Ser-Ser)-[B] induced higher levels of IgG2b and IgG3 but lower levels of IgG1 and IgG2a compared with s.c. immunization. This may indicate that the two routes of immunization result in the induction of somewhat different subsets of T cells providing help for Ab production, which may, in part, be due to the different populations of DCs encountered at different sites. It may also reflect the preference that DCs have for molecules with unusual geometries (44).
With the possible exception of Pam3Cys-Ser-Ser-[T]-[B], the secondary Ab titers elicited by each of the other immunogens were comparable. Nevertheless, only in those cases where the branched lipopeptides [T]-Lys(Pam3Cys)-[B] and [T]-Lys(Pam2Cys)-[B] were used was reproductive sterility achieved. In the cases of branched lipopeptides, the Ab titers observed in the primary response were higher than those achieved with the linear forms of the lipopeptides. Comparisons of the Ab titers induced during primary and secondary Ab responses of successful (i.e., branched lipopeptide [T]-Lys(Pam3Cys)-[B] and [T]-Lys(Pam2Cys)-[B]) and unsuccessful (i.e., linear lipopeptide Pam2Cys-[T]-[B] or Pam3Cys-Ser-Ser-[T]-[B]) vaccines suggested that there may be a correlation between infertility and primary Ab response. However, we have previously shown (27) that litters are still produced by animals in which primary Ab titers are similar to the secondary Ab titers of infertile animals, indicating that it is not a simple matter of Ab titer determining fertility or otherwise. Furthermore, the avidities of Abs generated in these two situations are comparable (27). These observations suggest that the success or otherwise of antifertility vaccines based on LHRH is related to effects other than quantitative (titer) or qualitative (affinity) properties of the Ab; contributing factors could include the length of time for which the endocrine system is bathed in Ab. For these reasons, it seems to be advantageous to use those antifertility vaccines that not only induce high Ab titers but induce high Ab titers as quickly as possible. Of course, this property applies to most vaccines.
The efficacy of the antifertility vaccines was also examined following i.n. administration. i.n. inoculation of the water-soluble peptide construct [T]-Lys(Pam2Cys-Ser-Ser)-[B] induced significantly higher anti-LHRH Ab titers 4 wk after the first dose of vaccine than did Pam2Cys-Ser-Ser-[T]-[B]. Fertility trials conducted with these mice demonstrated that only i.n. inoculation with [T]-Lys(Pam2Cys-Ser-Ser)-[B] was able to totally inhibit fertility. Although similar Ab titers were apparent in both groups of mice following the second dose of Ag, high titers of Ab were elicited only during the primary response to [T]-Lys(Pam2Cys-Ser-Ser)-[B]. These results indicate that i.n. administration of epitope-based lipopeptides can produce very high titers of Ab that exert a biological effect.
Taken together, the measurements of Ab titers and the results of the fertility trials demonstrate that placement of self-adjuvanting lipid moieties between the B cell epitope and the T helper epitope, at the approximate center of a totally synthetic peptide vaccine, increases the solubility and the immunogenicity of the vaccine. This improved immunogenicity can be further improved by the introduction of two serine residues between the lipid and the peptide portion of these branched peptide vaccines. The finding that incorporation of lipid, self-adjuvanting moieties into different positions of peptide-based vaccines profoundly alters physical, immunogenic, and biological properties provides another strategy for successful vaccine design.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David C. Jackson, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail address: d.jackson{at}microbiology.unimelb.edu.au
3 Abbreviations used in this paper: LHRH, luteinizing hormone-releasing hormone; TFA, trifluoroacetic acid; DCM, dichloromethane; DMAP, diaminopyridine; Pam3Cys, tripalmitoyl-S-glyceryl cysteine; Pam2Cys, dipalmitoyl-S-glyceryl cysteine; DC, dendritic cell; i.n., intranasal; RP, reversed phase.
Received for publication April 29, 2002. Accepted for publication August 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in macrophages by mycoplasmal lipopeptides. Eur. J. Immunol. 28:4207.[Medline]
-Fmoc protected derivatives of S-(2,3-dihydroxypropyl)-cysteine and their application in peptide synthesis. Int. J. Pept. Protein Res. 38:545.[Medline]
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  H. H. Alphs, R. Gambhira, B. Karanam, J. N. Roberts, S. Jagu, J. T. Schiller, W. Zeng, D. C. Jackson, and R. B. S. Roden Protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of L2 PNAS, April 15, 2008; 105(15): 5850 - 5855. [Abstract] [Full Text] [PDF]
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 I. Bettahi, A. B. Nesburn, S. Yoon, X. Zhang, A. Mohebbi, V. Sue, A. Vanderberg, S. L. Wechsler, and L. BenMohamed Protective Immunity against Ocular Herpes Infection and Disease Induced by Highly Immunogenic Self-Adjuvanting Glycoprotein D Lipopeptide Vaccines Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4643 - 4653. [Abstract] [Full Text] [PDF]
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 E. B. Day, W. Zeng, P. C. Doherty, D. C. Jackson, K. Kedzierska, and S. J. Turner The Context of Epitope Presentation Can Influence Functional Quality of Recalled Influenza A Virus-Specific Memory CD8+ T Cells J. Immunol., August 15, 2007; 179(4): 2187 - 2194. [Abstract] [Full Text] [PDF]
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 S. Khan, M. S. Bijker, J. J. Weterings, H. J. Tanke, G. J. Adema, T. van Hall, J. W. Drijfhout, C. J. M. Melief, H. S. Overkleeft, G. A. van der Marel, et al. Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells J. Biol. Chem., July 20, 2007; 282(29): 21145 - 21159. [Abstract] [Full Text] [PDF]
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 Y. F. Lau, G. Deliyannis, W. Zeng, A. Mansell, D. C. Jackson, and L. E. Brown Lipid-containing mimetics of natural triggers of innate immunity as CTL-inducing influenza vaccines Int. Immunol., December 1, 2006; 18(12): 1801 - 1813. [Abstract] [Full Text] [PDF]
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 M. J. McConnell, X. Danthinne, and M. J. Imperiale Characterization of a permissive epitope insertion site in adenovirus hexon. J. Virol., June 1, 2006; 80(11): 5361 - 5370. [Abstract] [Full Text] [PDF]
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X. Zhang, A. Issagholian, E. A. Berg, J. B. Fishman, A. B. Nesburn, and L. BenMohamed Th-Cytotoxic T-Lymphocyte Chimeric Epitopes Extended by N{varepsilon}-Palmitoyl Lysines Induce Herpes Simplex Virus Type 1-Specific Effector CD8+ Tc1 Responses and Protect against Ocular Infection J. Virol., December 15, 2005; 79(24): 15289 - 15301. [Abstract] [Full Text] [PDF]
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 R. K. Naz, S. K. Gupta, J. C. Gupta, H. K. Vyas, and a. G.P. Talwar Recent advances in contraceptive vaccine development: a mini-review Hum. Reprod., December 1, 2005; 20(12): 3271 - 3283. [Abstract] [Full Text] [PDF]
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D. C. Jackson, Y. F. Lau, T. Le, A. Suhrbier, G. Deliyannis, C. Cheers, C. Smith, W. Zeng, and L. E. Brown A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses PNAS, October 26, 2004; 101(43): 15440 - 15445. [Abstract] [Full Text] [PDF]
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 S G Hannesdottir, X Han, T Lund, M Singh, R van der Zee, I M Roitt, and P J Delves Changes in the reproductive system of male mice immunized with a GnRH-analogue conjugated to mycobacterial hsp70 Reproduction, September 1, 2004; 128(3): 365 - 371. [Abstract] [Full Text] [PDF]
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1 mg/ml and are in
saline.
) and in IFA
for the secondary (•) doses of vaccine. For administration of
[T]-[B] admixed with Pam3Cys-S-(Lys)4, peptide was
dissolved in saline and mixed with the lipopeptide in 1:1 or 1:5 molar
ratio. The dose of peptide immunogens used was 20 nmol. In all cases,
the control groups of animals received saline emulsified in CFA for
priming and saline emulsified in IFA for the secondary
inoculation.
) or i.n. (
) in saline.