Skip to main content

Full text of "USPTO Patents Application 09848616"

See other formats


Document AN2 
Appl.No. 09/848,616 

WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 



INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 5 : 
A61K 9/16 


Al 


(11) International Publication Number: WO 94/15585 
(43) International Publication Date: 21 July 1994 (21.07.94) 


(21) International Application Number: PCT/US93/10901 

(22) International Filing Date: 12 November 1993 (12.11 .93) 

(30) Priority Data: 

000,199 4 January 1993 (04.01 .93) US 

(71) Appficant: THE REGENTS OF THE UNIVERSITY OF 

CALIFORNIA [US/US]; 22nd floor, 300 Lakeside Drive, 
Oakland, CA 94612-3550 (US). 

(72) Inventors: KOSSOVSKY, Nir, 1820 Courtney Terrace, Los 

Angeles, CA 90046-2107 (US). GELMAN, Andrew, E.; 
418 N. Stanley Avenue, Los Angeles, CA 90036 (US). 
SPONSLER, Edward, R; 1921 Manning Street, Burbank, 
CA 91505 (US). 

(74) Agents: OLDENKAMP, David, J. et al.; Poms, Smith, Lande 
& Rose, 2121 Avenue of the Stars, Suite 1400, Los Angeles, 
CA 90067 (US). 


(81) Designated States: AU, BB. BG, BR, BY, CA, CZ, FT, HU, 
JP, KP, KR, KZ, LK, LV, MG, MN, MW, NO, NZ, PL, 
RO, RU, SD, SK, UA, VN, European patent (AT, BE, CH, 
DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, FT, SE), 
OAPI patent (BF, BJ, CF, CG, d, CM, GA, GN, ML, MR, 
NE, SN, TD, TG). 

Published 

With international search report. 



(54) Title: HUMAN IMMUNODEFICIENCY MRUS DECOY 




(57) Abstract 



A biologically active composition made up of core particles having diameters of less than about 1000 nanometers which are coated 
with a layer which is designed to allow attachment of biologically active proteins, peptides or pharmacological agents to the microparticles. 
When Human Immunodeficiency Virus (HIV) viral protein is attached to the core particles, the result is a viral decoy which accurately 
rnimics native HIV in size, structure and surface character while being entirely devoid of virulent activity due to the microparticle core. 
The HIV decoy is useful as a vaccine for treating mammals to elicit an immune response. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


GB 


United Kingdom 


MR 


Mauritania 


AU 


Australia 


GE 


Georgia 


MW 


MaUwi 


BB 


Barbados 


GN 


Guinea 


NB 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BF 


Burkina Paso 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


BJ 


Benin 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


FT 


Portugal 


BY 


Belarus 


KE 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Central African Republic 


* KP 


Democratic People's Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SB 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


St 


Slovenia 


CI 


Cote d'lvoire 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


CatDerooo 


U 




SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


LU 


Luxembourg 


TG 


Togo 


CZ 


Czech Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Germany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


Republic of Moldova 


CA 


Ukraine 


ES 


Spain 


MG 


Madagascar 


US 


United States of America 


FI 


Finland 


ML 


Mafi 


tJZ 




FR 


Prance 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











WO 94/15585 



PCT/US93/10901 



HUMAN IMMUNODEFICIENCY VIRUS DECOY 

BACKGROUND OF THE INVENTION 
This is a continuation-in-part of co-pending 
application Serial No. 07/690,601 which was filed on 
April 24, 1991 which is a continuation-in-part of co- 
5 pending application Serial No. 07/542,255 which was 
filed on June 22; 1990. 

1. Field of the Invention : 

The present invention relates generally to 
10 synthetic biologically active compositions which have a 
microparticulate core. More particularly, the present 
invention relates to a synthetic Human Immunodeficiency 
Virus (HIV) decoy which finds use as a vaccine for 
Acquired Immune Deficiency Syndrome (AIDS) . 

15 

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. 

20 These conjugated biological systems offer the promise of 
reduced toxicity, increased efficacy and lowered 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.): Polymers in Biology 

25 and Medicine . New York, Wiley, 2, 73-88 (1983). 
Ranney, D.F., Huf faker, H.H., in Juliano, R.L. (ed.): 
Biological Approaches to the Delivery of Drugs . Ann. 
N.Y. Acad. Sci . , 507, 104-119 (1987).) Nanocrystalline 
and micron sized inorganic substrates are the most 

30 common carriers and proteins are the most commonly 
conjugated agents. For example, gold/protein 

(principally immunoglobulin) conjugates measuring as 
small as 5 nm have been used in immunological labeling 
applications in light, transmission electron and 

35 scanning electron microscopy as well as immunoblotting. 



WO 94/15585 



PCT/US93/10901 



2 



(Faulk, W., Taylor, G., Immunochemistry 8, 1081-1083 
(1971). Hainfeld, J.F., Nature 333, 281-282 (1988).) 

Silanized iron oxide protein conjugates (again 
principally antibodies) generally measuring between 500 
5 and 1500 ran have proven useful in various in vitro 
applications where paramagnetic properties can be used 
advantageously. (Research Products Catalog, Advanced 
Magnetics, Inc., Cambridge, MA, 1988-1989.) Ugelstad 
and others have produced gamma iron oxides cores coated 
10 with a thin polystyrene shell. (Nustad, K. , Johansen, 
L., Schmid, R. , Ugelstad, J., Ellengsen, T., Berge, A.: 
Covalent coupling of proteins to monodisperse particles. 
Preparation of solid phase second antibody. Agents 
Actions 1982; 9:207-212 (id. no. 60).) The resulting 
15 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 
20 cores. For example, Davis and Ilium developed a 60 nm 
system comprised of polystyrene cores with the block 
copolymer poloxamer, polyoxyethy lene and 
polyoxypropylene, outer coats that showed a remarkable 
ability to bypass rat liver and splenic macrophages. 
25 (Davis, S.S., Ilium, L. , Biomaterials 9, 111-115 
(1988)) . Drug delivery with these particles has not yet 
been demonstrated. Ranney and Huf faker described an 
iron-oxide/albumin/drug system that yielded 350-1600 nm 
paramagnetic drug carriers. (Ranney, D.F., Huffaker, 
30 H.H., In, Juliano, R.L. (ed.): Biological approaches to 
the deli very of drugs r Ann. N.Y. Acad. Sci. 507, 104 5 119 
(1987).) Poznasky has developed an enzyme-albumin 
conjugate system that appears to decrease the 
sensitivity of the product to biodegradation while 
35 masking the apparent antigenicity of the native enzyme. 
(Poznasky, M.J.: Targeting enzyme albumin conjugates. 



WO 94/15585 PCT/US93/10901 



Examining the magic bullet. In, Juliano, R.L. (ed.): 
Biological approaches to the delivery of drugs . Annals 
New York Academy Sciences 1987; 507-211:219.) 

Shaw and others have prepared and characterized 
5 lipoprotein /drug complexes. (Shaw, J.M. , Shaw, K.V., 
Yanovich, S. , Iwanik, M. , Futch, W.S., Rosowsky, A., 
Schook, L.B.: Delivery of lipophilic drugs using 
lipoproteins. In, Juliano, R.L. (ed. ) : Biological 
a pproaches to the delivery of drugs . Annals New York 

10 Academy Sciences 1987; 507:252-271.) Lipophilic drugs 
are relatively stable in these carriers and cell 
interactions do occur although little detail is known. 

In any conjugated biological composition, it is 
important that the conformational integrity and 

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

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

25 or agent. For example, pharmacologic agents which 
exhibit in vitro activity may not exhibit in vivo 
activity owing to the loss of the molecular 
configuration formerly determined in part by the in 
vitro environment. Further, the size and associated 

30 ability 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 account when preparing a carrier particle. 

Although numerous different carrier particles have 

35 been developed, there is a continuing need to provide 
carrier particles for both in vivo and in vitro 



WO 94/15585 



PCT/US93/10901 



4 



application wherein a biologically active peptide, 
protein or pharmacological agent can be attached to the 
particles in a manner which promotes stabilization of 
the biologically active compound in its active 
5 configuration. With respect to in vivo applications , it 
would be desirable to develop synthetic decoy viruses 
which could be used as a vaccine to immunize individuals 
against such dreaded diseases as AIDS. 

10 SUMMARY OF THE INVENTION 

In accordance with the present invention, 
biologically active peptides, proteins or 
pharmacological agents are attached to a core particle 
to provide a wide variety of biologically active 

15 compositions. The invention is based on the discovery 
that the surface of ultrafine particles (nanocrystalline 
particles) can be modified with a surface coating to 
allow attachment of biologically active moieties to 
produce compositions wherein the naturally occurring 

20 structural environment of the moiety is mimicked 
sufficiently so that biological activity is preserved. 
The coating which provides for the attachment of 
biologically active moieties to nanocrystalline 
particles in accordance with the present invention can 

25 be composed of a basic or modified sugar or oligonucleo- 
tide. Coating nanocrystalline 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 

30 active moieties. 

In one embodiment of the present invention, 
nanocrystalline particles are used to prepare a decoy 
virus wherein the DNA or RNA core of the virus is 
replaced by the microparticle. The microparticle is 

35 chosen to be approximately the same size as the viral 
core so that the conformation of the surrounding protein 



WO 94/15SS5 



PCT/US93/10901 



5 



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 immune 
response and otherwise being antigenically bioreactive. 
5 In this embodiment, an ultrafine particle having a 

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

10 virus. This size of microparticle core is also well 
suited for carrying anchorage dependent pharmacological 
agents and other biologically active compounds which 
require a nanocrystalline particle anchor or core in 
order to maintain their activity. 

15 As a particular feature of the present invention, 

a decoy virus is provided which includes viral particles 
from HIV as at least part of the protein coat. It was 
discovered that this decoy virus was effective in 
eliciting cellular and humoral responses in animal 

20 models. 

The biologically active microparticles in 
accordance with the present invention have wide-ranging 
use depending upon the type of biologically active 
compound which is attached to the microparticle core. 

25 When viral protein from HIV is attached to the 
microparticle core, the result is a decoy virus which 
may be used as an AIDS vaccine, diagnostic tool or 
antigenic reagent for raising antibodies. Non-viral 
protein or antigen coatings may be selected and 

30 structured for use in raising specific antibodies or as 
a diagnostic tool. Further, the microparticles can 
function as a pharmacological agent when compounds 
having pharmacological activity are attached to the core 
particle. 

35 In accordance with the present invention, the 

utilization of a core microparticle around which HIV 



WO 94/15585 



PCT/US93/10901 



viral protein is attached provides an effective way to 
accurately mimic the antigenic reactivity of native HIV 
while totally eliminating any of the problems and risks 
associated with the presence of the viral genetic 
5 material. In addition, other proteins, peptides or 
pharmacological agents may be attached to the core 
particle to preserve and/ or enhance the activity of the 
compound . 

The above-discussed and many other features and 
10 attendant advantages of the present invention will 
become better understood by reference to the following 
detailed description. 

DETAILED DESCRIPTION OF THE INVENTION 

15 The present invention has wide application to 

immunologic procedures and methods wherein antigenic 
material or other biologically active moieties are 
utilized. These areas of application include 

vaccination agents, antigen agents used to raise 

20 antibodies for subsequent diagnostic uses and antigenic 
compounds used as diagnostic tools. The composition of 
the invention can also be used in a wide variety of 
other applications where there is a need to anchor a 
protein, peptide or pharmacological agent to a core 

25 particle in order to preserve and/or enhance 
bioreactivity . 

The compositions of the present invention include 
nanocrystalline core particles (diameters of less than 
1000 nm) which are coated with a surface energy 

30 modifying layer that promotes bonding of proteins, 
peptides or pharmaceutical agents to the particles. The 
coating modifies the surface energy of the 
nanocrystalline core particles so that a wide variety of 
immunogenic proteins, peptides and pharmaceutical agents 

35 may be attached to the core particle without significant 
loss of antigenic activity or denaturization. The 



WO 94/15585 



PCT/US93/10901 



7 



result is a biologically active composition which 
includes a biologically inert core. The end use for the 
compositions of the present invention will depend upon 
the particular protein, peptide or pharmacological agent 
5 which is attached to the coated core particle. For 
example, proteins or peptides having antigenic activity 
may be attached to provide compositions useful as 
immunodiagnostic tools. Viral fragments or protein 
coatings having immunogenic activity may be attached to 

10 provide a vaccine. Also, pharmacological agents may be 
attached to provide compositions which are useful in 
treating diseases. 

For preparing decoy viruses for use as vaccines, 
particles having diameters of between about 10 to 200 

15 nanometers are preferred since particles within this 
size range more closely mimic the diameter of DNA and 
RNA cores typically found in viruses. 

The core particles may be made from a wide variety 
of inorganic materials including metals or ceramics. 

20 Preferred metals include chromium, rubidium, iron, zinc, 
selenium, nickel, gold, silver, platinum. Preferred 
ceramic materials include silicon dioxide, titanium 
dioxide, aluminum oxide, ruthenium oxide and tin oxide. 
The core particles may be made from organic materials 

25 including carbon (diamond) . Preferred polymers include 
polystyrene, nylon and nitrocellulose. Particles made 
from tin oxide, titanium dioxide or carbon (diamond) are 
particularly preferred. 

Particles made from the above materials having 

30 diameters less than 1000 nanometers are available 
commercially or they may be produced from progressive 
nucleation in solution (colloid reaction) , or various 
physical and chemical vapor deposition processes, such 
as sputter deposition (Hayashi, C. , J . Vac. Sci. 

35 Technol. A5 (4), Jul/Aug. 1987, pgs. 1375-1384; Hayashi, 
C, Phvsics Today . Dec. 1987, pgs. 44-60; MRS Bulletin, 



WO 94/15585 



PCT7US93/10901 



8 



Jan 1990, pgs. 16-47)* Tin oxide having a dispersed (in 
H 2 0) aggregate particle size of about 140 nanometers is 
available commercially from Vacuum Metallurgical Co. 
(Japan). Other commercially available particles having 
5 the desired composition and size range are available 
from Advanced Refractory Technologies, Inc. (Buffalo, 

n.y.) • 

Plasma-assisted chemical vapor deposition (PACVD) 
is one of a number of techniques that may be used to 

10 prepare suitable microparticles. PACVD functions in 
relatively high atmospheric pressures (on the order of 
one torr and greater) and is useful in generating 
particles having diameters of up to 1000 nanometers. 
For example, aluminum nitride particles having diameters 

15 of less than 1000 nanometer can be synthesized by PACVD 
using Al (CHj) 3 and NH 3 as reactants. The PACVD system 
typically includes a horizontally mounted quartz tube 
with associated pumping and gas feed systems. A 
susceptor is located at the center of the quartz tube 

20 and heated using a 60 KHz radio frequency source. The 
synthesized aluminum 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 

25 flow rates of the N 2 /A1(CH 3 ) 3 and NH 3 gas into the 
chamber. A constant pressure in the reaction chamber of 
10 torr is generally maintained to provide deposition 
and formation of the ultrafine nanocrystalline aluminum 
nitride particles. PACVD may be used to prepare a 

30 variety of other suitable nanocrystalline particles. 

The core particles are coated with a substance that 
provides a threshold surface energy to the particle 
sufficient to cause binding to occur without that 
binding being so tight as to denature biologically 

35 relevant sites. Coating is preferably accomplished by 
suspending the particles in a solution containing the 



WO 94/15585 



PCT/US93/10901 



9 



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 
5 invention include cellobiose, trehalose, isomaltose, 
maltose, nystose, maltotriose, related basic sugars, and 
modified sugars such as nitrocellulose. Disaccharides 
and sugars with relatively high glass transition 
temperatures are preferred. The glass transition 

10 temperature is preferably on the order of about 77 °C. 
Cellobiose is a preferred coating material. 

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 

15 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 containing from 
0.1 to 10 weight/volume percent are suitable. 

20 Suspensions of approximately 1 weight /volume percent 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 

25 preferred method for maintaining the dispersion. 
Dispersion 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. 

30 Thicknesses of the coating may vary provided that the 
final core particles include a uniform coating over 
substantially all of the particle surface. 

The particles are separated from the suspension 
after coating and may be stored for future use or 

35 redispersed in a solution containing the protein or 
peptide to be attached to the particles. Alternatively, 



WO 94/15585 



PCT/US93/10901 



10 



the coated particles may be left in the suspension for 
further treatment involving attachment of the desired 
protein or peptide. 

The protein or peptide which is applied to the 
5 coated particles may be selected from a wide variety of 
proteins or peptides. Those having antigenic properties 
are preferred when a vaccine is required. The protein 
can be the viral protein coat from a selected virus or 
immunogenic portion thereof. The viral protein coat is 

10 isolated according to known separation procedures for 
isolating and separating viral proteins. The viral 
coating is the preferred protein because the viral 
coating is where the antigenic activity of viruses is 
known to be located. Typically, the virus is digested 

15 or solubilized to form a mixture of viral proteins. The 
viral proteins are then separated by liquid 
chromatography or other conventional process into the 
various protein particle fractions and dialyzed to 
remove impurities. 

20 Suitable viruses from which viral protein particles 

can be separated and isolated include Epstein-Barr 
virus, human immunodeficiency virus (HIV) , human 
papilloma virus, herpes simplex virus and pox-virus. 
Preparations of a wide variety of antigenic protein 

.25 materials may also be purchased commercially from supply 
houses such as Microgene Systems, Inc. (400 Frontage 
Road, West Haven, Connecticut 06516), Amgen Corporation 
(1900 Oak Terrace Lane, Thousand Oaks, California 91320- 
1789) and Cetus Corporation (1400 53rd Street, 

30 Emeryville, California 94608 and Advanced Biotechnology, 
Inc. (Columbia, Maryland) . Synthetic peptides and/or 
proteins which correspond to naturally occurring viral 
particles may also be utilized. 

With respect to HIV, any of the viral fragments 

35 which are known to elicit an immune response can be 
used. Suitable viral fragments include gpl20, gpl60, 



WO 94/15585 



PCT/US93/10901 



11 



gp41, and core proteins (p24) . Any of the known 
techniques for preparing HIV fragments may be used 
including recombinant methods. 

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

10 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 

15 the particle surface is often not advantageous. For 
example, the dispersion agents present in the solution 
may create an undesirable coating on the suspended 
particles prior to protein attachment. Water miscible 
solvents such as methanol or ethanol may be used. The 

20 aqueous solution of coated microparticles 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. 

25 Sonication is a preferred method for providing a uniform 
suspension of the coated particles in solution. 

The suspension of coated particles and antigens 
must be within certain parameters for protein attachment 
and self assembly to occur. The temperature of the 

30 particle solution should be between 1°C to 45 °C. 
Certain 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 pharmaceutical agents 

35 which are unstable or will not disperse readily in 
distilled water. In general, the salt solutions should 



WO 94/15585 



PCT/US93/10901 



12 



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 
5 sparged initially by helium and then gassed with helium, 
nitrogen and carbon dioxide. The pH of the solution is, 
advantageously, slightly acidic (relative to blood) , 
with a value, preferably, of between 6.8 to 7.2. An 
exemplary solution for dispersion of the coated 

10 microparticles and for protein attachment is an aqueous 
solution containing: 0.0360 milligrams MgSo 4 per liter, 
0.0609 milligrams MgCl 26 H 2 0, 0.0441 milligram CaCl 22 H 2 0, 
22.823 grams K^HPC^, 13.609 grams KH 2 P0 4 , 7.455 grams KC1, 
and 4 . 101 gram sodium acetate. The pH of this solution 

15 is adjusted to 6.8.' 

The coated particle cores with the attached protein 
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 typically used for 

20 storing antigenic compounds or antibodies. For example, 
the coated particles may be freeze dried or stored as a 
suspension in a compatible solution. When used as a 
vaccine, the particles coated with a viral protein coat 
are injected or otherwise administered to the individual 

25 according to conventional procedures. Any 
pharmaceutically acceptable carrier solution or other 
compound may be used in administering the coated 
particles to the individual. When used for diagnostic 
purposes in vitro, the protein coated particles are 

30 suspended in solution and used in the same manner as 
other antigenic compounds. The same is true for use of 
the protein coated particles for raising antibodies. 
The same protocol and procedures well known for using 
antigens to produce antibodies may be used wherein the 

35 protein coated particles of the present invention are 
substituted for normally used antigenic compounds. 



WO 94/15585 



PCT/US93/10901 



The following non-limiting examples describe 
certain aspects of the present invention in greater 
detail. 

5 Example 1, Preparation of nanocrvstalline tin 

oxide microparticles : 1.5 to 2.0 rog of ultrafine 
(nanocrystalline) metal powder was placed in a 1.7 ml 
screw-cap microcentrifuge with 1.5 mis of double 
distilled water (ddH 2 0) . The ddH 2 0 was filtered through 

10 a rinsed 0.45 micron filter-sterilizing unit or acrodisc 
(Gelman Scientific) . The metal powder was tin oxide 
with a mean diameter (by photon correlation spectrosco- 
py) of 140 nm. The mixture was vortexed for 30 seconds 
and placed into a water sonicating bath overnight. The 

15 sonication bath temperature was stabilized at y 60°C. 
After a 2 4 -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 

20 carried out on a Coulter N4MD sub-micron particle 
analyzer. 

The coating was applied to the tin oxide particles 
by suspending the particles in a stock solution of 
cellobiose. The cellobiose stock solution was a 292 mM 

25 solution made by dissolving 1.000 gram of cellobiose in 
9.00 mis of ddH 2 0. Solution was accomplished at 
approximately 70°C in order to promote quick 
dissolution. The resulting cellobiose solution was 
filter sterilized through a rinsed 0.45 micron filter 

30 with the final volume being adjusted to 10.00 ml. 

Sufficient cellobiose stock solution was added to 
150 microliters of ultrafine tin oxide dispersion so 
that the final concentration of the tin oxide was 1.00 
percent (w/v) or 29.2 mM. A typical volume for 

35 preparation was 2.0 mis which was mixed four or five 
times by the action of a micro-pipetor. After mixing, 



WO 94/15585 



PCT/US93/10901 



14 

the dispersion was allowed to equilibrate for two hours. 
Demonstration of successful coating of the particles was 
provided by measuring the mobility of the particles 
(coated and uncoated) on a Coulter DELSA 440 doppler 
5 energy light scatter analyzer. The coated tin oxide 
particles exhibited a relatively low mobility compared 
to the non-coated tin oxide particles. Measurements 
were also taken at various dilute salt concentrations to 
ensure that the observations with respect to mobility 
10 were not artif actual. The tests demonstrate that the 
particles were coated with the cellobiose. 

The coated particles are then used to attach 
antigenic proteins , peptides or pharmacological agents 
to prepare bioreactive particles. 

15 

Example 2 . Preparation of nanocrystalline 

ruthenium oxide particles : The same procedure was 
carried out in accordance with Example 1, except that 
ruthenium oxide microparticles were substituted for the 
20 tin oxide particles. The ruthenium oxide particles were 
obtained from Vacuum Metallurgical Company (Japan) . 

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

25 Nanocrystalline silicon dioxide was acguired 
commercially from Advanced Refractory Technologies, 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 

30 tin in an argon-oxygen mixture and collected on cooled 
substrates. Nanocrystalline tin oxide was .also 

synthesized by D.C. reactive Magnetron sputtering 
(inverted cathode) . A3" diameter target of high purity 
tin was sputtered in a high pressure gas mixture of 

35 argon and oxygen. The ultrafine particles formed in the 
gas phase were collected on copper tubes cooled to 77 °K 



WO 94/15585 



PCMJS93/10901 



with flowing liquid nitrogen. All materials were 
characterized by X-ray diffraction crystallography, 
transmission electron microscopy, photon correlation 
spectroscopy, and Doppler electrophoretic light scatter 
5 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 
diffractometer. The spectrum obtained was compared with 
ASTM standard data of tin oxide. (Powder Diffraction 

10 File, Card #21-1250. Joint Committee on Power 

Diffraction Standards, American Society for Testing and 
Materials, Philadelphia 1976.) The specimens for (TEM) 
were collected on a standard 3 mm diameter carbon coated 
copper mesh by dipping into a dispersion of the (UFP's) 

15 in 22-propanol. The samples were examined on a JEOL- 
STEM 100 CX at an acceleration voltage of 60-80 KV. 

To create working dispersions of these metal 
oxides, 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 

20 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 

25 15 seconds. Approximately 1.3 ml of supernatant was 
then removed and placed in another dust-free screw top 
microcentrifuge tube. A sample was prepared for photon 
correlation spectroscopy (Coulter N4MD) and Doppler 
electrophoretic light scattering (Coulter delsa 440) 

30 analysis by removing 50 to 100 /il of the dispersion and 
placing it in a polystyrene cuvette and diluting it to 
a final volume of 1.00 ml with ddH 2 0. The stability of 
the dispersion was determined by sequential measurements 
over a 24-hour period and was found to be stable. The 

35 stability of the dispersion with respect to progressive 
salinity of the solvent (increasing conductivity) was 



WO 94/15585 



PCTYUS93/10901 



similarly determined. The stability increased with 
progressive salinity of the solvent. 

1.00 ml of the dispersion was combined and stirred 
with 8.00 ml of ddH 2 0 and 1.00 ml of 29.2 mM cellobiose 
5 stock in a 15.0 ml capacity ultrafiltration stir cell 
(Spectra) which has been fitted with a pre-rinsed 5x10 s 
molecular weight cutoff type F membrane (Spectra) . The 
sample was then left to stir for 15 minutes. After 
stirring, the excess cellobiose was removed by flushing 

10 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 established by the removal of 500 ul of 

15 the treated dispersion by N4MD analysis. The mean 
dispersion diameter 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 

20 progressive salinity of the solvent (increasing 
conductivity) was similarly determined. 

The resulting coated nanocrystalline particles are 
suitable for attachment of various proteins, peptides 
and pharmaceutical agents. 

25 

Example 4. Preparation, isolation and surface 
adsorption of human serum transferrin proteins ; 
Nanocrystalline tin oxide was synthesized by D.C. 
reactive Magnetron sputtering (inverted cathode). A3" 

30 diameter target of high purity tin was sputtered in a 
high pressure gas mixture of argon and oxygen. The 
ultra-fine particles formed in the gas phase were 
collected on copper tubes cooled to 77 °K with flowing 
liquid nitrogen. All materials were characterized by x- 

35 ray diffraction crystallography, selected area electron 
diffraction, transmission electron microscopy, photon 



WO 94/15585 



PCTAJS93/10901 



17 



correlation spectroscopy, and energy dispersive x-ray 
spectroscopy. X-ray diffraction samples were prepared 
by mounting the powder on a glass slide using double- 
sized Scotch tape. CuK {alpha) radiation was used on a 
5 Norelco diffractometer. The spectrum obtained was 
compared with ASTM standard data of tin oxide. The 
specimens for transmission electron microscopy and 
selected area diffraction were collected on a standard 
3 mm diameter carbon coated copper mesh by dipping into 

10 a dispersion 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- 
propanol suspension of particles was also characterized 
by photon correlation spectroscopy at 22.5°C, 600 s run 

15 time on a Coulter N4MD. Energy dispersive x-ray 
spectroscopy was performed on a JEOL JSM-T330A scanning 
electron microscope using Kevex quantex V software. 

To create working dispersions of these metal oxides 
for the synthesis of compositions in accordance with the 

20 present invention, 0.5 mg of metal oxide powder was 
added to 1.0 ml of a 29.2 mM cellobiose-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 

25 macroparticulates by microcentrifugation at 16, OOOxg for 
30 seconds. Approximately 900 ftl of 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 

30 electrophoretic light scattering (Coulter DELSA 440) 
analysis. Aliquots were also removed for characterizing 
the stability of the coated dispersion over time and 
with respect to progressive salinity of the solvent 
(increasing conductivity) . 

35 To adsorb protein to the cellobiose coated metal 

oxide nanocrystalline cores, the core sample was diluted 



WO 94/15585 



PCT/US93/10901 



18 



to 10.0 ml with Ca ++ and Mg ++ free phosphate buffered 
saline (Gibco) . Forty (40*0) /zg of purified human serum 
transferrin (4/ig/^l) (Gibco), whose antigenicity was 
verified by ELISA, was then added to a 10 ml stir cell 
5 (Spectra) . The sample was then left to stir slowly for 
30 minutes, taking great care not to allow 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 

10 head. After washing, the sample was again concentrated 
to 1.00 ml under N 2 and a 500 pi sample was removed for 
analysis by photon correlation spectroscopy, Doppler 
electrophoretic light scatter and transmission electron 
microscopy as detailed below. 

15 Conformational integrity was assessed by measuring 

the retained antigenicity of the bound protein. To the 
sample cell, 50.0 ^1 of rabbit polyclonal anti-human 
transferrin antibody (Dako) , whose antigenicity was 
confirmed by ELISA, was added to the concentrated 1.0 ml 

20 reaction product at 37.5°C with gentle stirring. After 
a 30 minute incubation 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 and the reaction volume was again reduced to 1.0 

25 ml. 

A 200 /il aliquot of blocking agent, 1% w/v bovine 
serum albumin in divalent free saline, was added 
followed by a 10 minute equilibration period. The 
secondary antibody, 30 nm gold conjugated goat anti- 

30 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 transmission electron 
microscopy grid, and vacuum dried. The mixture was 
again washed with 15 ml of divalent free saline under a 

35 nitrogen pressure head and then fixed with 
glutaraldehyde. One ml of 3% solid bovine collagen 



WO 94/15585 



PCT/US93/10901 



19 



(Collagen Corp.) was then added to the mixtures and the 
composite was ultracentrif uged at 10 6 xg for 30 minutes 
yielding a pellet that was then routinely processed as 
a biological specimen for transmission electron 
5 microscopy. Ten nm thick sections were viewed on a 
Zeiss transmission electron microscopy. Control samples 
were prepared as above without the cellobiose 
intermediate bonding layer. 

Transmission electron micrographs showed that the 

10 D.C. magnetron sputtered tin oxide was composed of 
individual particles measuring 20-25 nm in diameter 
which aggregated into clusters measuring 80 to 120 nm in 
diameter. By photon correlation spectroscopy, these 
same particles when dispersed in distilled water 

15 produced agglomerates measuring 154 ± 55 nm. The tin 
oxide particles were fully crystalline as characterized 
by electron and x-ray diffraction. Energy dispersive x- 
ray spectroscopy showed no other elements present as 
impurities. 

20 By Doppler electrophoretic light scatter analysis, 

tin oxide exhibited a mean mobility of 2.177 ± 0.215 /tm- 
cm/V-s in aqueous solutions ranging from 10.8 to 20.3 /*M 
NaCl. Following cellobiose surface coating in a 1% 
solution, tin oxide exhibited a mean mobility of 1.544 

25 ± 0.241 /im-cm/V-s in aqueous solutions ranging from 0.0 
to 21.0 /zM NaCl. The oxide agglomerated in salt concen- 
trations of greater than 40.0 (M and in solutions of 
increasing cellobiose concentration. 

Following transferring binding, the crude tin ox- 

30 ide/cellobiose/protein conjugates measured 350 ± 84 nm 
by photon correlation spectroscopy and transmission 
electron microscopy. Vacuum dried dropped samples with 
low concentration gold antibody measured 35-50 nm. 
Without the cellobiose bonding layer, vacuum dried 

35 sections measured 400 to > 1000 nm. Occasional antibody 
bonding was noted. Following high concentration 



WO 94/15585 



PCT/US93/10901 



20 



immunogold labeling and filtering, the thin section 
cellobiose treated specimens measured 50-100 nm. 
Positive gold binding was identified in approximately 
20% of the appropriately coated samples whereas negative 
5 controls (prepared as above but lacking the primary 
rabbit antibody) exhibited approximately 1% nonspecific 
binding. 

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

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

15 Nanocrystalline tin oxide particles were 

synthesized by D.C. reactive Magnetron sputtering as 
previously described in Example 1. 

Elutriated sucrose gradient purified Epstein-Barr 
virus (EBV) acquired from the B95-8 cell line were 

20 purchased from Advanced Biotechnologies, Inc., Columbia 
MD. Each viral aliquot contained approximately 5.00 x 
10 10 virus particles/ml suspended in lOmM TRIS-150mM NaCl 
ph 7.5 buffer (approximately 0.94 mg/ml protein). The 
virions were solubilized 0.75% (v/v) Triton X100 and 

25 then ultracentrifuged at 150,000xg for 60 minutes to 
pellet the DNA core using a modification of the method 
described by Wells. (Wells A, Koide N, Klein G: Two 
large virion envelope glycoproteins mediate EBV binding 
to receptor-positive cells. J Virology 1982; 

30 41:286-297.) Following dialysis, the supernatant EBV 
extract was characterized by both SDS-PAGE (denatured) 
[Biorad Mini Gel 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 

35 autoinjector and 720 photodiode array detector, 0.5 



WO 94/15585 



PCT7US93/10901 



21 



ml/minute over a Waters SW300 GFC column using a lOOmM 
NaCl/20mM TRIS pH 9.4 gradient mobile phase]. 

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

Aliquots of the tin oxide powder weighing 
approximately 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 

10 Industries, Bohemia, NY] . The resultant brownish cloudy 
suspension was then sonified at 175 W for 10.0 minutes 
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 

15 16,000xg for 15 seconds. The remaining pellet was then 
discarded in favor of the supernatant. Unadsorbed 
cellobiose was removed by ultrafiltration against 20 mis 
of 25 mM phosphate reaction buffer (pH 7.40 25mM 
HPO^/HjPCV") in a 10 kD nominal molecular weight filtered 

20 stir cell [Pharmacia] under a 7.5 psi N 2 gas head at 
37.5°C. Aliquots of the intermediate product were 
characterized by photon correlation spectroscopy and, 
following dialysis as described below, by doppler 
electrophoretic light scatter analysis. 

25 The process of viral protein adsorption was 

initiated by the removal of the mild triton surfactant 
from 250 fil 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 

30 adjusted to a concentration of 1.0 jig/ pi 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 dispersion prewarmed to 

35 37.5°C. The mixture was then slowly stirred while being 
incubated at 37.5°C for 2.0 hours. After incubation the 



WO 94/15585 



PCT/US93/10901 



22 



unabsorbed EBV extract was removed by ultrafiltration 
against 25 mis of phosphate reaction buffer. 

Control (non-EBV) decoys fabricated with lambda 
phage viral protein extracts were synthesized using the 
5 same process described above. 

Intermediate components, the final assembled 
decoys, and whole Epstein-Barr virions were 
characterized by doppler electrophoretic light scatter 
analysis [DELSA 440, Coulter Electronics Inc., Hialeah, 

10 FL] to determine their electrophoretic mobility (surface 
charge) in a fluid phase. Nine phosphate buffer 
solutions having at 25°C pH's ranging between 4.59 and 
9.06 and corresponding conductivities ranging between 
2.290 and 4.720 mS/cm were prepared. Aliquots of raw 

15 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 

20 respectively. The mobility values acquired 

simultaneously by the 4 angled detectors of the 
instrument were averaged and the means of 3 measurements 
per dispersion were recorded. 

The synthesized EBV decoys and control decoys were 

25 characterized by immunoagglutination photon correlation 
spectroscopy to determine the antibody reactivity of 
their surfaces. Positive reactivity was assessed by 
incubating the EBV decoy for 60 minutes at 37.5°C with 
a cocktail of anti-EBV murine monoclonal antibodies (1 

30 ug each of anti-EBV-VCA, anti-EBV EA-R, anti-EBV MA, and 
anti EBV EA-D) in 15% lactose, 0.9% NaCl, 10 mM HEPES 
buffer, and 0.2% NaN3 [DuPont, Wilmington, DE]). 
Background reactivity was assessed by incubating the EBV 
decoy with irrelevant murine IgG,. Specificity was 

35 assessed by reacting the lambda phage decoy with 
monoclonal anti-EBV murine antibodies. Agglutination 



WO 94/15585 



PCT/US93/10901 



23 



was measured by photon correlation spectroscopy at a 90° 
angle [N4MD, Coulter]. 

Antibody affinity intensity was assessed by 
immunogold transmission electron microscopy using the 
5 particulates and antibodies listed above and then adding 
secondary anti-murine 30 nm gold-labeled antibodies 
(Faulk W, Taylor G. Immunocolloid method for electron 
microscopy, Jmmunocheraistry 8:1081-1083, 1971). 

Labeling of the EBV decoy (positive reaction) was 

10 accomplished by incubating a 20 pi mixture of murine 
monoclonals (1 fig anti-EBV-VCA and 1 fig anti-EBV EA-R in 
15% lactose, 0.9% NaCl, 10 mM HEPES buffer, 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 

15 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 /il of goat anti-murine antibody covalently fused to 
30 nm gold spheres (10 6 particles/ml [Zymed Laboratories, 

20 San Francisco, CA]) were incubated with 200 /ils of the 
labeled particles in a 1M nominal molecular weight stir 
cell at 37.5°C for 30 minutes. Unbound secondary 
antibody was removed by ultrafiltration against 10 mis 
of phosphate reaction buffer. 

25 Labeling of the EBV decoy (negative reaction) was 

accomplished by incubating 2.5 fil of murine polyclonal 
nonspecific IgGl (l-/xg//xl in 15 mM NaCl 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 

30 same washing and gold-labeling steps. Labeling of the 
lambda phage control decoy (negative reaction) was 
accomplished by incubating a 20 /tl mixture of murine 
monoclonal anti-EBV antibodies with the lambda phage 
virus coated decoy using the same procedure detailed 

35 above. 



WO 94/15585 



PCT/US93/10901 



24 



Immunolabeled particles were prepared for electron 
microscopy in two ways. A direct immersion technique 
where a carbon coated copper viewing grid [Ted Pella 
Inc., Redding, CA] was submersed into sample for 
5 approximately 5 seconds and then fixed in 5% 
glutaraldehyde for 1 minute, was used for all reactions 
as a fast screening technique. A more involved method 
adding glutaraldehyde directly to the reaction solution, 
then pelleting the product at 16,000xg for 5 minutes 

10 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 pm 
sheets for viewing. 

Analysis of both the positive and negative controls 

15 was performed by examining pelleted samples of the 
labeled reaction products by transmission electron 
microscopy. The relative intensity of antibody binding 
was determined by counting the number of tin oxide based 
particles observed to have bound gold spheres (% 

20 positive) and then noting the number of gold spheres 
bound to a given particle (intensity, number/event) . 

The ultrafine tin oxide particles measured 20-25 nm 
in diameter and formed aggregates measuring 80 to 120 nm 
in diameter by transmission electron microscopy. By 

25 photon correlation spectroscopy, these same particles 
when dispersed in distilled water produced agglomerates 
measuring 154 ± 55 nm. The tin oxide particles were 
fully crystalline as characterized by electron and x-ray 
diffraction. Energy dispersive x-ray spectroscopy 

30 showed no other elements present as impurities. 

Characterization of the EBV proteins by SDS-PAGE 
showed two distinct protein bands. The first, existing 
as a dimer suggesting variable glycosylation, exhibited 
a molecular weight of approximately 350 kd which is 

35 consistent with the predominant envelope glycoprotein of 
EBV. The second exhibited a molecular weight of 



WO 94/15585 



PCT/US93/10901 



25 



approximately 67 kd consistent with serum albumin which 
apparently adsorbs avidly to the viral surface. HPLC 
confirmed the presence of two distinct bands that 
exhibited spectrophotometry absorption maxima at 280 nm 
5 consistent with proteins. The 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 15.75 minutes similar 

10 to bovine serum albumin standards. 

The previously described Doppler electrophoretic 
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 

15 identical mobilities of approximately -1.4 jim-cm/V-s 
throughout the pH range. Second, untreated tin oxide 
exhibited a mobility of approximately -1.0 fim-cm/V-s at 
a pH of 4-5 which then rose rapidly to -3.0 /xm-cm/V-s at 
pH values of 5.0 and higher. Third, surface modified 

20 tin oxide treated with cellobiose retained a mobility of 
approximately -1.5 /im-cm/V-s until it increased 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 

25 approximately 102 +/ -32 nm and the synthesized EBV 
decoy measured approximately 154 + /- 52 nm. 
Synthesized EBV decoy, when reacted with the monoclonal 
anti-EBV cocktail, agglutinated to form 1534 +/ -394 nm 
masses. Synthesized EBV decoy, when reacted with 

30 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. 

35 The previously described transmission electron 

microscopy of anti-EBV antibody labeled EBV decoy 



WO 94/15585 



PCT/US93/10901 



26 



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-specific 
mouse IgG antibody labeled EBV decoy particles revealed 
5 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 antibody labeled 
lambda phage decoy particles revealed a positive gold 
staining frequency of 7.21% +/-1.26 with an average 
10 staining intensity of 1.06 gold labels per event. 

Example 6: In Vivo Elicitation of Antibodies Bv 
Epstein-Barr Virus Decoy : Four sensitization solutions 
were prepared and delivered once every other week by 

15 intramuscular injection in three 250 fil aliquots to New 
Zealand rabbits aged approximately 8 weeks. The first 
f our animals received approximately 10 9 whole EBV virions 
(approximately 32 fig of gp350 estimated by integration 
of the spectrophotometry absorption curve at 280 nm 

20 against a 25 fig bovine serum albumin standard) dispersed 
in phosphate reaction buffer per injection. The second 
four animals received 32 fig per injection of isolated 
and purified gp350 using the same injection protocol. 
The third group received EBV viral decoys (Example 5) 

25 synthesized from a starting aliquot of 32 fig of gp350 
per injection. The last group received cellobiose 
coated in tin oxide dispersed in phosphate reaction 
buffer. Injections were free of adjuvant. Whole blood 
was removed using aseptic techniques via cardiac 

30 puncture 2 weeks following each of the three injections 
and the animals were terminated by cardiac puncture 
followed by lethal sedation at 6 weeks. Serum was 
extracted by microcentrifugation at 16 kg of whole blood 
for 1 minute and then stored frozen at -70 °C pending 

35 analysis. 



WO 94/15S85 



PCT/US93/10901 



27 

Immunospecific antibody against whole EBV virions 
(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 
5 4°C in polycarbonate assay plates (Falcon) - Rabbit 
serum affinity for the bound EBV virions was determined 
by the colorimetric reaction of goat anti-rabbit IgG 
alkaline phosphatase (Sigma) developed with 
para-nitrophenyl phosphate. The concentration of 

10 immunospecific 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 containing serum from the 
rabbits stimulated with tin oxide only. 

15 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 

20 the 6 week period. Animals sensitized with purified EBV 
proteins alone showed a maximum of approximately 0.05 
ug//il anti-EBV IgG at six weeks. In contrast, animals 
sensitized with either whole EBV or decoy EBV exhibited 
a statistically significant four fold greater response 

25 with approximately 0.20 ng/ul of anti-EBV IgG at six 
weeks. The immunospecific responses to decoy EBV and 
whole EBV were virtually identical. 

As is apparent from Examples 5 and 6, the 
synthesized EBV decoy in accordance with the present 

30 invention possesses the same surface charge as native . 
virus, is recognized specifically and avidly by 
monoclonal antibodies, and evokes immunospecific 
antibodies with the same effectiveness as whole virus. - 
Using photon correlation spectroscopy, the number of 

35 particles that agglutinated in the three reaction 
conditions were calculated from the measured diameters 



WO 94/15585 



PCT/US93/10901 



28 



of the aggregates. These calculations indicate that 
monoclonal anti-EBV antibodies produce agglutinated 
masses consisting of an average 988.0 decoy EBV 
particles. Non-specific mouse IgG antibodies produce 
5 agglutinated masses consisting of an average 3.33 decoy 
EBV particles, while monoclonal anti-EBV antibodies 
produce agglutinated masses consisting of an average 
1.35 decoy control lambda phage particles. These 
measured results show that the measured agglutination 

10 potential of the EBV decoy in accordance with the 
present invention is almost three orders of magnitude 
greater than controls. The immunogold transmission 
electron microscopy shows that the gold labeled antibody 
staining of anti-EBV labeled EBV decoys is 25 to 30 

15 times greater than controls. The ELISA analysis of the 
immunospecif 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 elicited by isolated purified proteins. 

20 Examples 5 and 6 are summarized in Kossovsky, N. et al., 
Nanocrystalline Epstein-Bar r Virus Decoys, Journal of 
Applied Biomaterial, Vol. 2, 251-259, (1991). 

Example 7. Preparation of HIV Decoys : 
25 The following procedure was used to adsorb HIV 

membrane antigens onto diamond nanocrystalline particles 
to provide HIV decoys. 

HIV Workup . 1.0 ml of HIV (TCID 50 titre which 
varied between 10 s - 75 to 10 717 as determined by the producer 
30 Advanced Biotechnology, Inc.) was dialyzed into PBS by 
100 KD ultrafiltration and frozen down to -70°C until 
needed. On injection day the viral stock was thawed on 
ice and diluted to 1:25 in PBS. 100 ul of this 
preparation was used for injection. 1.0 ml of HIV (10 5,75 
35 transforming units per ml) [ABI] was added to 0.5 ml of 
envelope extraction buffer [1.0% of Triton X 100\0.25 mM 



WO 94/15585 



PCT/US93/10901 



29 



DTT\10 mM Tris pH 7. 4 \ 1.0 mM MgCl] and was allowed to 
incubate for 1.0 hr at room temp* The extract was then 
ultracentrifuged at 100K*g for 2.0 hrs[35krpm SW50.1 
Beckman rotor] at 4.0°C to remove nucleocapsid. Removal 
5 of Triton X and envelope protein enrichment was 
accomplished by incubation with a 300 ul slurry of 
polystyrene micro beads [Spectra Gel D2] and subsequent 
100 kD ultra filtration into PBS. For a 100 ul 
injection the extract volume was corrected to a 1.0 ml 

10 volume and diluted 1:25 in PBS or to a protein 
concentration of around 2.5 ug/100ul/ injection volume. 
Protein quantization was conducted by HPLC HPLC 
conditions were as follows: Waters GFC SW300/Mobile 
phase: 300mM NaCl, 20mM phosphates pH 7.4/ one major peak 

15 with a retention time of around 8.9 minutes at a flow 
rate of 0.5 ml per min/ Integration was done against BSA 
standards. 

Preparation of HIV Decoy . HI Vex was adjusted to 
1.0 ml volume after being ultraf iltered against pH 7.40 

20 20 mM phosphate buffer and was incubated with 1.0 ml of 
diamond particles which had been coated with 500 mM 
cellobiose at 4.0°C for 24 hours. The diamond particles 
had an average particle size on the order of 50 nra. 
After adsorption the decoy dispersion was prepared for 

25 injection by 300 kD ultrafiltration against PBS to 
remove unadsorbed protein and was adjusted to 1.0 ml 
with PBS and parceled out for ten 100 fil injections. 
Immunological Activity o f HIV Decoy 
Rabbits, guinea pigs, and mice were injected with 

30 either live virus, protein extract, protein extract 
mixed with Freund's adjuvant, or the HIV decoy virus. 
Antibody titres against whole virus were measured by 
ELISA and characterized by western blotting. Cell 
mediated reactivity was assessed in the guinea pigs by 

35 dermal skin challenge with live virus follows by biopsy. 



WO 94/15585 



PCT/US93/10901 



30 



At physiological pH, the mean electrophoretic 
mobility and average dispersion diameter (50 nra) of 
these synthetic carriers closely mimicked that of their 
infectious counterparts. Vaccination of mouse, guinea 
5 pig, and rabbit with the HIV decoy elicited the 
production of antisera which exhibited specific binding 
to whole HIV preparation as measured by ELISA. The 
histological analysis of earprick sites for animal 
sensitized to decoy virus and whole virus showed similar 
10 (quantitative and quantitative) reactions which 
differed significantly from both Freund's-sensitized ' 
animals and purified protein-sensitized animals at l, 2, 
7 and 24 weeks. Binding specificity was confirmed by 
Western blots. 

15 As shown in the above example, the HIV decoy of the 

present invention has a number of characteristics which 
are shared with native whole HIV virus. These 
characteristics include: size, surface charge, 
immunorecognition, ability to elicit comparable antibody 

20 titers, and the magnitude and character of cellular 
response. These attributes show that the decoy virus in 
accordance with the present invention can function 
effectively as a vaccinating agent. 

The entire contents of all references cited 

25 hereinabove are hereby incorporated by reference. 

Having thus described exemplary embodiments of the 
present invention, it should be noted by those skilled 
in the art that the within disclosures are exemplary 
only and that various other alternatives, adaptations 

30 and modifications may be made within the scope of the 
present invention. Accordingly, the present invention 
is not limited to the specific embodiments as 
illustrated herein, but is only limited by the following 
claims. 



WO 94/15585 



PCTAJS93/10901 



31 
CLAIMS 

What is Claimed is : 

1. A vaccine for use in treating an animal to 
elicit an immune response against the human 
immunodeficiency virus, said vaccine comprising: 

a decoy virus comprising: 
5 a core particle having a diameter of between 

about 10 to 200 nanometers; 

a coating comprising a substance that provides 
a threshold surface energy to said core particle which 
is sufficient to bind immunologically active fragments 
10 of the human immunodeficiency virus without denaturing 
said immunologically active fragments, said substance 
covering at least a part of the surface of said core 
particle; 

at least one immunologically reactive fragment 
15 of the human immunodeficiency virus bound to said coated 
core particle to form said decoy virus; and 

a pharmaceutical^ acceptable carrier for said 
decoy virus. 

2. A vaccine according to claim 1 wherein said 
substance is a disaccharide. 

3 . A vaccine according to claim 2 wherein said 
coating is cellobiose. 

4. A vaccine according to claim 1 wherein said 
core particle comprises a metal, ceramic or polymer. 

5. A vaccine according to claim 4 wherein said 
metal is selected from the group consisting chromium, 
rubidium, iron, zinc, selenium, nickel, gold, silver and 
platinum. 



WO 94/15585 



PCT/US93/10901 



32 

6. A vaccine according to claim 4 wherein said 
ceramic is selected from the group consisting of silicon 
dioxide , aluminum oxide, ruthenium oxide, carbon and tin 
oxide. 

7. A vaccine according to claim 1 wherein said 
core particle consists essentially of diamond. 

8. A vaccine according to claim 1 wherein said 
fragments of human immunodeficiency virus are selected 
from the group of fragments consisting of gp 120, gp 
160, and gp 41. 

9. A method for vaccinating an animal to raise 
antibodies against the human immunodeficiency virus, 
said method comprising the step of administering to said 
animal an amount of the decoy virus according to claim 

5 1 sufficient to elicit an immune response which raises 
said antibodies to said human immunodeficiency virus. 

10. A method for vaccinating an animal according 
to claim 9 wherein said core particle comprises a metal, 
ceramic or polymer. 

11. A method for vaccinating an animal according 
to claim 9 . wherein said core particle consists 
essentially of tin oxide. 

12. A method for vaccinating an animal according 
to claim 9 wherein said core particle consists 
essentially of diamond. 

13. A method for vaccinating an animal according 
to claim 12 wherein said coating consists essentially of 
cellobiose. 



INTERNATIONAL SEARCH REPORT 



International application No. 
PCT/US93/10901 



A. CLASSIFICATION OF SUBJECT MATTER 
IPC(5) :A61K9/16 

US CL :424/88, 89, 93, 493, 494; 514/934 
According to International Patent Classification (IPC) or to both national classification and IPC 

B. FIELDS SEARCHED , 

Minimum documentation searched (classification system followed by classification symbols) 

U.S. : 424/88.89,93.493.494:514/934 
Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


A 
A 


US, A, 4,501,726 (SCHRODER ET AU 

26 FEBRUARY 1985. See column 2, lines 29-68. Column 

3, lines 5-65. Examples 1-15. 

US, A, 4,904,479 (ILLUM) 27 FEBRUARY 1990; 
See column 1, lines 60-68. Column 10, lines 1-49. 


1-13 
1-13 


[""I Furtl 


icr documents are listed in the continuation of Box C. [_j See patent family annex. 


Special categories of cued documents: 'T later document publiihed after ihc oUernationsl ftlin* date or prionty 
special can? ones 01 cw» oocwdcius. dale and not in conflict with the application but chad to understand the 
"A" document defining tbe icncrml itale of the art which U nof considered principle or theory underlying the invention 
to be part of particular relevance 

*X* document or particular retevnnco; the churned invention cannot be 
*E* earlier document published oo or after the international fdint dote conaidered novel or cannot be considered to involve an inventive atcp 

"L" donimfBT which may throw doubts on priority cUimd) or which a when *" documcnl u **** * looc 

cited to establish tbe publkauoo date of another citation or other document of particular relevance; the claimed mvention cannot be 
special reaaon (a* specified) considered id involve an inventive step when the document b 

•O- document referrinf to an oral disclosure, use. exhibition or other combined whh ooe or nx>« od»er «ucb documents, such combiiuaioa 
ujejjjj beini obvious to a person skilled m tbe an 

•p- document published prior to the tntemmiotml filing date but btcr than document member of tbe same patent family 


Date of the actual completion of the international search 
25 JANUARY 1994 


Date of mailing of the. international search report 

28 FEB W 


Name and mailing address of the ISA/US 
Commissioner of Patents and Trade marts 
Box PCT 

Washington. D.C. 20231 
Facsimile No. NOT APPLICABLE 


JAMES M.3PEAR 1/ 
Telephone No. (703) 308-235 1 



Form PCT/ISA/210 (second sheet)(July 1992)*