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BRIEF SUMMARY OF THE INVENTION
This invention relates to a process of enhancing immunogenic response in
mammals. More particularly it relates to a process of enhancing the
immunogenic response in mammals by the administration of a synthetic
glycolipid adjuvant not later than the time of administration of an
immunogenic agent.
Although the complete structure of endotoxic lipopolysaccharide (LPS) is
not known, some salient features have been detected. The most unusual
structural property is in the liquid moiety of the LPS, which has a
phosphorylated D-glucosamine backbone, and this backbone has ester- and
amide-bound long-chain carboxylic acids, thus forming a new class of
glycolipids which has been named phosphomucolipid (1,2). The long-chain
carboxylic acids of the lipid moiety were first studied by reversed phase
paper chromatography and it was found that a hydroxy-acid, probably
3-hydroxymyristic acid, is one of the major components (3). These findings
were confirmed by several laboratories and many further details were
investigated (4-9).
During our efforts to synthesize model compounds structurally similar to
known parts of the lipid moiety of endotoxic LPS, we prepared, among
others, N-acylated D-glucosamine derivatives. One of the compounds,
N-palmitoyl-D-glucosamine, was found by us to be active in B cell
mitogenesis, although it is inactive in a number of characteristic
endotoxicity reactions, such as mouse lethality, Limulus lysate clotting
assay, pyrogenicity, and local Shwartzman skin assay in rabbits (10).
As a logical next step, homologs such as N-octanoyl-(NOG),
N-decanoyl-(NDG), N-lauroyl-(NLG), N-myristoyl-(NMG), N-oleyl-(NOLG), and
N-stearoyl-(NSG) D-glucosamines were prepared.
We have now found that the administation of N-acylated D-glucosamine
compounds not later than the time of administration of an immunogen to a
mammel enhances the immunogenic response of the mammal.
DETAILED DESCRIPTION OF THE INVENTION
Synthesis of N-acylated D-glucosamine derivatives
The procedure of Fieser et al (12) was followed. Ten mMole acid chloride
(caprylyl, capryl, lauroyl, myristoyl, palmitoyl, oleyl, or stearoyl
chloride) dissolved in 10 ml tetrahydrofuran were added dropwise to 10
mMole D-glucosamine.HCl dissolved in 20 ml 10% Na.sub.2 CO.sub.3 solution
under constant stirring at room temperature. The stirring was continued
for 60 min, then 10 volumes of water were added. The white precipitate was
filtered on a Buchner funnel and recrystallized twice from 50% hot
propanol. The preparations were analyzed for C, H, and N, and their
melting points were determined.
Endotoxic LPS preparation
LPS were isolated from Serratia marcescens 08 by the Boivin procedure, as
modified earlier (13).
Preparation of liposomes from synthetic glycolipids
Two milligrams of the synthetic glycolipid were dissolved in 20 ml ethanol
by refluxing in a 500-ml round bottom flask for 30 min in a water bath.
With a rotating vacuum distillation apparatus such as the Buchi type (made
in Switzerland), the alcohol was slowly removed while the flask was
rotating in a warm water bath. Upon complete removal of ethanol, the
glycolipid will form a thin layer on the inside wall of the 500-ml flask.
The flask was briefly chilled in an ice bath before the addition of 20 ml
double distilled water, followed by vigorous shaking for several minutes.
The suspension was sonicated for 2 min at 1.7 amp. in a 50-ml beaker. At
this time a droplet of this suspension placed under a light microscope
will reveal the presence of liposomes as small spherical bodies. After a
dry weight determination, the suspension was adjusted to a final
concentration of 50 .mu.g/ml.
Application of liposomal glycolipid suspension for enhancement of anti-SRBC
response
Washed SRBC were brought to a final concentration of 5.times.10.sup.7
SRBC/ml in a 1.8% NaCl solution. Equal volumes of the SRBC and liposome
suspensions were gently mixed to obtain isotonicity. This mixture was
allowed to incubate, with occasional careful shaking, at room temperature
for 30 min. Four-to 6-week old female ICR mice were used throughout these
experiments. A total of 0.4 ml, containing 1.times.10.sup.7 SRBC and 10
.mu.g liposomes was injected i.p. per mouse.
Application of liposomal glycolipid suspension for enhancement of anti-HGG
response
Lyophilized HGG (Pentex Cohn fraction II) was slowly dissolved in a 1.8% pH
adjusted (7.4) NaCl solution with minimal agitation to a final
concentration of 1 mg/ml. Equal volumes of the HGG solution and the
liposomal suspension were combined and allowed to incubate for 30 min, as
described above. Each mouse was injected i.p. with a total volume of 0.4
ml containing 0.2 mg HGG and 10 .mu.g liposomes in an isotonic NaCl
solution.
Use of LPS for enhancement of anti-SRBC and anti-HGG response
The same procedure was followed as described above by replacing the
equivalent concentration of glycolipid with distilled water-dissolved LPS.
Assays in the determination of adjuvancy
Spleen cell rosetts formation (RFC), plaque formation (PFC), and
hemagglutination (HA) were carried out for the determination of the immune
response to SRBC. The passive hemagglutination (PHA) assay was employed
for the serum titer determination of the anti-HGG immunoglobulins. For all
assays, pooled anti-sera and spleen cell suspensions were obtained from
individual groups of six mice.
RFC assay
The method of Zaalberg (14) was used for RFC determination with the
following modifications. Ten days after immunization and/or treatment, a
spleen cell suspension was obtained by means of a tissue grinder
containing 2 ml per spleen of Hanks' balanced salt solution (HBSS) and
adjusted to pH 7.2 with NaHCO.sub.3. After the cell suspension was passed
through gauze and the cells were washed twice with HBSS, the cell
suspension was adjusted to a concentration of 6.times.10.sup.7 cells/ml.
From these pooled spleen cell suspensions, triplicate samples of 100 .mu.l
were added to 100 .mu.l of washed SRBC suspension (approximately
3.times.10.sup.7 cells) in saline, pH 7.2. To each of these samples, 0.8
ml HBSS was added to bring the final volume to 1.0 ml. The samples were
thoroughly agitated for 1 min by means of a Vortex before incubation at
4.degree. C. for 3 hr. The test tubes containing the samples were gently
rotated and hand-shaken to ensure a homogeneous cell suspension with
minimum stress before analysis in a hemocytometer. Lymphocytes with more
than five attached SRBC were classified as rosettes.
PFC assay
A modified technique of the hemolytic plaque assay first described by Jerne
and Nordin (15) was used. Pooled, washed spleen cells from mice 4 days
after immunization were obtained as described above. Appropriate cell
suspensions (by volume: 1:10, 1:100, 1:500) were obtained for plating.
However, accurate cell concentrations were determined for each sample by
counting stained cells (Turk's stain) with a hemocytometer. To individual
test tubes containing 2-ml aliquots of a 1% noble agar solution kept in
liquid state at 50.degree. C. in a water bath, the following were added:
(1) 0.1 ml DEAE dextran (1%); (2) 0.1 ml 20% washed SRBC suspended in
saline; (3) 0.1 ml of any of the various spleen cell concentrations. The
contents of the tubes were quickly swirled and evenly layered onto the
previously prepared base layer plates containing 15 ml 1.4% noble agar in
HBSS. The plates were incubated for 1 hr. before guinea pig complement
(Cappel Labs., State College, Pa.) was added. Incubation was continued for
30 min. At this time the complement was decanted and the plates were ready
to be analyzed. Plates for each of the three concentrations of spleen
cells were done in triplicate and analyzed randomly in a blind manner.
Direct HA and PHA
For direct HA, the procedure using the micro-titer system was used. Pooled
anti-SRBC serum was obtained by heart puncture 10 days after treatment.
The micro-titer system was also used for the PHA assay involving tannic
acid-treated SRBC coated with HGG. The procedure followed was that of
Boyden (16, 17).
Determination of endotoxicity
Shwartzman skin assays were carried out in albino rabbits by earlier
described routine procedures (17). Chick embryo lethality was done on
11-day-old embryos and the preparations to be tested were given i.v.,
according to the method of Smith and Thomas (18). Mortality was recorded
24 hr. later and the LD.sub.50 was calculated. The Limulus lysate assay
was performed by the procedure of Levin et al. (19).
Protection from lethal irradiation
Mice in groups of 10 were irradiated with a total exposure dose of 700 R in
individual perforated plastic centrifuge tubes mounted on a plywood
rotating platform to ensure uniform exposure. The radiation source was a
General Electric Maxitron 300 x-ray machine, operated at 20 ma, 300 Kvp,
with added filtration of 0.26 mm Cu and 1.05 mm Al, yielding a half-value
layer of 1.10 mm Cu. At a target distance of 60 cm, the air exposure dose
rate was 235 R per minute as determined by a Victoreen air ionization
chamber (model 154).
Glycolipid synthesis
The chemical synthesis of the glycolipids gave an approximately 50 to 65%
yield of the calculated, measuring the amount of twice recrystallized
products. The C, H, and N analyses were carried out by Micro-Analysis,
Inc., laboratories, Wilmington, Del. 19808. Table I gives the results of
these microanalyses as well as the melting point determinations. The close
agreement between the theoretical and empirical values indicate the high
degree of purity of the synthetic glycolipids.
Adjuvant effect of synthetic glycolipids and LPS on the anti-SRBC response
Before investigating the possible adjuvant properties of the liposomal
glycolipids, we established the effect of the antigenic dose and its
influence on the immune response enhancement by LPS, a known adjuvant.
Table II shows the PFC response of normal and LPS-treated mice for
increasing doses of SRBC. A better than 20-fold increase in the number of
PFC was observed for the LPS-treated group receiving 5.times.10.sup.6
SRBC. This observation is in agreement with those of Sjoberg et al. (20),
who observed endotoxic adjuvancy only for low immunogenic doses of SRBC.
In view of the low PFC response for the control group at this antigenic
dose level, the antigenic dose of 1.times.10.sup.7 SRBC was chosen for all
the adjuvant determinations of the subseqent experiments.
The number of plaque-forming or antibody-producing cells in response to
1.times.10.sup.7 SRBC/mouse was significantly enhanced for all the treated
groups, except for the group treated with NDG. Similarly, a quantitative
enhancement in serum anti-SRBC antibody levels was observed for the
glycolypids and LPS relative to the control (Table III). In contrast to
the PFC and serum antibody response, both clearly involving the B cell,
the RFC response is thought to involve the T cell (Wilson, 21). Although
all the glycolipids tested gave various degrees of enhancement, LPS was
clearly the strongest adjuvant involving the resetting cell (Table IV).
Adjuvancy of the anti-HGG response
Our investigations of the effects of synthetic glycolipids and endotoxin on
the antibody response to a serum protein, HGG, by means of PHA,
demonstrated a low but significant enhancement of antibody titer for LPS
and at least two of the glycolipids (NLG and NOLG). The results are
summarized in Table IV.
Endotoxic reactions of the glycolipids
In the Shwartzman skinn assay, none of the preparations gave a positive
response at the 50 .mu.g level, but 2.5 .mu.g endotoxin gave clearly
detectable hemmorrhage in the skin of the rabbits. In chick embryo
lethality of the synthetic glycolipids, none of them killed more than 20%
at a 10-.mu.g dose level given i.v. By way of comparison, the LD.sub.50 of
endotoxin in the same assay was found to be 0.006 .mu.g. The Limulus
lysate clotting assay was the only one that showed a slight activity by
NPG and NSG preparations. According to the findings, these two
preparations were 10.sup.4 and 10.sup.5 times less active than the control
endotoxin preparation. Since this activity was maintained even after
repeated recrystallization, one may exclude the possibility of endotoxin
contamination.
Protection against lethal doses of radiation
In my copending application Ser. No. 659,423 filed Feb. 19, 1976, it is
disclosed that endotoxin and its polysacchariderich fraction (PS) obtained
from endotoxin by acid hydrolysis offered excellent to good radiation
protection, respectively, when it preceded irradiation during specific
time intervals (22). Treatment of mice 2 days before lethal irradiation
exposure not only delayed the time of death but also increased the number
of survivors to nearly the same degree as the PS for at least two of the
glycolipids tested (Table V).
After several attempts to disperse the glycolipids mechanically or by
solvent exchanges, the most consistent results were obtained by the
procedure described here designed for the preparation of liposomes. This
method gave not only a rather fine dispersion of the glycolipids, suitable
for aqueous injection, but also significant adjuvant effects.
It should be emphasized that proper dispersion of the sample is absolutely
essential for adjuvancy. Dispersions obtained by sonication at room or
elevated temperatures, in the presence or absence of colloid stabilizers
such as PS or proteins, yielded suspensions that appeared to be finely
dispersed, but the adjuvant effect of these samples was marginal and for
the most part not always reproducible. The possible reason for this may
lie in the amorphous nature of the sonicated glycolipid aggregates. In the
liposomes, the molecules are oriented and their even distribution on the
surface of the spherical liposomes may present them in the most suitable
way to obtain reproducible adjuvant effect.
The admixture of the glycolipid liposomes with the immunogen before
injection is similarly important. In the case of both SRBC or HGG, a
minimum of at least 30 min was found to be essential for elevated immune
response. A reason for this time requirement has not been determined.
The proper dose of immunogen is also quite critical for the determination
of adjuvant effect. A larger than optimal immunogenic dose of SRBC may
result in an already high PFC or RFC response, which cannot be further
augmented more than 1.5- or 2-fold with the best adjuvant. Similarly, the
health of the mice is also a critical factor. If the animals are, or
recently have been infected by some microorganism, their response in RFC
may already be at a higher than normal level, most probably due to a
nonspecific immunostimulation elicited by the infecting microbes. In such
cases, hardly any adjuvant effect can be observed.
The immune response to 1.times.10.sup.7 SRBC/mouse was clearly enhanced for
nearly all treated groups, as expressed by the three separate assays
involving anit-SRBC agglutination titer, PFC and RFC (Table III). Although
the degree of adjuvancy in the three assays varied somewhat, it must be
kept in mind that these assays represent a different stage and/or
component of the immune response. The anti-SRBC serum obtained from mice
10 days after treatment/immunization most likely consists of a mixture of
IgM and IgG antibodies, whereas the plaque formation seen 4 days after
treatment/immunization is known to be an IgM response. Furthermore,
although the PFC and serum antibody responses are known to involve the B
cell, the RFC is thought to involve the T cell (21).
In the experiments reported here, the adjuvant was given simultaneously
with the immunogen. Studying the optimal conditions for endotoxin-induced
adjuvant effect, we found that endotoxin given 10 to 13 days before SRBC
gives a better RFC adjuvant effect than that given together with the
immunogen. Endotoxin given 5 days before SRBC has a definite
immunosuppressive effect on the RFC response.
TABLE I
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Chemical analyses of the synthetic glycolipids
C H N
Cal- Cal- Cal-
Pre- cu- cu- cu-
para- lated Found lated
Found lated
Found
tions % % % % % % M.P. .degree.C.
______________________________________
NOG 55.04 54.92 8.91 8.80 4.59 4.60 193-197
NDG 55.61 57.66 9.38 9.25 4.20 4.04 194-196
NLG 59.77 59.74 9.76 9.68 3.88 3.83 190-191
NMG 61.65 61.64 10.10
10.01 3.60 3.59 195-197
NPG 63.26 63.31 10.38
10.26 3.35 3.45 202-204
NSG 64.66 64.63 10.64
10.55 3.14 3.11 192-194
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TABLE II
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Adjuvant effect of lipopolysaccharide (LPS) as a function of the
antigenic dose (sheep red blood
cell, SRBC) in the plaque-forming cell (PFC) response.sup.a in ICR mice
Antigenic Dose of SRBC
Treatment
1 .times. 10.sup.6
5 .times. 10.sup.6
1 .times. 10.sup.7
5 .times. 10.sup.7
1 .times. 10.sup.8
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Control
4.07 .+-. 0.32
4.62 .+-. 0.49
54.62 .+-. 6.11
255.65 .+-. 16.19
503.88 .+-. 42.77
LPS (10 .mu.g)
7.55 .+-. 0.49
95.33 .+-. 7.1
209.44 .+-. 14.22
881.66 .+-. 53.14
719.44 .+-. 41.38
(1.85 .+-. 0.19).sup.b
(20.63 .+-. 2.66).sup.b
(3.83 .+-. 0.50).sup.b
(3.90 .+-. 0.36).sup.b
(1.42 .+-. 0.16).sup.b
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.sup.a PFC response is expressed in the number of PFC/10.sup.6 spleen
cells
.sup.b
##STR1##
TABLE III
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Adjuvant effect of various preparations on the anti-SRBC
immune response of ICR mice
Adjuvant Assays
Preparations
HA.sup.a PFC.sup.b RFC.sup.c
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NOG 2.sup.7 1.8 1.7
NDG 2.sup.6 0.7 3.2
NLG 2.sup.7 6.3 3.1
NMG 2.sup.5 1.6 1.0
NPG 2.sup.9 2.4 6.0
NOLG 2.sup.8 1.3 6.4
NSG 2.sup.6 1.9 2.5
LPS 2.sup.7 8.2 11.0
None 2.sup.4 1.0.sup.d 1.0.sup.e
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.sup.a Hemagglutination titer.
.sup.b Plaque-forming cells, adjuvant index.
.sup.c Rosette-forming cells, adjuvant index.
.sup.d Control PFC response: 42.3 .+-. 4.9 PFC/10.sup.6 spleen cells.
.sup.e Control RFC response: 6.1 1.1 RFC/10.sup.3 spleen cells.
TABLE IV
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Adjuvant effect of various preparations on the anti-HGG immune
response of BALB/c mice.
Preparation Passive Hemagglutination Titer
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NOG 2.sup.0
NDG 2.sup.0
NLG 2.sup.5
NMG 2.sup.3
NPG 2.sup.4
NOLG 2.sup.6
NSG 2.sup.3
LPS 2.sup.3
None 2.sup.1
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TABLE V
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Survival of lethally irradiated.sup.a ICR female mice treated with:
(1) LPS (Serratia marcescens 10 .mu.g); (2) PS (Serratia marcescens
10 .mu.g); (3) NDG (50 .mu.g); (4) NLG (50 .mu.g); (5) NMG (50 .mu.g);
(6) NPG (50 .mu.g)
Percent Survivors after Irradiation
Treatment
Day 1 Day 6 Day 12
Day 18
Day 24
Day 30
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LPS 100 90 80 80 80 80
PS.sup.b 100 70 50 50 40 40
NDG 100 90 60 40 40 40
NLG 100 90 30 10 10 10
NMG 100 90 40 40 40 40
NPG 100 90 50 30 20 20
Control 100 80 0
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.sup.a Radiation exposure (700 R wholebody) occurred 2 days after
treatment.
.sup.b Polysaccharide-rich fraction obtained by acid hydrolysis of LPS (1
HCl, 30 min. 100.degree. C.).
The synthetic glycolipids described had no activity in chick embryo
lethality. Shwartzman skin assay, and Limulus lysate tests, which are
characteristic as well as sensitive assays of endotoxicity. The absence of
these activities rules out the possibility that the glycolipid
preparations are active because they are contaminated with endotoxin.
(Abbreviations used in this paper: CSF, colony stimulating factor; HA,
hemagglutination; HBSS, Hanks; balances salt solution; HGG, human
.gamma.-globulin; NDG, N-decanoyl-D-glucosamine; NOG,
N-octanoyl-D-glucosamine; NOLG, N-oleyl-D-glucosamine; NSG,
N-stearol-D-glucosamine; PHA, passive hemagglutination; PS,
polysaccharide-rich fraction of hydrolyzed lipopolysaccharide; RFC,
rosette-forming cell.)
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