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Document AOl 
Appl.No. 09/848,616 



J 



Europais h s Patentamt 
Europ an Patent Office 
Office uropeen do brev ts 





(Tj) Publication number: 0 465 081 B1 



@ 



EUROPEAN PATENT SPECIFICATION 



@ Date of publication of patent specification : 
20.04.94 Bulletin 94/16 



@ int. ci. 5 : A61K 9/16, A61K 39/12 



@ Application number: 91305706.3 



(§) Date of filing : 24.06.91 



(S) Viral decoy vaccine. 



(§) Priority : 22.06.90 US 542255 
24.04.91 US 690601 



@ Date of publication of application : 
08.01.92 Bulletin 92/02 



@ Publication of the grant of the patent : 
20.04.94 Bulletin 94/16 



(§4) Designated Contracting States : 

AT BE CH DE DK ES FR GB GR IT U LU NL SE 



References cited : 
EP-A- 0 142 193 
EP-A- 0 420 785 
WO-A-83/03102 
DE-B- 1 203 912 
GB-A- 1 198 513 
GB-A- 2 144 331 
US-A- 4 251 509 



CO 

00 
O 

ID 
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a. 

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@ Proprietor : THE REGENTS OF THE 
UNIVERSITY OF CAUFORNIA 
300 Lakeside Drive, 22nd Floor 
Oakland, California 94612-3550 (US) 



Inventor : Kossovsky, Nir 

1820 Courtney Terrace 

Los Angeles, California 90046-2107 (US) 

Inventor : Bunshah, Rointan F. 

8138 Zitola Terrace 

Playa del Rey, California 90293 (US) 



Representative : MacGregor, Gordon et at 
ERIC POTTER & C LARKS ON St Mary's Court 
St Mary's Gate 
Nottingham, NG1 1LE (GB) 



Note : Within nine months from the publication of the mention of the grant of the European patent, any 
person may give notice to the European Pat nt Office of opposition to the European patent granted. 
Notice of opposition shall be fil d in a writt n reasoned statement. It shall not be deemed to have been 
filed until the opposition fee has been paid (Art. 99(1) European patent convention). 



Jouve, 18, rue Saint-Denis, 75001 PARIS 



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EP 0 465 081 B1 



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D scrlptl n 

1. Field of the Invention 

The present invention relates generally to a bio- 
logically active composition having a microparticulate 
core. The composition can be used as a vaccine, irrv 
munodiagnostic or as a pharmaceutical, depending 
upon the nature of the particular biologically active 
moiety. 

2. Description of Related Art 

The attachment of biologically active proteins, 
peptides or pharmacologic agents to various carrier 
particles has been an area of intense investigation. 
These conjugated biological systems offer the prom- 
ise of reduced toxicity, increased efficacy and low- 
ered cost of biologically active agents. As a result, 
many different carrier models are presently available. 
(Varga, J.M., Asato, N., in Goldberg, E.P. (ed.): Poly- 
mers in Biology and Medicine . New York, Wiley, 2, 73- 
88 (1983). Ranney, D.F., Huffaker, H.H., in Juliano, 
R.L (ed.): Biological Approaches to the Delivery of 
Drugs , Ann. NY Acad. ScL, 507, 104-119 (1987).) 
Nanocrystalline and micron sized inorganic sub- 
strates are the most common carriers and proteins 
are the most commonly conjugated agents. For ex- 
ample, gold/protein (principally immunoglobulin) con- 
jugates measuring as small as 5 nm have been used 
in immunological labeling applications in light trans- 
mission electron and scanning electron microscopy 
as well as immunoblotting. (Faulk, W., Taylor, G., /m- 
munochemistry 8, 1081-1083 (1971). Hainfeld, J.F., 
Nature 333, 281-282 (1988).) 

Silanized iron oxide protein conjugates (again 
principally antibodies) generally measuring between 
500 and 1500 nm have proven useful in various in vi- 
tro applications where paramagnetic properties can 
be used advantageously. (Research Products Cata- 
log, Advanced Magnetics, Inc., Cambridge, MA, 
1988-1989.) Ugelstad and others have produced 
gamma iron oxides cores coated with a thin polystyr- 
ene shell. (Nustad, K., Johansen, L, Schmid, R., 
Ugelstad, J., Ellengsen, T., Berge, A.: Covalent cou- 
pling of proteins to monodisperse particles. Prepara- 
tion of solid phase second antibody. Agents Actions 
1982; 9:207-212 (id. no. 60).) The resulting 4500 nm 
beads demonstrated both the adsorption capabilities 
of polystyrene latex beads as well as the relatively 
novel benefit of paramagnetism. 

Carrier systems designed for in vivo applications 
have been fabricated from both inorganic and organic 
cores. For example, Davis and Ilium developed a 60 
nm system comprised of polystyrene cores with the 
block copolymer poloxamer, polyoxyethylene and 
polyoxypropylene, outer coats that showed a remark- 
able ability to bypass rat liver and splenic macro- 



phages. (Davis, S.S., Ilium, L, Biomaterials 9, 111- 
115 (1988)). Drug delivery with th s particles has 
noty t been demonstrated. Ranney and Huffak r de- 
scribed an iron-oxide/albumin/drug system that yield- 

5 ed 350-1600 nm paramagnetic drug carriers. (Ran- 
ney, D.F., Huffaker, H.H., In, Juliano, R.L. (ed.): Bio- 
logical approaches to the delivery of drugs , Ann. N. Y 
Acad. Sci. 507, 104-119(1987).) Poznasky has devel- 
oped an enzyme-albumin conjugate system that ap- 

10 pears to decrease the sensitivity of the product to bio- 
degradation while masking the apparent antigenicity 
of the native enzyme. (Poznasky, M.J.: Targeting en- 
zyme albumin conjugates. Examining the magic bul- 
let In, Juliano, R.L. (ed.): Biological approaches to 

15 the delivery of drugs , Annals New York Academy Sci- 
ences 1987; 507-211:219.) 

Shaw and others have prepared and character- 
ized lipoprotein/drug complexes. (Shaw, J.M., Shaw, 
K.V., Yanovich, S., Iwanik, M., Futch, W.S., Rosows- 

20 ky, A., Schook, LB.: Delivery of lipophilic drugs using 
lipoproteins. In, Juliano, R.L(ed.): Biological ap- 
proaches to the delivery of drugs , Annals New York 
Academy Sciences 1987; 507:252-271.) Lipophilic 
drugs are relatively stable in these carriers and cell 

25 interactions do occur although little detail is known. 
EP-A-142193 discloses an immunogenic composi- 
tion comprising a glycoside core particle, with the gly- 
coside having a hydrophilic part and a hydrophobic 
part, a hydrophobic compound which coats the core 

30 particle, and an antigen or antigenic determinant or 
derivative thereof bonded to the hydrophobic core 
coating. 

In any conjugated biological composition, it is im- 
portant that the conformational integrity and biologi- 

35 cal activity of the adsorbed proteins or other biologi- 
cal agents be preserved without evoking an untoward 
immunological response. Spacial orientation and 
structural configuration are known to play a role in de- 
termining the biological activity of many peptides, 

40 proteins and pharmacological agents. Changes in the 
structural configuration of these compounds may re- 
sult in partial or total loss of biological activity. 
Changes in configuration may be caused by changing 
the environment surrounding the biologically active 

45 compound or agent. For example, pharmacologic 
agents which exhibit in vitro activity may not exhibit in 
vivo activity owing to the loss of the molecular con- 
figuration formerly determined in part by the in vitro 
environment. Further, the size and associated ability 

so of the carrier particle to minimize phagocytic trapping 
is a primary concern when the composition is to be 
used in vivo. All of these factors must be taken into ac- 
count when preparing a carrier particle. 

Although numerous different carrier particles 

55 have been developed, there is a continuing need to 
provide carrier particles for both in vivo and in vitro 
application wherein a biologically active peptid , pro- 
tein or pharmacological agent can b attached to the 



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particles in a manner which promot s stabilisation of 
the biologically active compound in its active config- 
uration. 

In accordance with th pr sent invention there is 
provided a composition of matter comprising a core 
particle; a coating which at least partially covers the 
surface of said core particle; and at least one biolog- 
ically active agent in contact with said coated core 
particle, characterised in that the core particle com- 
prises a metal, ceramic or polymer, and has a diame- 
ter of less than about 1000 nanometres, and the coat- 
ing comprises a basic sugar, modified sugar or oligo- 
nucleotide. 

The invention also resides in use in the manufac- 
ture of a vaccine for the purpose of vaccinating an an- 
imal to elicit an immune response to raise antibodies 
to Epstein-Barr virus, human immunodeficiency vi- 
rus, human papilloma virus, herpes virus or pox-vi- 
rus, the decoy virus comprising a composition ac- 
cording to any one of Claims 1 to 7 wherein the bio- 
logically active agent is at least one immunologically 
reactive viral protein or peptide bound to the coated 
core particle. 

In accordance with a preferred embodiment, bio- 
logically active peptides, proteins or pharmacological 
agents are attached to a core particle to provide a 
wide variety of biologically active compositions. The 
composition formulation is based on the discovery 
that the surface of ultraf ine particles (nanocrystalline 
particles, i.e, particles having a diameter of less than 
about 1000 nanometers,) can be modified with a sur- 
face coating to allow attachment of biologically active 
moieties to produce compositions wherein the natur- 
ally occurring structural environment of the moiety is 
mimicked sufficiently so that biological activity is pre- 
served. The coating which provides for the attach- 
ment of biologically active moieties to the particles is 
composed of a basic or modified sugar or oligonu- 
cleotide. Coating the core particles with a basic sugar 
or oligonucleotide produces changes in the surface 
energy and other surface characteristics which make 
the particles well suited for attachment of biologically 
active moieties. 

In another embodiment, the particles are used to 
prepare a decoy virus wherein the DNA or RNA core 
of the virus is replaced by the core particle. The core 
particle is chosen to be the same size as the viral core 
so that the conformation of the surrounding protein 
coat accurately mimics the native virus. The resulting 
viral decoy is incapable of infectious behavior while at 
the same time being fully capable of effecting an im- 
mune response and otherwise being antigenically 
bioreactive. 

A core particle having a diameter of less than 
about 1000 nanometers (a nanocrystalline particle) is 
chosen so as to mimic the DNA or RNA core. Viral 
peptides attached to the coating surrounding the cor 
have a structure which mimics at least a portion of the 



native virus. This size of core particle is als well suit- 
d for carrying anch rag dependent pharmacologh 
cal agents and ther biologically activ compounds 

5 which require a nanocrystallin particl anchor or 
core in order to maintain their activity. 

Examples of appropriate core particle materials 
include chromium, rubidium, iron, zinc, selenium, 
nickel, gold, silver, platinum, silicon dioxide, alumi- 

10 num oxide, ruthenium oxide, tin oxide and polystyr- 
ene. 

The disclosed compositions have wide-ranging 
use depending upon the type of biologically active 
agent which is in contact with the coated core partic- 

15 le. When the agent is viral protein, the result is a de- 
coy virus which may be used as a vaccine, diagnostic 
tool or antigenic agent for raising antibodies. Non-vi- 
ral protein or antigen coatings may be selected and 
structured for use in raising specific antibodies or as 

20 a diagnostic tool. Further, the composition can func- 
tion as a pharmacological agent when compounds 
having pharmacological activity are in contact with 
the coated core particle. 

The utilization of a core particle around which a 

25 viral protein is attached provides an effective way to 
accurately mimic the antigenic reactivity of a native vi- 
rus while totally eliminating any of the problems and 
risks associated with the presence of the viral genetic 
material. In addition, other proteins, peptides or phar- 

30 maco logical agents may be attached to the core par- 
ticle to preserve and/or enhance the activity of the 
compound. 

The present invention has wide application to im- 
munologic procedures and methods wherein a biolog- 
35 ically active agent is utilized. These areas of applica- 
tion include vaccination agents, antigen agents used 
to raise antibodies for subsequent diagnostic uses 
and antigenic compounds used as diagnostic tools. 
The composition disclosed can also be used in a wide 
40 variety of other applications where there is a need to 
anchor a protein, peptide or pharmacological agent to 
a core particle in order to preserve and/or enhance 
bioreactivity. 

The compositions include nanocrystalline core 
45 particles (diameters of less than 1000 nm) which are 
coated with a surface energy modifying layer that 
promotes bonding of proteins, peptides or pharma- 
ceutical agents to the particles. The coating modifies 
the surface energy of the nanocrystalline core par tie- 
so les so that a wide variety of immunogenic proteins, 
peptides and pharmaceutical agents may be attached 
to the core particle without significant loss of antigen- 
ic activity ordenaturization. The result is a biologically 
active composition which includes a biologically inert 
55 core. The end use for the compositions will depend 
upon the particular protein, peptide or pharmacolog- 
ical agent which is attached to the coated core par- 
ticle. For example, proteins or p ptides having anti- 
genic activity may be attached to provide compost- 



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tions useful as immunodiagnostic tools. Viral frag- 
ments or prot in coatings having immunogenic activ- 
ity may b attached to provid a vaccin . Also, phar- 
macological ag nts may be attach d to provide com- 5 
positions which are useful in treating diseases. 

For preparing decoy viruses for use as vaccines, 
particles having diameters of between about 10 to 
200 nanometers are preferred since particles within 
this size range more closely mimic the diameter of 10 
DlSlA and RNA cores typically found in viruses. 

The core particles are made from a metal or cer- 
amic or polymer The core material may be organic or 
inorganic. Preferred metals include chromium, rubi- 
dium, iron, zinc, selenium, nickel, gold, silver, plati- 15 
num. Preferred ceramic materials include silicon diox- 
ide, titanium dioxide, aluminum oxide, ruthenium ox- 
ide and tin oxide. Preferred polymers include poly- 
styrene, nylon and nitrocellulose. Particles made 
from tin oxide or titanium dioxide are particularly pre- 20 
ferred. 

Particles made from the above materials having 
diameters less than 1000 nanometers are available 
commercially or they may be produced from progres- 
sive nucleation in solution (colloid reaction), or van- 25 
ous physical and chemical vapor deposition process- 
es, such as sputter deposition (Hayashi, C, J. Vac. 
Sci. Technol. A5 (4), Jul/Aug. 1987, pgs. 1375-1384; 
Hayashi, C, Physics Today , Dec. 1987, pgs. 44-60; 
MRS Bulletin, Jan 1 990, pgs. 1 6-47). Tin oxide having 30 
a dispersed (in H 2 0) aggregate particle size of about 
140 nanometers is available commercially from Va- 
cuum Metallurgical Co. (Japan). Other commercially 
available particles having the desired composition 
and size range are available from Advanced Refrac- 35 
tory Technologies, Inc. (Buffalo, N.Y.). 

Plasma-assisted chemical vapor deposition 
(PACVD) is one of a number of techniques that may 
be used to prepare suitable microparticles. PACVD 
functions in relatively high atmospheric pressures (on 40 
the order of one torr and greater) and is useful in gen- 
erating particles having diameters of upto 1000 nano- 
meters. For example, aluminum nitride particles hav- 
ing diameters of less than 1000 nanometer can be 
synthesized by PACVD using Al (CH 3 ) 3 and NH 3 as re- 45 
a eta nts. The PACVD system typically includes a hor- 
izontally mounted quartz tube with associated pump- 
ing and gas feed systems. A susceptor is located at 
the center of the quartz tube and heated using a 60 
KHz radio frequency source. The synthesized alumi- so 
num nitride particles are collected on the walls of the 
quartz tube. Nitrogen gas is used as the carrier of the 
Al (CH 3 ) 3 . The ratio of Al (CH 3 ) 3 : NH 3 in the reaction 
chamber is controlled by varying the flow rates of the 
N 2 /AI(CH 3 ) 3 and NH 3 gas into the chamber. A constant 55 
pressure in the reaction chamber of 10 torr is gener- 
ally maintained to provide deposition and formation of 
the ultraf ine nanocrystalline aluminum nitrid partic- 
les. PACVD may be us d to prepare a variety of other 



suitabl nan crystallin particles. 

The cor particles ar coated with a substance 
that provides a threshold surfac nergy to the par- 
ticle sufficient to caus binding to occur without that 
binding being so tight as to denature biologically rel- 
evant sites. Coating is preferably accomplished by 
suspending the particles in a solution containing the 
dispersed surface modifying agent. It is necessary 
that the coating make the surface of the particle more 
amenable to protein or peptide attachment. Suitable 
coating substances in accordance with the present 
invention include basic sugars, and modified sugars, 
or Oligonucleotides. Suitable oligonucleotides in- 
clude polyadenosine (polyA). Cellobiose is a prefer- 
red coating material. Suitable modified sugars include 
nitrocellulose. 

The coating solution into which the core particles 
are suspended contains, for example, from 1 to 30 
weight/volume percent of the coating material. The 
solute is preferably double distilled water (ddH 2 0). 
The amount of core particles suspended within the 
coating solution will vary depending upon the type of 
particle and its size. Typically, suspensions contain- 
ing from 0.1 to 1 0 weight/volume percent are suitable. 
Suspensions of approximately 1 weight/volume per- 
cent of particles are preferred. 

The core particles are maintained in dispersion in 
the coating solution for a sufficient time to provide 
uniform coating of the particles. Sonication is the pre- 
ferred method for maintaining the dispersion. Disper- 
sion times ranging from 30 minutes to a few hours at 
room temperature are usually sufficient to provide a 
suitable coating to the particles. The thickness of the 
coating is preferably less than 5 nanometers. Thick- 
nesses of the coating may vary provided that the final 
core particles include a uniform coating over substan- 
tially all of the particle surface. 

The particles are separated from the suspension 
after coating and may be stored for future use or re- 
dispersed in a solution containing the protein or pep- 
tide to be attached to the particles. Alternatively, the 
coated particles may be left in the suspension for fur- 
ther treatment involving attachment of the desired 
protein or peptide. 

The biologically active agent which is in contact 
with the coated particles may be selected from a wide 
variety of proteins or peptides. Those having antigen- 
ic properties are preferred when a vaccine is re- 
quired. The protein can be the viral protein coat from 
a selected virus or immunogenic portion thereof. The 
viral protein coat is isolated according to known sep- 
aration procedures for isolating and separating viral 
proteins. The viral coating is the preferred protein be- 
cause the viral coating is where the antigenic activity 
of viruses is known to b located. Typically, the virus 
is digested or solubilized to form a mixture of viral pro- 
teins. The viral proteins are then separated by liquid 
chromatography or other conventional process into 



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the various prot in particle fractions and dialyz d to 
r move impurities. 

Suitabl viruses from which viral pr tein particles 
can be separat d and isolat d includ Epstein-Barr 
virus, human immunodeficiency virus (HIV), human 
papilloma virus, herpes simplex virus and pox-virus. 
Preparations of a wide variety of antigenic protein ma- 
terials may also be purchased commercially from sup- 
ply houses such as Microgene Systems, Inc. (400 
Frontage Road, West Haven, Connecticut 06516), 
Amgen Corporation (1900 Oak Terrace Lane, Thou- 
sand Oaks, California 91320-1789) and Cetus Cor- 
poration (1400 53rd Street, Emeryville, California 
94608). Synthetic peptides and/or proteins which cor- 
respond to naturally occurring viral particles may also 
be utilized. 

Other biologically active proteins and peptides 
that can be attached include enzymes, hormones, 
transport proteins and protective proteins. Human 
serum transferrin, plasminogen activator and coagu- 
lation factors, in addition to the pharmacologic agents 
amphotericin and insulin, are examples. 

The procedure for attaching the antigens or other 
protein to the coating on the core particles involves 
suspending the coated core particles in an aqueous 
solution containing the antigen. The presence in the 
solution of materials that may preferentially attach to 
the particle surface is often not advantageous. For 
example, the dispersion agents present in the solu- 
tion may create an undesirable coating on the sus- 
pended particles prior to protein attachment Water 
miscible solvents such as methanol or ethanol may 
be used. The aqueous solution of coated micropar ti- 
des can be agitated sufficiently to provide a uniform 
suspension of the particles. Typically, the amount of 
particles in solution will be between about 0.5 mg per 
milliliter of solution and 5 mg per milliliter of solution. 
Son i cation is a preferred method for providing a uni- 
form suspension of the coated particles in solution. 

The suspension of coated particles and antigens 
must be within certain parameters for protein attach- 
ment and assembly to occur. The temperature of the 
particle solution should be between 1°C to45°C. Cer- 
tain proteins and pharmaceutical agents may be 
bound to the coated particles in distilled water. Salts 
may be added to the solution for reactions between 
coated particles and proteins and other pharmaceut- 
ical agents which are unstable or will not disperse 
readily in distilled water. In general, the salt solutions 
should be formulated so that the ionic balance (in 
mM) does not exceed: K=300-500; Na=30-70; Cl=40- 
150; Ca=0.0003-0.001; and Mg=0.0003-0.001 . The 
oxygen tension of the solution is, advantageously, 
less than 10% in a solution sparged initially by helium 
and then gassed with helium, nitrogen and carbon di- 
oxide. The pH of the solution is, advantageously, 
slightly acidic (relative to blood), with a value, prefer- 
ably, of between 6.8 to 7.2. An exemplary solution for 



dispersion of the coated micropar ticl s and for pro- 
tein attachment is an aqueous soluti n containing: 
0.0360 milligrams MgSo 4 per liter, 0.0609 milligrams 

5 MgCI 2 . 6 H 2 0, 0.0441 milligram CaCl2. 2 H 2 0, 22.823 
grams K 2 HP0 4 , 13.609 grams KH 2 P0 4 , 7.455 grams 
KCI, and 4.101 gram sodium acetate. The pH of this 
solution is adjusted to 6.8. 

The coated particle cores with the attached pro- 

10 tein can be separated from the ionic growth medium 
and stored for further use. The coated particles may 
be stored by any of the conventional methods typical- 
ly used for storing antigenic com pounds or antibodies. 
For example, the coated particles may be freeze 

15 dried or stored as a suspension in a compatible solu- 
tion. When used as a vaccine, the particles coated 
with a viral protein coat are injected or otherwise ad- 
ministered to the individual according to conventional 
procedures. Any pharmaceutical^ acceptable carrier 

20 solution or other compound may be used in adminis- 
tering the coated particles to the individual. When 
used for diagnostic purposes in vitro, the protein coat- 
ed particles are suspended in solution and used in the 
same manner as other antigenic compounds. The 

25 same is true for use of t he protein coated particles for 
raising antibodies. The same protocol and procedures 
well known for using antigens to produce antibodies 
may be used wherein the protein coated particles of 
the present invention are substituted for normally 

30 used antigenic compounds. 

Example 1. Preparation of nanocrystalline tin oxide 
microparticles : 

35 1 .5 to 2.0 mg of ultraf ine (nanocrystalline) metal 

powder was placed in a 1.7 ml screw-cap microcen- 
trifuge with 1.5 mis of double distilled water (ddH 2 0). 
The ddH 2 0 was filtered through a rinsed 0.45 micron 
filter-sterilizing unit or acrodisc (Gelman Scientific). 

40 The metal powder was tin oxide with a mean diameter 
(by photon correlation spectroscopy) of 140 nm. The 
mixture was vortexed for 30 seconds and placed into 
a water sonicating bath overnight. The sonication 
bath temperature was stabilized at 60°C. After a 24- 

45 hour sonication, the samples were vortexed once 
more for 30 seconds with the resulting dispersion 
clarified by microcentrifugation at approximately 
16,000 rpm for 15 seconds. The analysis of particle 
size was carried out on a Coulter N4MD sub-micron 

50 particle analyzer. 

The coating was applied to the tin oxide particles 
by suspending the particles in a stock solution of cel- 
lobiose. The cellobiose stock solution was a 292 mM 
solution made by dissolving 1.000 gram of cellobiose 

55 in 9.00 mis of ddH 2 0. Solution was accomplished at 
approximately 70°C in order to promot quick disso- 
lution. The resulting cellobiose solution was filter ster- 
ilized through a rinsed 0.45 micron filter with the final 
volume being adjusted to 10.00 ml. 



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EP 0 465 081 B1 



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Sufficient cellobiose stock solution was added to 
150microlit rs f ultraf ine tin oxid dispersion so that 
th final concentration of th tin oxide was 1 .00 p r- 
cent (w/v) or 29.2 mM. A typical volum for prepara- 5 
tion was 2.0 mis which was mixed four or five times 
by the action of a micro-pipetor. After mixing, the dis- 
persion was allowed to equilibrate for two hours. 
Demonstration of successful coating of the particles 
was provided by measuring the mobility of the par tic- 10 
les (coated and uncoated) on a Coulter DELSA 440 
doppler energy light scatter analyzer. The coated tin 
oxide particles exhibited a relatively low mobility com- 
pared to the non-coated tin oxide particles. Measure- 
ments were also taken at various dilute salt concen- 15 
trations to ensure that the observations with respect 
to mobility were not artif actual. The tests demonstrate 
that the particles were coated with the cellobiose. 

The coated particles are then used to attach an- 
tigenic proteins, peptides or pharmacological agents 20 
to prepare bioreactive particles. 

Example 2. Preparation of nanocrystalline 
ruthenium oxide particles : 

25 

The same procedure was carried out in accor- 
dance with Example 1, except that ruthenium oxide 
micropar tides were substituted for the tin oxide par- 
ticles. The ruthenium oxide particles were obtained 
from Vacuum Metallurgical Company (Japan). 30 

Example 3. Preparation of the nanocrystalline 
silicon dioxide and tin oxide particles : 

Nanocrystalline silicon dioxide was acquired 35 
commercially from Advanced Refractory Technolo- 
gies, Inc. (Buffalo, N.Y.) and tin oxide was acquired 
commercially from Vacuum Metallurgical Co. (Japan). 
The tin oxide particles were also prepared by reactive 
evaporations of tin in an argon-oxygen mixture and 40 
collected on cooled substrates. Nanocrystalline tin 
oxide was also synthesized by D.C. reactive Magne- 
tron sputtering (inverted cathode). A 3" diameter tar- 
get of high purity tin was sputtered in a high pressure 
gas mixture of argon and oxygen. The ultraf ine par- 45 
tides formed in the gas phase were collected on cop- 
per tubes cooled to 77°K with flowing liquid nitrogen. 
All materials were characterized by X-ray diffraction 
crystallography, transmission electron microscopy, 
photon correlation spectroscopy, and Doppler elec- so 
trophoretic light scatter analysis. X-ray diffraction 
samples were prepared by mounting the powder on 
a glass slide using double-sized Scotch tape. CuKa 
radiation was used on a Norelco drffractometer. The 
spectrum obtained was compared with ASTM stan- 55 
dard data of tin oxide. (Powder Diffraction File, Card 
#21-1250. Joint Committee on Power Diffraction 
Standards, American Society for Testing and Mat ri- 
als, Philadelphia 1976.) The specimens for (TEM) 



were collected on a standard 3 mm diam ter carbon 
coated copper mesh by dipping int a disp rsion of 
the (UFP's) in 22-propanol. The samples wer exam- 
in d on a JEOL-STEM 100 CX at an acceleration vol- 
tage of 60-80 KV. 

To create working dispersions of these metal ox- 
ides, 1.5 to 3.0 mg of metal oxide powder was added 
to 1.5 ml double distilled H 2 0 in a dust-free screw top 
microcentrifuge tube (Sarsted) and vortexed for 30 
seconds. The mixture was then sonicated for 16 to 24 
hours followed by a second 30 seconds vortex. The 
submicron fraction was then isolated by pelleting 
macroparticulates by microcentrifugation 16,000 xg 
for 15 seconds. Approximately 1.3 ml of supernatant 
was then removed and placed in another dust-free 
screw top microcentrifuge tube. A sample was pre- 
pared for photon correlation spectroscopy (Coulter 
N4MD) and Doppler electrophoretic light scattering 
(Coulter delsa 440) analysis by removing 50 to 1 00 pJ 
of the dispersion and placing it in a polystyrene cuv- 
ette and diluting it to a final volume of 1.00 ml with 
ddH 2 0. The stability of the dispersion was deter- 
mined by sequential measurements over a 24-hour 
period and was found to be stable. The stability of the 
dispersion with respect to progressive salinity of the 
solvent (increasing conductivity) was similarly deter- 
mined. The stability increased with progressive salin- 
ity of the solvent. 

1 .00 ml of the dispersion was combined and stir- 
red with 8.00 ml of ddH 2 0 and 1.00 ml of 29.2 mM cel- 
lobiose stock in a 15.0 ml capacity ultrafiltration stir 
cell (Spectra) which has been fitted with a pre- rinsed 
5x1 0 5 molecular weight cutoff type F membrane 
(Spectra). The sample was then left to stir for 15 min- 
utes. After stirring, the excess cellobiose was re- 
moved by flushing through the cell chamber 250 ml 
of ddH 2 0 by the action of a peristaltic pump at a rate 
that does not exceed 10.0 ml/min. After washing, the 
filtrate was concentrated by the means of pressurized 
N 2 gas to approximately 1 .0 ml. Character was estab- 
lished by the removal of 500 ul of the treated disper- 
sion by N4MD analysis. The mean dispersion diame- 
ter was re-established at this step. The stability of the 
coated dispersion was determined by sequential 
measurements over a 24-hour period. The stability of 
the coated dispersion with respect to progressive sal- 
inity of the solvent (increasing conductivity) was sim- 
ilarly determined. 

The resulting coated nanocrystalline partides 
are suitable for attachment of various proteins, pep- 
tides and pharmaceutical agents. 

Example 4. Preparation, isolation and surface 
adsorption of human serum transferrin proteins : 

Nanocrystalline tin oxide was synthesized by 
D.C. reactive Magn tron sputtering (inverted cath- 
ode). A 3" diameter target of high purity tin was sput- 



6 



11 



EP 0 465 081 B1 



12 



tered in a high pressure gas mixtur of argon and oxy- 
gen. Th ultra-fine particles formed in the gas phas 
wer collected on copper tubes cool d to 77°K with 
flowing liquid nitrogen. All materials were character- 5 
ized by x-ray diffraction crystallography, selected 
area electron diffraction, transmission electron micro- 
scopy, photon correlation spectroscopy, and energy 
dispersive x-ray spectroscopy. X-ray diffraction sam- 
ples were prepared by mounting the powder on a 10 
glass slide using double-sized Scotch tape. CuK(a/- 
pha) radiation was used on a Norelco diffractometer. 
The spectrum obtained was compared with ASTM 
standard data of tin oxide. The specimens for trans- 
mission electron microscopy and selected area dif- 15 
fraction were collected on a standard 3 mm diameter 
carbon coated copper mesh by dipping into a disper- 
sion of the nanocrystalline materials in 2-propanol. 
The samples were examined on a JEOL-STEM 100 
CX at an acceleration voltage of 60-80 KeV. The 2- 20 
propanol suspension of particles was also character- 
ized by photon correlation spectroscopy at 22.5°C, 
600 s run time on a Coulter N4MD. Energy dispersive 
x-ray spectroscopy was performed on a JEOL JSM- 
T330A scanning electron microscope using Kevex 25 
quantex V software. 

To create working dispersions of these metal ox- 
ides for the synthesis of compositions in accordance 
with the present invention, 0.5 mg of metal oxide pow- 
der was added to 1.0 ml of a 29.2 mM cellobiose- 30 
phosphate buffered saline solution in a dust free 
screw top glass vial and sonicated for 20 minutes at 
22.5-35°C. The submicron fraction was then isolated 
by pelleting macro particulates by microcentrifugation 
at 16,000xg for 30 seconds. Approximately 900 ul of 35 
supernatant was then removed and placed in a dust 
free screw top microcentrifuge tube. An aliquot was 
removed for photon correlation spectroscopy (Coulter 
N4MD) and Doppler electrophoretic light scattering 
(Coulter DELSA 440) analysis. Aliquots were also re- 40 
moved for characterizing the stability of the coated 
dispersion over time and with respect to progressive 
salinity of the solvent (increasing conductivity). 

To adsorb protein to the cellobiose coated metal 
oxide nanocrystalline cores, the core sample was di- 45 
luted to 10.0 ml with Ca ++ and Mg ++ free phosphate 
buffered saline (Gibco). Forty (40.0) ug of purified hu- 
man serum transferrin (4jag/(il) (Gibco), whose anti- 
genicity was verified by ELISA, was then added to a 
1 0 ml stir cell (Spectra). The sample was then left to so 
stir slowly for 30 minutes, taking great care not to al- 
low foaming. After the addition period, 15 ml of Ca ++ 
and Mg"^ free phosphate buffered saline (Gibco) was 
then washed through the cell under a 2 psi nitrogen 
gas pressure head. After washing, the sample was 55 
again concentrated to 1.00 ml under N 2 and a 500 ul 
sample was removed for analysis by photon correla- 
tion spectroscopy, Doppl r electrophoretic light scat- 
ter and transmission electron microscopy as detailed 



below. 

Conformational int grity was assess d by meas- 
uring the retained antigenicity of th bound protein. 
To the sampl cell, 50.0 u.l of rabbit polyclonal anti- 
human transferrin antibody (Dako), whose antigenici- 
ty was confirmed by ELISA, was added to the con- 
centrated 1.0 ml reaction product at 37.5°C with gen- 
tle stirring. After a 30 minute incubation period, 15 ml 
of Ca ++ and Mg^free phosphate buffered saline (Gib- 
co) was then washed through the cell under a 2 psi 
nitrogen gas pressure head and the reaction volume 
was again reduced to 1 .0 ml. 

A 200 uJ aliquot of blocking agent, 1 % w/v bovine 
serum albumin in divalent free saline, was added fol- 
lowed by a 10 minute equilibration period. The sec- 
ondary antibody, 30 nm gold conjugated goat anti- 
rabbit polyclonal IgG (Zymed), was then added and 
the reaction mixture was allowed to incubate for 30 
minutes. A sample was removed, chopped on a trans- 
mission electron microscopy grid, and vacuum dried. 
The mixture was again washed with 15 ml of divalent 
free saline under a nitrogen pressure head and then 
fixed with glutaraldehyde. One ml of 3% solid bovine 
collagen (Collagen Corp.) was then added to the mix- 
tures and the composite was ultracentrifuged at 1 0 6 xg 
for 30 minutes yielding a pellet that was then routinely 
processed as a biological specimen for transmission 
electron microscopy. Ten nm thick sections were 
viewed on a Zeiss transmission electron microscopy. 
Control samples were prepared as above without the 
cellobkfee intermediate bonding layer. 

Transmission electron micrographs showed that 
the D.C. magnetron sputtered tin oxide was com- 
posed of individual particles measuring 20-25 nm in 
diameter which aggregated into clusters measuring 
80 to 1 20 nm in diameter. By photon correlation spec- 
troscopy, these same particles when dispersed in dis- 
tilled water produced agglomerates measuring 154 ± 
55 nm. The tin oxide particles were fully crystalline as 
characterized by electron and x-ray diffraction. Ener- 
gy dispersive x-ray spectroscopy showed no other 
elements present as impurities. 

By Doppler electrophoretic light scatter analysis, 
tin oxide exhibited a mean mobility of 2.177 ± 0.215 
um-cm/V-s in aqueous solutions ranging from 10.8 to 
20.3 |iM NaCI. Following cellobiose surface coating in 
a 1% solution, tin oxide exhibited a mean mobility of 
1 .544 ± 0.241 um-cm/V-s in aqueous solutions rang- 
ing from 0.0 to 21 .0 uM NaCI. The oxide agglomerated 
in salt concentrations of greater than 40.0 uM and in 
solutions of increasing cellobiose concentration. 

Following transferring binding, the crude tin ox- 
ide/cellobiose/protein conjugates measured 350 ± 84 
nm by photon correlation spectroscopy and transmis- 
sion electron microscopy. Vacuum dried dropped 
samples with low concentration gold antibody meas- 
ured 35-50 nm. Without the cellobiose bonding layer, 
vacuum dried s ctions measured 400 to > 1000 nm. 



7 



13 



EP 0 465 081 B1 



14 



Occasional antibody bonding was noted. Following 
high concentration immunogold lab ling and filt ring, 
the thin section c llobiose treat d sp cim ns meas- 
ured 50-100 nm. Positiv gold binding was id ntified 5 
in approximately 20% of the appropriately coated 
samples whereas negative controls (prepared as 
above but lacking the primary rabbit antibody) exhib- 
ited approximately 1% nonspecific binding. 

As can be seen from the above examples, the 10 
biological activity of protein absorbed to the surface 
of carbohydrate-treated nanocrystalline metal oxide 
particles is preserved. 

Example 5. Preparation and Characterization of 15 
Epstein-Barr Virus Decoys : 

Nanocrystalline tin oxide particles were synthe- 
sized by D.C. reactive Magnetron sputtering as previ- 
ously described in Example 1 20 

Elutriated sucrose gradient purified Epstein-Barr 
virus (EBV) acquired from the B95-8 cell line were 
purchased from Advanced Biotechnologies, Inc., Co- 
lumbia MD. Each viral aliquot contained approximate- 
ly 5.00 x 10 10 virus particles/ml suspended in 10mM 25 
TRIS-150mM NaCI ph 7.5 buffer (approximately 0.94 
mg/ml protein). The virions were solubilized 0.75% 
(v/v) Triton X100 and then ultracentrifuged at 
1 50,000xg for 60 minutes to pellet the DNAcore using 
a modification of the method described by Wells. 30 
(Wells A, Koide N, Klein G: Two large virion envelope 
glycoproteins mediate EBV binding to receptor-posi- 
tive cells. J Virology 1982; 41:286-297.) Following di- 
alysis, the supernatant EBV extract was character- 
ized by both SDS-PAGE (denatured) [Biorad Mini Gel 35 
II, 4-20% gradient gel, 200V x 45 minutes and stained 
with silver] and size exclusion HPLC (non-denatured) 
[Waters 620 system with a WISP autoinjector and 720 
photodiode array detector, 0.5 ml/minute over a Wa- 
ters SW300 GFC column using a 1 0OmM NaCI/20mM 40 
TRIS pH 9.4 gradient mobile phase]. 

Control (non-EBV) proteins were extracted from 
aliquots of Lambda phage virus [Pharmacia, Milwau- 
kee Wl] using the same methods as described above. 

Aliquots of the tin oxide powder weighing approx- 45 
imately 1.5 mg were initially suspended in 3.0 ml of 
29.2 mM cellobiose solution in a dust free glass vial 
by liberal vortexing [Vortex Genie, Scientific Indus- 
tries, Bohemia, NY]. The resultant brownish cloudy 
suspension was then sonified at 175 Wfor 10.0 min- 50 
utes at a frequency of approximately 20 kHz at 
25°C[Branson 2" Cup Horn, Branson Ultrasonics 
Corp., Danbury CT]. The dispersion was clarified by 
microcentrifugation at 16,000xg for 15 seconds. The 
remaining pellet was then discarded in favor of the su- 55 
pernatant Unadsorbed cellobiose was removed by 
ultrafiltration against 20 mis of 25 mM phosphate re- 
action buffer (pH 7.40 25mM HPO^/HaPO* 1 ") in a 10 
kD nominal molecular weight filtered stir cell Phar- 



macia] under a 7.5 psi N 2 gas head at 37. 5°C. Aliquots 
of th int rmediat product w r characteriz d by 
photon correlation sp ctroscopy and, f (lowing dialy- 
sis as described below, by doppler lectrophoretic 
light scatter analysis. 

The process of viral protein adsorption was initi- 
ated by the removal of the mild triton surfactant from 
250 uJ aliquots of EBV extract by ultrafiltration against 
25 mis of phosphate reaction buffer at 4°C in a 10 kD 
nominal molecular weight stir cell and then adjusted 
to a concentration of 1 .0 ug/uJ or approximately 1 .0 ml 
final volume. Then 500 ul of the triton free EBV extract 
was quickly added to a MD nominal molecular weight 
stir cell with 2.0 ml of the surface treated tin oxide dis- 
persion prewarmed to 37.5°C. The mixture was then 
slowly stirred while being incubated at 37.5°C for 2.0 
hours. After incubation the unabsorbed EBV extract 
was removed by ultrafiltration against 25 mis of phos- 
phate reaction buffer. 

Control (non-EBV) decoys fabricated with lamb- 
da phage viral protein extracts were synthesized us- 
ing the same process described above. 

Intermediate components, the final assembled 
decoys, and whole Epstein-Barr virions were charac- 
terized by doppler electrophoretic light scatter analy- 
sis [DELSA440, Coulter Electronics Inc., Hialeah, FL] 
to determine their electrophoretic mobility (surface 
charge) in a fluid phase. Nine phosphate buffer solu- 
tions having at 25°C pH's ranging between 4.59 and 
9.06 and corresponding conductivities ranging be- 
tween 2.290 and 4.720 mS/cm were prepared. Ali- 
quots of raw tin oxide, surface modified cellobiose 
covered tin oxide, synthesized EBV decoy, and whole 
EBV were dialyzed against each of the nine solutions 
and the mobilities of the particulates in dispersion 
were then measured at field strengths of 4.0, 5.5, 5.5, 
and 8.0 mA respectively. The mobility values acquired 
simultaneously by the 4 angled detectors of the in- 
strument were averaged and the means of 3 meas- 
urements per dispersion were recorded. 

The synthesized EBV decoys and control decoys 
were characterized by immunoagglutination photon 
correlation spectroscopy to determine the antibody 
reactivity of their surfaces. Positive reactivity was as- 
sessed by incubating the EBV decoy for 60 minutes 
at 37.5°C with a cocktail of anti-EBV murine monoclo- 
nal antibodies (1 ug each of anti-EBV-VCA, anti-EBV 
EA-R, anti-EBV MA, and anti EBV EA-D) in 15% lac- 
tose, 0.9% NaCI, 10 mM HEPES buffer, and 0.2% 
NaN3 [DuPont, Wilmington, DE]). Background reac- 
tivity was assessed by incubating the EBV decoy with 
irrelevant murine IgG^ Specificity was assessed by 
reacting the lambda phage decoy with monoclonal 
anti-EBV murine antibodies. Agglutination was meas- 
ured by photon correlation spectroscopy at a 90° an- 
gle [N4MD, Coulter]. 

Antibody affinity intensity was assessed by im- 
munogold transmission electron microscopy using 



8 



15 EPO 

the particulates and antibodi s listed above and then 
adding secondary anti-murin 30 nm gold-labeled an- 
tibodies (Faulk W, Taylor G. Immunocolloid m thod 
for lectron microscopy, Immunochomistry 8:1081- 
1083, 1971). 

Labeling of the EBV decoy (positive reaction) was 
accomplished by incubating a 20 uJ mixture of murine 
monoclones (1 ug anti-EBV-VCA and 1 ug anti-EBV 
EA-R in 15% lactose, 0.9% NaCI, 10 mM HEPES buf- 
fer, and 0.2% NaN3 [DuPont] ) with a fresh 0.5 ml 
sample of EBV decoy at 37.5°C for 30 minutes in a 
300 kD nominal molecular weigh stir cell. Unbound 
antibody was then removed by ultrafiltration against 
20 mis of phosphate reaction buffer under a 5.0 psi N 2 
pressure head. After washing, 50 ul of goat anti- 
murin e antibody covalently fused to 30 nm gold 
spheres (10 6 particles/ml [Zymed Laboratories, San 
Francisco, CA]) were incubated with 200 uis of the 
labeled particles in a 1 M nominal molecular weight stir 
cell at 37. 5° C for 30 minutes. Unbound secondary an- 
tibody was removed by ultrafiltration against 1 0 mis of 
phosphate reaction buffer. 

Labeling of the EBV decoy (negative reaction) 
was accomplished by incubating 2.5 pJ of murine poly- 
clonal nonspecific lgG1 (1-ug/ul in 15 mM NaCI pH 
7.4 [Sigma Chemical Corp., St. Louis, MO]) with a 
fresh 0.5 ml sample of EBV decoy as described above 
followed by the same washing and gold-labeling 
steps. Labeling of the lambda phage control decoy 
(negative reaction) was accomplished by incubating a 
20 uJ mixture of murine monoclonal anti-EBV antibo- 
dies with the lambda phage virus coated decoy using 
the same procedure detailed above. 

Immunolabeled particles were prepared for elec- 
tron microscopy in two ways. A direct immersion tech- 
nique where a carbon coated copper viewing grid [Ted 
Pel I a Inc., Redding, CA] was submersed into sample 
for approximately 5 seconds and then fixed in 5% glu- 
taraldehyde for 1 minute, was used for all reactions as 
a fast screening technique. A more involved method 
adding glutaraldehyde directly to the reaction solu- 
tion, then pelleting the product at 16,000xg for 5 min- 
utes into 0.5 ml soft agar preparation (0.7% agarose 
[Sea Kern, Temecula, CA] in H 2 0). Then the resultant 
agar plugs were embedded in plastic and sectioned 
into 0.1 \im sheets for viewing. 

Analysis of both the positive and negative con- 
trols was performed by examining pelleted samples 
of the labeled reaction products by transmission elec- 
tron microscopy. The relative intensity of antibody 
binding was determined by counting the number of tin 
oxide based particles observed to have bound gold 
spheres (% positive) and then noting the number of 
gold spheres bound to a given particle (intensity, 
number/event). 

The ultrafine tin oxide particles measur d 20-25 
nm in diameter and formed aggregates measuring 80 
to 1 20 nm in diameter by transmission electron micro- 



65 081 B1 16 

scopy. By photon correlation spectroscopy, these 
same particl s when disp rsed in distilled water pro- 
duced agglomerates measuring 154 1 55 nm. The tin 

s oxide particles w re fully crystalline as charact rized 
by electron and x-ray diffraction. Energy dispersive x- 
ray spectroscopy showed no other elements present 
as impurities. 

Characterization of the EBV proteins by SDS-PA- 

10 GE showed two distinct protein bands. The first, ex- 
isting as a dimer suggesting variable glycosylate n, 
exhibited a molecular weight of approximately 350 kd 
which is consistent with the predominant envelope 
glycoprotein of EBV. The second exhibited a molecu- 

15 lar weight of approximately 67 kd consistent with ser- 
um albumin which apparently adsorbs avidly to the vi- 
ral surface. HPLC confirmed the presence of two dis- 
tinct bands that exhibited spectrophotometric absorp- 
tion maxima at 280 nm consistent with proteins. The 

20 predominant peak had a chromatographic retention 
time of 10.30 minutes and could be suppressed 90% 
by monoclonal anti VCA. The second and relatively 
minor peak exhibited a chromatographic retention 
time of 1 5.75 minutes similar to bovine serum albumin 

25 standards. 

The previously described Dopplerelectrophoretic 
mobility studies conducted between the pH range of 
4.5 to 9.0 demonstrated 3 distinct patterns. First, both 
the decoy and native EB virus retained virtually iden- 

30 tical mobilities of approximately -1.4 um-cm/V-s 
throughout the pH range. Second, untreated tin oxide 
exhibited a mobility of approximately -1.0 um-cm/V-s 
at a pH of 4.5 which then rose rapidly to -3.0 um- 
cm/V-s at pH values of 5.0 and higher. Third, surface 

35 modified tin oxide treated with cellobiose retained a 
mobility of approximately -1 .5 um-cm/V-s until it in- 
creased rapidly to -2.5 um-cm/V-s at a pH of 7.5. 

The previously described photon correlation 
spectroscopy showed that native EBV measured ap- 

40 proximately 1 02 +/ -32 nm and the synthesized EBV 
decoy measured approximately 154 + /- 52 nm. Syn- 
thesized EBV decoy, when reacted with the monoclo- 
nal anti-EBV cocktail, agglutinated to form 1534 +/ - 
394 nm masses. Synthesized EBV decoy, when re- 

45 acted with non-specific mouse IgG, only increased 
slightly in size with agglutination diameters of 230 +/ - 
76 nm. Lambda phage decoy, when reacted with the 
monoclonal anti-EBV cocktail, only increased slightly 
in size with agglutination diameters of 170 +/ -35 nm. 

so The previously described transmission electron 
microscopy of anti-EBV antibody labeled EBV decoy 
particles revealed a positive gold staining frequency 
of 23.51% +/ -5.53 with an average staining intensity 
of 7.41 gold labels per event Examination of non-spe- 

55 cific mouse IgG antibody labeled EBV decoy particles 
revealed a positive gold staining frequency of 5.53% 
+/ -2.04 with an average staining intensity of 1.00 
gold labels per event. Examination of anti-EBV anti- 
body labeled lambda phage decoy particles revealed 



9 



17 



EP 0 465 081 B1 



18 



a positive gold staining frequency of 7.21% +/-1.26 
with an average staining intensity of 1 .06 gold labels 
per vent. 

Example 6: In Vivo Elicitation of Antibodies By 
Epstein-Barr Virus Decoy : 

Four sensitization solutions were prepared and 
delivered once every other week by intramuscular in- 
jection in three 250 ul aliquots to New Zealand rabbits 
aged approximately 8 weeks. The first four animals 
received approximately 10 9 whole EBV virions (ap- 
proximately 32 ug of gp350 estimated by integration 
of the spectrophotometric absorption curve at 280 nm 
against a 25 \xq bovine serum albumin standard) dis- 
persed in phosphate reaction buffer per injection. The 
second four animals received 32 jig per injection of 
isolated and purified gp350 using the same injection 
protocol. The third group received EBV viral decoys 
(Example 5) synthesized from a starting aliquot of 32 
u-g of gp350 per injection. The last group received cel- 
lobiose coated in tin oxide dispersed in phosphate re- 
action buffer. Injections were free of adjuvant Whole 
blood was removed using aseptic techniques via car- 
diac puncture 2 weeks following each of the three in- 
jections and the animals were terminated by cardiac 
puncture followed by lethal sedation at 6 weeks. Ser- 
um was extracted by microcentrifugation at 16 kg of 
whole blood for 1 minute and then stored frozen at - 
70°C pending analysis. 

Immunospecif ic antibody against whole EBV vir- 
ions (ABI) was assayed by ELISA. Approximately 10 9 
virions/ml in phosphate reaction buffer were diluted 
1:10 in coating buffer and then allowed to adsorb 
overnight at 4°C in polycarbonate assay plates (Fal- 
con). Rabbit serum affinity for the bound EBV virions 
was determined by the colorimetric reaction of goat 
anti-rabbit IgG alkaline phosphatase (Sigma) devel- 
oped with para-nitrophenyl phosphate. The concen- 
tration of immunospecif ic IgG were determined by 
comparison to a calibration curve using nonspecific 
rabbit IgG as the adsorbed antigen and by subtracting 
the baseline values recorded from the wells contain- 
ing serum from the rabbits stimulated with tin oxide 
only. 

Serum collected from the 4 rabbits sensitized 
with tin oxide showed no increased anti-EBV activity 
over pre- immune serum at any of the three two week 
sampling intervals. The remaining 3 groups showed 
a progressive rise in the concentration of anti-EBV 
specific IgG over the 6 week period. Animals sensi- 
tized with purified EBV proteins alone showed a max- 
imum of approximately 0.05 ug/uJ anti-EBV IgG at six 
weeks. In contrast animals sensitized with either 
whole EBV or decoy EBV exhibited a statistically sig- 
nificant fourfold greater response with approximately 
0.20 ug/ul of anti-EBV IgG at six weeks. The immuno- 
specific responses to decoy EBV and whole EBV 



were virtually identical. 

As is apparent from Exampl s 5 and 6, th syn- 
thesized EBV decoy possess s th same surface 
charge as nativ virus, is recognized specifically and 
avidly by monoclonal antibodies, and evokes immu- 
nospecif ic antibodies with the same effectiveness as 
whole virus. Using photon correlation spectroscopy, 
the number of particles that agglutinated in the three 



10 reaction conditions were calculated from the meas- 
ured diameters of the aggregates. These calculations 
indicate that monoclonal anti-EBV antibodies pro- 
duce agglutinated masses consisting of an average 
988.0 decoy EBV particles. Non-specific mouse IgG 

15 antibodies produce agglutinated masses consisting 
of an average 3.33 decoy EBV particles, while mono- 
clonal anti-EBV antibodies produce agglutinated 
masses consisting of an average 1.35 decoy control 
lambda phage particles. These measured results 

20 show t hat t he measured agglutination potential of t he 
EBV decoy in accordance with the present invention 
is almost three orders of magnitude greater than con- 
trols. The immunogold transmission electron micro- 
scopy shows that the gold labeled antibody staining 

25 of anti-EBV labeled EBV decoys is 25 to 30 times 
greater than controls. The ELISA analysis of the im- 
munospecif icity of anti-EBV IgG elicited in the rabbits 
by the EBV decoy is similar to the response elicited 
by native virus and is 4 fold greater than the response 

30 elicited by isolated purified proteins. 

Claims 

35 1 . A composition of matter comprising a core partic- 
le, a coating which at least partially covers the 
surface of said core particle, and at least one bio- 
logically active agent in contact with said coated 
core particle, characterised in that the core par- 

40 tide comprises a metal, ceramic or polymer, and 

has a diameter of less than about 1000 nanome- 
ters, and the coating comprises a basic sugar, 
modified sugar or oligonucleotide. 

45 2. A composition of matter according to claim 1 
wherein the diameter of said core particle is be- 
tween about 10 to 200 nanometers. 

3. A composition of matter according to Claim 1 or 
so 2, wherein said coating is cellobiose. 

4. A composition of matter according to any preced- 
ing claim, wherein said biologically active agent is 
a pharmacologic agent. 

55 

5. A composition according to any preceding claim 
wherein said metal is selected from chromium, 
rubidium, iron, zinc, selenium, nickel, gold, silver 
and platinum. 



10 



19 



EP 0 465 081 B1 



20 



6. A composition according to any on of Claims 1 
to 4 wherein said ceramic is selected from silicon 
dioxid , aluminum oxid , ruth nium xid , car- 
bon and tin oxid . 

7. A composition according to any one of Claims 1 
to 4, wherein said polymer is polystyrene. 

8. A composition according to any preceding claim 
for use as viral decoy vaccine for use in treating 
an animal to elicit an immune response, wherein 
said coating comprises a substance that provides 
a threshold surface energy to said core particle 
which is sufficient to bind immunologically active 
proteins or peptides without denaturing said pro- 
teins or peptides, and said biologically active 
agent is an at least one immunologically reactive 
viral protein or peptide bound to said coated core 
particle, and comprising a pharmaceutically ac- 
ceptable carrier for said viral decoy vaccine. 

9. A composition according to Claim 8 wherein said 
viral peptide or protein is isolated from Epstein- 
Barr virus, human immunodeficiency virus, hu- 
man papilloma virus, herpes virus or pox-virus. 

1 0. Use of a decoy virus in t he manufacture of a vac- 
cine for the purpose of vaccinating an animal to 
elicit an immune response to raise antibodies to 
Epstein-Barr virus, human immunodeficiency vi- 
rus, human papilloma virus, herpes virus or pox- 
virus, the decoy virus comprising a composition 
according to any one of Claims 1 to 7 wherein the 
biologically active agent is at least one immuno- 
logically reactive viral protein or peptide bound to 
the coated core particle. 



4. Zub r itung nach einem der vorhergehenenden 
Anspruche, wobei das biologisch aktiv Mittel 
aus einem pharmakologischen Mittel besteht. 

5 

5. Zubereitung nach einem der vorhergehenden An- 
spruche, wobei das M eta II aus Chrom, Rubidium, 
Eisen, Zink, Selen, Nickel, Gold, Silber Oder Pla- 
tin ausgewahlt ist. 

10 

6. Zubereitung nach einem der Anspruche 1 bis 4, 
wobe i die Keramik aus Siliciumdioxid, Alum inium- 
oxid, Rutheniumoxid, Kohlenstoff und Zinnoxid 
ausgewahlt ist. 

15 

7. Zubereitung nach einem der Anspruche 1 bis 4, 
wobei das Polymer aus Polystyrol besteht. 

8. Zubereitung nach einem dervorhergehenden An- 
20 spruche zur Verwendung als viraler Lockimpf- 

stoff bei der Behandlung eines Tiers zur Ent- 
lockung einer Immunantwort, wobei der Uberzug 
eine Substanz umfaftt, die dem Kernteilchen eine 
zur Bindung immunologisch aktiver Proteine Oder 

25 Peptide ohne Denaturierung der Proteine oder 

Peptide ausreichende Schwellenoberflachen- 
energie verleiht, und wobei das biologisch aktive 
Mittel aus mindestens einem an das beschichtete 
Kernteilchen gebundenen immunologisch reakti- 

30 onsfahigen viralen Protein oder Peptid besteht, 
mit einem Gehalt an einem pharmazeutisch ak- 
zeptablen Tragerfur den viralen Lockimpfstoff. 

9. Zubereitung nach Anspruch 8, wobei das virale 
35 Peptid oder Protein aus Epstein- Barr- Virus, Hu- 

manimmundefektvirus, Human pappiloma Virus, 
Herpes Virus oder Pockenvirus isoliert ist. 



Patentanspruche 

1. Zubereitung, umfassend ein Kernteilchen, einen 
die Oberflache dieses Kernteilchens zumindest 
teilweise bedeckenden Uberzug und mindestens 
ein mit dem beschichteten Kernteilchen in Beruh- 
rung stehendes biologisch aktives Mittel, da- 
durch gekennzeichnet, daR das Kernteilchen ein 
Metall, eine Keramik oder ein Polymer umfa&t 
und einen Durchmesser von weniger als etwa 
1000 nm aufweist und dad der Uberzug einen 
Grundzucker, einen modifizierten Zucker oder 
ein Oligonucleotid umfa&t. 

2. Zubereitung nach Anspruch 1, wobei der Durch- 
messer des Kernteilchens zwischen etwa 10 und 
200 nm liegt. 

3. Zubereitung nach Anspruch 1 oder 2, wobei der 
Uberzug aus Cellobiose besteht. 



10. Verwendung eines Lockvirus bei der Herstellung 
40 eines Impfstoffs zum Impfen eines Tiers, urn eine 

Immunantwort zur Bildung von Antikdrpern ge- 
gen den Eppstein-Barr-Virus, Humanimmunde- 
fektvirus, Humanpappiloma Virus, Herpes Virus 
oder Pockenvirus zu entlocken, wobei das Lock- 
45 virus eine Zubereitung nach einem der Anspru- 
che 1 bis 7 mit einem aus mindestens einem an 
das beschichtete Kernteilchen gebundenen im- 
munologisch reaktionsfahtgen viralen Protein 
oder Peptid bestehenden biologisch aktive n Mit- 
50 tel umfa&t. 



Revendications 

55 1. Une composition de matiere comprenant une 
particule nucleaire, un enrobage qui recouvre au 
moins partiellement la surface de cett particule 
nucleair , et au moins un agent biologiquement 
act if en contact avec cette particule nucleaire en- 



11 



21 



EP 0 465 081 B1 



22 



robee, caractensee en ce que la particule nu- 
cleair comprend un m6tal, un ceramique ou un 
polymer , et a un diametre inferieur a environ 
1 000 nanometres, tl' nrobag compr ndun Su- 
cre basique, un sucre modifie ou un oligonucleo- 
tide. 

2. Une composition de matiere selon la Revendica- 
tion 1 dans laquelle le diametre de la particule nu- 
cleate est compris entre 10 et 200 nanometres. 

3. Une composition de matiere selon la Revendica- 
tion 1 ou 2, dans laquelle I'enrobage est de la cel- 
lobiose. 

4. Une composition de matiere selon Tune ou I'autre 
des revendications qui precedent, dans laquelle 
I'agent biotogiquement actif est un agent pharma- 
cologique. 

5. Une composition selon Tune ou I'autre des reven- 
dications qui precedent, dans laquelle le metal 
est selection ne parmi le chrome, le rubidium, le 
fer, (e zinc, le selenium, le nickel, Tor, I'argent et 
le platine. 

6. Une composition selon Tune ou I 'autre des Re- 
vendications 1 a 4, dans laquelle la ceramique est 
selectionnee parmi le dioxyde desilicium, I'oxyde 
d'aluminium, I'oxyde de ruthenium, le carbone et 
I'oxyde d'6tain. 

7. Une composition selon Tune ou I'autre des Re- 
vendications 1 a 4, dans laquelle le polymere est 
du polystyrene. 

8. Une composition selon Tune ou I'autre des reven- 
dications qui precedent a utiliser comme vaccin 
comportant un leu r re viral dans le traitement d'un 
animal pour provoquer une reponse immune, 
dans laquelle I'enrobage comprend une substan- 
ce qui fournit a la particule nucleaire une energie 
superficielle de seuil qui est suffisante pour Iter 
des proteines ou des peptides immunologique- 
ment actifs sans d6naturer ces proteines ou pep- 
tides, et I'agent biologiquement actif est au moins 
une proteine ou un peptide viral immunologique- 
ment reactif lie a la particule nucleaire enrobee, 
et comprenant un vecteur pharmaceutiquement 
acceptable pour ce vaccin comportant un leurre 
viral. 



10. ('utilisation d'un leurre viral dans la fabrication 
d'un vaccin d stine a vacciner un animal pour 
provoquer un r6pons immun af in d suscit r 

5 des anticorps contr le virus Epstein-Barr, le vi- 

rus I'immunodei icience humaine, le virus du pa- 
pillome humain, le virus herpetique ou le poxvi- 
rus, le leurre viral comprenant une composition 
selon Tune ou ('autre des Revendications 1 a 7 

10 dans laquelle I'agent biologiquement actif est au 
moins une proteine ou un peptide viral immuno- 
logiquement reactif lie a la particule nucleaire en- 
robee. 

15 



20 



25 



30 



35 



40 



45 



50 



9. Une composition selon la Revendication 8 dans 
laquelle le peptide ou la proteine viral est isole du 55 
virus Epstein-Barr, du virus de I'immunodef icien- 
ce humaine, du virus du papillome humain, du vi- 
rus herpetique ou du poxvirus. 



12