Note: Descriptions are shown in the official language in which they were submitted.
~0 92/10203 PCr/~591/09268
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A Vaccine Aeainss Cholesterol to Prcvent Hv~ercholes~erolemia
And Atherosç~rosis
1. GOVERNhlE~'T INTEREST
The invention described herein may be manufactured. Iicensed and
used by or for governmcntal purposes without the payment of any royalties
to us thereon.
II. RELATED APPLICA ~
~o This applicadon is a condnuadon-in-part of U.S. Patent Applicadon
Scrial No. 071444,214 filcd Dccember 1, 1989, which in turn is a
condnuadon-in-part of U.S. Patent Applicadon Serial l~.umber 06/875.0~8
filcd June 2, 1988. Addidonally, thc applicadon is a condnuadon-in-part of
U.S. Patent Applicadon Serial No. 07/6t)1,090 filcd October 2', 1990.
which in turn is a cantinuation~ part of U.S. Patent Application Serial No.
07n02,S99 filcd Junc 2, 1988 now U.S. Patent No. 4,88S,2S6 issued
Decanbe,r S, 1989.
I11. F1ELD OF THE INVE~mON
This invcntion relates to immunoreactive compositions for
in~nunizing or hypcrimmunizing humans against cholesterol and more
panicularly to the usc of thesc composidons in mcthods for reducing the
scrum choksterol levels of the immunized individuals and to retard or
rcduce the scvcrity of athcrosclcrosis or atherosclerosis plaques caused b-
ingesdon of dietary cholcstcrol or by other factors.
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IV. BACKGRO~'~'D OF THE I~ ~10~
It is widely believed that high levels of serum cholestcrol arc a
significam causadve effect in the pathogenesis of atherosclerosis and
associatcd diseases such as atherosclerotic coronary hcar~ disease
atherosclerodc ccrebral vascular diseasc, renal disease, e~c. It is alsc
believed that lowcring of blood cholesterol levels is associated with
amelioration of atherosclerotic vascular diseases (Goodman~ D.S. er al
o Rcport of the national cholesterol education program expert panel on
detection, evaluadon, and ~rcatment of high blood cholesterol in adults
Arch. Intern. Mcd. 148:36-69, 1988; Kromhout, D. et al.. Serum
cholesterol and 25-year incidcnce of and mortality from mvocardial
infracdon and canca. Thc Zutphen Study. Arch. Intern. Med. 148:10SI-
1055, 1988). In 1984, a Nadonal Institutes of Health consensus
dcvelopment conference panel recommended a framcwork of detection and
treattnent of hypercholcsterolemia. Based on this the National Cholesterol
Education Prograrn has tnade the well-known recommendation to adults:
"Know your cholesterol number" (Luepker, R.V. et al., Recommendations
regatding public screening for measuring blood cholesterol. Summary of a
Nadonal Heart, Lung, and Blood Institute Workshop, October 1988. Arch.
Intern. Med. 149:26S0-26S4, 1989).
The major methods recommended for achieving reduced serum
chole~ol kvds re throul~h reducdon of dietary intake of cholcsterol and
other hts, and treatment of hypercholesterolemic individuals with drugs
designed to lower blood cholesterol. The blood cholesterol levels are
p nicul rly associated with homeostadc mechanisms related to levels of
pl sma lipoproteins that serve as carriers of cholesterol. The dangerous
lipoproteins, from the standpoint of atherosclerodc risk are the low density
lipoproteins (LDL), and the levels of LDL are regulated by the functional
acdvida of LDL receptors on the surfaces of cells, particularly in the liver
(Lrown, M.S. and Goldstein, J.L. A receptor-mediated pathway for
cholesterol hon~costasis. Science 232:3~47, 1986). Many of the strategies
for using drugs to reduce blood cholcsterol involve interference ~ i~h the
processing of cholesterol derived frorn LDL (Brown and Goldstcin. 19g6).
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The exIenmhat cholesterol can be reduced by diet is linmed by numerous
factors, and the reducdon of cholesterol bv drugs could be associated with
side effects of the drugs as well as cost. In any case. a vanetv of additional
variables can influence cholestcrol levels, such as genetic background.
s stress effccts. and age. Additional methods for reduc~ion of cholesterol
might be expcc~ed to havc beneficial health effects. particularly in
individuals who might receive such treatrnent before signif~cant progrcssion
of atherosclerotic disease has occurred.
o The present invendon describes the use of a vaccine forrnuladon that
would be used to immunize humans against cholesterol and thereby lower
the concentradon of serum cholesterol, either by itself or in combination
with other methods commonly used to lower cholesterol. A variety of
immunization procedures might be used to induce antibodies to cholesterol,
and the presence of antibodies to choJesterol would be detected either by a
solid-phase immunoassay using crystalline cholesterol or a cholesterol
conjugate or by a complement-dependent assay such as complemem-
dependent immune damage to liposomes containing cholesterol as taught by
Swanz et al. lAntibodies to cholesterol. Proc. Nat. Acad. Sci. 85: 190~-
1906, 1988] and Alving et al. [U.S. Pa~ent No. 4,885,'S6 issued S
December 19891-
To our knowledge, humans have never been acdvely itnmunized
against cholesterol and the safety of doing this, as well as the potential
consequences relating to serum cholesterol levels or progression of
atherosclerosis due to intake of dictary lipids, has not been established. It
has been demonstrated that human sera usually do contain varying
quanddes, depending on thc individual serum, of "naturally-occurring"
andbodies to cholestol l~lving et al., Naturally occurring autoandbodies
to cholesterol in humans. Biochem. Soc. Trans. 17:637-639 (1989)].
However, there has not been any correladon of such naturally-occurring
anibodies with sen m chokstaol lewls or with atherosclerosis.
The possibility has been discussed that naturally-occu~ing
3S andbodies to cholesterol might be a normal part of the aging p ocess and
might contribute to (ratha than ameliorate) atherosclerosis (Alving. C.R.
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Antibodies ~o liposomes, phospholipids, and cholesterol: Implications for
au~oimmunity. atherosclcrosis, and aging. In: Horizons in .Uembrane
Biotechnology, ~'icolau, C. and Chapman, D., editors. ~'ilev-Liss. pp. ~1-
41, 1990). The possible dangers of injecting liposomes containing
cholesterol into animals containing antibodies to cholesterol have been
illustrated by anaphylactoid effects observed by Wassef et al.
[Anaphylactoid reacdons mediated by autoantibodies to cholesterol in
miniature pigs. J. Imrnunol. 143:2990 2995 (1989)]. Therefore it is not
obvious that this invemion could have practical use in humans.
Nonesheless. the potential feasibility of this invendon as a possible safe and
effecdve vaccine against choksterol has been demonstrated bv experiments
in humans in which repeated injecdons of a candidate liposomal anti-
malarial vaccine thu contained cholesterol did result in thc production of
antibodies to cholesterol. This is cleuly indicated in a U.S. Patent
Application Serial No. 07/601,090 entitled: "Encapsulated High-
Concentradon Lipid A Composidons as Immunogenic Agents To Produce
Human Andbodies To Prevent Or Treat Gram-negadve Bacterial Infcc~ions"
by Alving and Swartz filed on 22 October 1990. In that disclosure, thc
example shown in Figure 9 thaein cleuly dernonstrates that antibodies to
cholestaol cun be safely induced in certain individuals. The present
invention utilizes an antigen that produces higher and more consistent
andbodies than in the previous and-malarial disclosure, and produce such
antdbodies for the purpose of preventdng diet-inducet serum cholesterol
elevuions and athaosclerosis,
2S
~Ithough we have not found in the prior art any teaching relating to
immunization of humans with choleste~l, in the literature there has been
one anempl described to try to ameliorate hypercholesterolemia and
athosclerosis in r bbits by immunological means. Bailey et al.
Ilmmunizadon with a synthedc cholesterol-ester antigen and induced
athe osclerosis in r bbits. Nature 201:407-408 (1964)1 immunized rabbits
with an andgen consisting of cholesterol conjugated to bovine serum
albumin. Bailey et al. stated that the "mean antibody itre measured by an
interfacial precipitaion technique was 1 :70û0", but the was no attempt to
prodwe or to me#ure andbodies that had specificity against cholesterol.
The ssay andgen consisted of the original conjugate, not cholesterol either
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alone or as part of ano~her conju~ate. At no place did Bailey el al. teach that
they had induced amibodies to cholesterol. and the- did not teach th31
antibodies to cholesterol could have been produced or that such amibodies
might have played a role in the lowering of serum cholesterol levels or
amelioration of atherosclerosis.
Bailey et al. did observe a reduced hypercholesterolemia and less
aor~ic plaque formation in the immunized animals th~t were fed a
cholesterol-rich diet. However. in the absence of funher information the
0 antibody titer could have been entirely directed against the bovine serumalbumin component and the cholesterol-albumin conjugate might simpl!
have lowered cholesterol through nonspecific mechanisms. such as by
nonspecific adsorption or serum cholesterol by the albumin. This latter
explanation could be supponed by the fact that albumin has a considerable
degree of hydrophobicity and can be used as a reagent to prornote solubility
of cholesterol in an aqueous medium such as serum. The disclosure by
Bailey et al. is too insufficient to draw any immunological conclusion
regarding the role. if any, that antibodies to cholesterol may have played in
the experimental results. It is probably because of this that Baile,v et al. didnot teaeh any such role.
V. SUMMARY OF THE INVEl~,' llO~
This invendon eonsists of a vaecine which is effective in
2S immunizing hurnans against eholesterol. The purpose of this would be to
reduee the sen~m eholesterol levéls of the imrnunized individuals and to
red or reduee the severity of atheroselerosis or atherosclerosis plaques
eauset by ingesdon of dietary eholesterol or by other faetors. The vaecine
would eonsist of a fonnulatdon eontaining eholesterol or eholesterol and
phosphatidyl ehoUne; or eholesterol and dimyristoyl phosphatidyl eholine
together with a suitable delivery vehiele and may also eontain a suitable
adjuvant. The reladve molar rado between the eholesterol and phosphatidyl
eholine or dimyristoyl phosphatidyl eholine is within the range of 1:1 to
I :~.S. An exarnple of a suitable fonnulation would be liposomes containing
phosphatidyleholine, eholesterol. and lipid A in molar ratios of liposomes
containing phosphatidylcholine. cholesterol. and lipid A in lar r~tios of
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2/5/0.0~ (where the molafil~ of lipid A is based on the mol3ril- of
phosphate in native lipid A). This ra~io is not necessaril~ cri~ical. bccause
other ra~ios might be successful in accomplishing the sarne result Deliver~
vehicles other than liposomes would also be suitable, includins
microcapsules, microspheres, lipospheres, polvmers. and slow release
devices could serve instead of liposomes. An experiment in rabbits ha:.
demonstrated that the stipulated vaccine does ameliorate diet-induced
elevations of serum cholesterol,
Vl. BR~EF ~?ES~RI~I-ION' OF THE DRAWI~G
A more complete appreeiation of the invention and many attendant
advantages theof will be readily obtained by reference to the following
s DETAILED DESCRIPllON OF THE INVENTION when considered in
conjunetion with the aeeompanying drawing, wherein:
Figure 1 shows IgG respon#s 2 weeks after initial immuniza~ion in
the 6 human volunteers that eonstituted Group IV in the experimental
immunization eholesterol from the above patent applieation. The individuals
were immunized with 43% eholesterol liposomes as taught in the presenl
diselosure and in the previous patent applieation. Each of the components
was individually tested by ELISA for the appearanee of IgG antibodies
agunst the purified individud eomponent. In the ease of lipid A, the
2S indiv du ls were injeeted with liposomes eontaining monophosphoryl lipid
A. Tl# d-ta ue shown with preimmunization values, if any, subtraeted
from the postimmunization vdues. Eaeh serum was diluted Ill00 for
ELISA andysis. Three of the six immunized individuals developed
signific ntly increased levels of antibodies to eholesterol. Figure i
conesponds to figure 9 from the U.S. Pa~ent Applieation Serial No.
07/601,090, entitled: UEnc-psulaled High-Coneentration Lipid A
Compositions as Inununogenic Agents To Produee Human Antibodies To
Prevent Or Treat Grun-negative Bacterial Infeetions" by Alving and Swartz
filed on 22 Oetober 1990.
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VII. DETAILED DESCRIPTlO!~ OF THE I~VE~TIO~ A~D
EXAMPLES
The working example set forth below illustrate. withou~ anv implied
limitation a vaccine useful for the immunization of humans agains~
cholesterol. This vaccine is useful for immunizing or hvpenmmunizing ~
human against cholesterol. which vaccine comprises as an active ingredient
A. a delivery vehicle and B. either. (i) cholesterol; or (ii) cholesterol and ~nadjuvant: or (iii) cholesterol. phosphatidyl choline on an adjuvann or ( i- ~
0 cholesterol, dimyristoyl phosphatidyl choline and an adjuvann or (v)
choles~erol and phosphatidyl choline; or (vij cholesterol and dim~Tisto~l
phosphatidyl choline
EXAMPLE
An experiment is currently underway to determine the possible
feasibility of ameliorating diet-induced hypercholesterolemia and
atherosclcrosis in rabbits. Groups of rabbits are being immunized while
oth groups are not being immunized against cholesterol; at least one eroup
~o of immunized and one group of nonimmunizcd rabbits will be fed a diet rich
in cholesterol. It is our prediction that the immunization process will
ameliorate the hypercholesterolemia and athosclerosis that is cxpected lo
be produced by the cholesterol-rich diet and that this will reduce to practice
the inwndon that is herein disclosed. The experimental results from the
rabbit expenment described below provides substantive evidence in support
of our prediction by demonstrating that the 1% cholesterol diet causes a
dramadcally increased serum choksterol level within I week (6 weeks sfter
immunizadon in those rsbbits thst were immunized), and the cholesterol
condnues to rise ov the second week (7 weeks sfter initial immunization
was started in the irnmunized snimals). Howev. the increased level of
diet-induced choles~erol was the 30% less elevated in the snim~ls (Gtwp 11 !
thst were irntnunized against cholesterol (sce the Table herein).
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METHODS
Liposomes
Liposomes are being manufactured bv standard methods in which
liposomes loaded with cholesterol (containing ~19c cholesterol) and also
containing lipid A as an adjuvant are prepared for injection as taught b~
Swartz et al. [Amibodies to cholesterol. Proc. Nau Acad. Sci. 85:190'-
1906, 1988] and Alving et al. [U.S. Patent No. 4,885.256 issued 5
December 1989].
The liposomes used for immunization contained dimyristovl
phosphaddylcholine (DMPC~/cholesterol (chol)/dimyristoyl phosphadd~l
glycerol (DMPG)llipid A (molar ratio 0.9/2.5/0.110.0' for rabbits, or
0.910.75/0.1/0.02 for humans, where the molarity of lipid A refers to lipid
A phosphate). The total dose of lipid A injected as part of the 71*
cholcsterol liposomes was 50 ug lipid A. Thc liposomal cholesterol
concentradon is described as a percen~age. and this is calculated as mol c~
with reference to (DMPC + DMPG); e.g., a cholesterol/(DMPC + DMPG)
ratio of 0.7S/1 is 43 mol%, and 2.5/1 is 71 mol'rc.
Enzy~ne-lip~ orbent Assav (ELISA).
ELISAs were performed by using crystalline cholesterol as an
andgen on the bottoms of the wells of microtiter plates. Chrystalline
cholcstesol w s coated onto the surface of wells in polystyrene plates
~Immunlon 96, "U" bottom, Dynatech Laboratories, Alexandria, VA) by
addition of an etlunolic solution and evaporation of the solvent by air under
a fume hood. Pl ces were funhcr dried under high vacuum and stored at
-30C when not used the same day. Plalcs werc blocked by addition of
phosphate-buffercd saline (PBS: 137 mM NaC112.7mM KCI/9.6mM
phosphate, pH7.2) containing 10% heat-inactivated fctal bovinc serum
(FBS) (M.A. Bioproducts, WallccrsvUlc, MD). This was accomplishcd by
washing thc wclls thrce titnes for 10 min cach. Fifty microliters of ascites
fluid containing monoclonal antibodies, diluted in PBS containing 10~
FBS, was added to thc wclls and incubated I hr at room tcmperaturc. Plates
were thcn washcd three timcs for 5 minutes each with PBS. Fifty
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microliters of goat and-mouse Ig~ (micro-chain) alkaline phosphatase
conjugate (Kirkegaard and Perry Laboratones, Gaithersburg. ~D) al 1
rnicrogram ml in PBS containing 10- FBS was added to the wells and
incubated 1 hour a~ room temperature. Plates werc again washed thrce dmes
for 5 minutes each PBS. Fifty mioliters of the substrate, p-nitrophen-l
phosphate at 2 mglml in diethanolamine buffer (Kirkegaard and Perry
Laboratories) was added to the well and incubated 30 minutes at room
temperature. Plates were scanned for optical acdvity at 405 nm using a
Titenek Multiscan (Flow Laboratories). Values reponed wcre adjusted by
subtracting value in blank wells that lackcd both antigen and monoclonal
antibody.
Imrnunizanon Protocol
Four groups of rabbits were either immunized with liposomes
containing 71 1% chol, or were not immunized. Immunization was
performed eith intrarnuscularly or intravenously every two weeks for 6
weeks~ The immunization procedure routinely induced andbodies to
cholesterol in rabbits. as determined by ELISA or by complement-induced
im nune damage to high-cholesterol liposomes as taught by Swartz et al.,
Antibodies tocholesterol. Proc. Nat. Acad. Sci. 85:190~-1906, 1988, and
Alving et al., U.S. Patent No. 4,885,256 issued 5 December 1989.
The immunizadon of human subjects with 43% cholestcrol
2S liposomes w s conducted as part of the testing of the efficacy of a vaccine
for ioducdon of andbodies to rnalaTia andgen and andbodies to lipid A, as
taught by the previous disclosure entitled: "Encapsulated High-
Conccntruion Lipid A Composidons as Imtnunogenic Agents To Produce
Hum n ~dbodies To Prevent Or Tre t Gnun-negadvc Bacterial Infccdons"
by Alving and Sw rtz that is cur ently being prepared as a U.S. patcnt
applicatdon, the htter of which is a continuation-in-part of U.S. Patcnt
Applicadon Serial Number 07n~.sos filcd lune ~, 1988 (A Vaccine For
Inducdon of bnmunity to Mal ria. And-cholcstcrol andbodics induccd wcre
deteacd in Group IV and are illustrated in thc accompan,ving Figure.
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Ex~enmen~LDiets
At week 6. the experimental diets were ini~ated. The diets consisted
either of ordinary rabbit chow or a 1% cholesterol die~ (obtained from
Bioserve). Four groups and two subgroups of animals were emploved
Group I, 4 rabbits, not immunized, fed normal die~: Group lla. 1 rabbi~s.
immunized intramuscularly, fed 1% cholestcrol diet; Group llb, ~ rabbits.
irnmunized inrravenously, fed 1% cholesterol diet: Group III, 4 rabbi~s. not
immunized, fed normal diet; Group IVa, 4 rabbi~s, immunized
0 intramuscularly, fed normal diet; Group IVb, ~ rabbits, immunized
intravenously, fcd normal diet.
In addition to the above teaching with rabbits, i~ is now evident that
even liposomes containing 43% cholesterol (using liposomes as taught
above and in the prior att describcd above) also can induce antibodies to
cholesterol in a limited numbcr of individual humans.
It appcars tha~ the 71% cholesterol liposomes will bc superior to the
43% cholesterol liposomes as the basis of a vaccine to induce antibodies to
cholcstc ol. This conclusion is drawn from the fact tha~ only a small number
of thc individual humans immunized with liposomes containing 43'~t
chokstcrol developed andbodics to cholesterol Isec Fig. 1. which is derived
from tttc prcvious U.S. Patcnt Application Serial No. 07/601,090~ entitled:
"Enc psulatcd High-Conccntradon Lipid A Compositions as Immunogenic
2S Agcnts To Ploduce Hum n Anibodies To Prevent Or Treat Grarn-negative
B c~ial Wccions" by Alving and Swartz filed on 22 October 19~0, the
lattcr of which is a continuation-in-pan of U.S. Patent Application Serial
No.07nO2,S09 filed June 2, 1988 (A Vaccine For Inducdon of Immunin
to M l~ia)l. Thc contrast, approximately 70% of splccn cells from mice
immunizcd with 71% cholestcrol wcrc producing antibodies to cholesterol
Swanz ct al., Andbodics to cholcsterol. Proc. Nat. Acad. Sci. 85:1902-
1906, 1988] ant Alving ct al., lU.S. Patent No. 1.885.256 issued
December 1989~.
Bascd on the prior art it is evident that cholesterol is highl
imrnunogenic and the immunogenicity is enhanced both bv adjuvanls (e.
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lipid A or other adjuvanls) and by thc epitope densitv of cholesterol used for
immunization. Il should be possible to achieve the combination of hiVh
epitopc densities of cholesterol togethcr with adju~ants b~ a vanet- Ot
carrier mechanisms, including microcapsules, microspheres. Iipospheres.
high density conjugation or association of cholesterol with proteins or orher
macromolecules, natural sources of high cholesterol (such as organisms
such as mycoplasma that have the capacity to accumulate cholesterol). It is
presumed that any established me~hod for inducing antibodies to particulale
substances or macromolccules sheoretically could be adapted to inducing
~0 antibodies to choleslcrol.
E2~ncntal Results
Table 1. Reduction of Diet-lnduced Hypcrcholesterolemia in Rabbits
Immunizcd Against Cholesterol.
High Bk~SngSum Inae~e Rod~
Chobs~ munized Time Chdestelol Canpar~d l~lase
G~ Dict~- -- (Wed~s~tm~/dl)~ Weck 5 t~
76
n - ~ 5 6'
m - - 5 ~3
IV - ~ 5 83
- 6 775 699
n + , 6 797 731
m - - 6 64
IV - ~ 6 68
- 7 1205 1129
n ~ , 7 952 790 30
m - - 7 74
7 62
~Data shown are mcans of rcsults (Group 1, 4 rabbits; Il, 6 rabbits: 111, 4
rabbits; IV, 6 rabbits).
e lS~ cholcstcrol diet was initiated at the 5 week time poin~ after
starting the cxperiment.
~The immunization againQ cholesterol was ini~ialed at O weel;s.
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The above resul~s demonstrate ~ha~ ~he hi~h cholcsterol diet
invariably caused elevated serum cholesterol values. However, two eeks
aft inidating the diet (week 7) the elevation of cholesterol in the imrnunized
group (Group II) was 30% lcss than thc clevation of cholestcrol in the
nonirnmunized group (Group I).
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