GROUP 1	6	Test Vaccine	5/6 ducks remained DHBV
			negative following challenge
GROUP 2	4	Sham Vaccine [Glutaraldehyde-	4/4 ducks became DHBV
		inactivated DHBV (chemical kill)]	positive following challenge.
GROUP 3	6	Mock Vaccine	6/6 ducks became DHBV
(Control)		[Phosphate Buffered Saline (PBS)]	positive following challenge.

1. Glutaraldehyde Inactivation

Glutaraldehyde inactivation was achieved as known by those of ordinary skill in the art by fixation with a dilute solution of glutaraldehyde at about 1:250. Glutaraldehyde is a well known cross linking agent.

2. Delipidation Procedure

An organic solvent system was employed to perform delipidation of serum. The solvent system consisted of a ratio of 40% butanol (analytical reagent grade) and 60% diisopropyl ether and was mixed with the serum in a 2:1 ratio. Accordingly, 4 ml of the organic solvent was mixed with 2 ml of the serum and rotated for 1 hour. This mixture was centrifuged at approximately 400×g for 10 minutes followed by removal of the aqueous phase. The aqueous phase was then mixed with an equal volume of diethyl ether and centrifuged at 400×g for 10 minutes. Next, the aqueous phase was removed and mixed with an equal volume of diethyl ether and rotated end-over-end at 30 rpm for about 1 hour, and centrifuged at 400×g for 10 minutes. The aqueous phase was removed and the residual diethyl ether was removed through evaporation in a fume cabinet for approximately 10 to 30 minutes. The treated serum remained following removal of diethyl ether and was used to produce the vaccine. The delipidation procedure control involved subjecting the DHBV negative serum to the same delipidation procedure as the DHBV positive serum.

3. Vaccine Production

TABLE 2

	First Dose	Second Dose	Third Dose
	(injected with 200 μl of	(injected with 300 μl	(injected with 300 μl of
	respective vaccine into	of respective vaccine	respective vaccine
	peritoneal cavity on Day 8	intramuscularly on	intramuscularly on Day 22
Vaccine Type	post-hatch)	Day 16 post-hatch)	post-hatch)

TEST	A 40 μl aliquot of the	A 40 μl aliquot of the	A 200 μl aliquot of the
	delipidated serum was mixed	delipidated serum	delipidated serum was mixed
	with 1960 μl of phosphate	was mixed with	with 1800 μl of PBS and then
	buffered saline (PBS)	1960 μl of PBS and	emulsified in 1000 μl of Freund's
		then emulsified in	Incomplete Adjuvant.
		1000 μl of Freund's
		Incomplete
		Adjuvant.
SHAM	A 200 μl aliquot of DHBV	A 200 μl aliquot of	A 200 μl aliquot of DHBV positiv$$
(DHBV	positive serum pool #4	DHBV positive serum	serum pool #4 (20.4.99) was
SERUM	(20.4.99) was mixed with	pool #4 (20.4.99) was	mixed with 300 μl of PBS and
CONTROL)	300 μl of PBS and 100 μl of a	mixed with 300 μl of	100 μl
	2% glutaraldehyde solution	PBS and 100 μl	Aidal Plus (Whiteley Chemicals)
	(Aidal Plus from Whiteley	Aidal Plus (Whiteley	and incubated for 10 minutes to
	Chemicals) and incubated for	Chemicals) and	inactivate the DHBV. A 40μl
	10 minutes to inactivate the	incubated	aliquot of the inactivated
	DHBV. A 40 μl aliquot of the	for 10 minutes to	serum/PBS mixture was added to
	inactivated serum/PBS	inactivate the DHBV.	1960 μl PBS and emulsified in
	mixture was added to 1960 μl	A 40 μl aliquot of the	1000 μl Freunds Incomplete
	PBS.	inactivated serum/PBS	Adjuvant.
		mixture was added to
		1960 μl PBS and
		emulsified in 1000 μl
		Freunds Incomplete
		Adjuvant.
MOCK	PBS	A 2000 μl aliquot of	A 2000 μl aliquot of PBS was
(DHBV		PBS was emulsified	emulsified in 1000 μl Freunds
NEGATIVE		in 1000 μl Freunds	Incomplete Adjuvant.
CONTROL)		Incomplete
		Adjuvant.

4. Experimental Procedure

Ducks were challenged with 1000 μl of DHBV positive serum (serum pool 20.1.97) on day 29, post-hatch. Serum pool 20.1.97 was shown to have 1.8×10¹⁰genome equivalent (gev)/ml by dot-blot hybridization. One genome equivalent (gev) is approximately one viral particle. Ducks were bled prior to full vaccination ondays 1 and 10, prior to challenge on days 17 and 23, and post challenge on days 37, 43 and 52. Their serum was tested for DHBV DNA by dot-blot hybridization as described by Deva et al. (1995). Ducks were euthanized on day 58 and their livers removed, the DNA extracted and tested for the presence of DHBV by dot-blot hybridization as described by Deva et al. (1995).

5. Analysis of Results

- a. Test Ducks
  - i. Five of the 6 test ducks vaccinated with the test vaccine remained negative for DHBV DNA in the serum and liver following challenge. One test duck became positive for DHBV following challenge.
- b. Sham Vaccinated Ducks
  - i. All 4 of the ducks vaccinated with glutaraldehyde inactivated serum became DHBV positive following challenge with DHBV.
- c. Mock Vaccinated Ducks
  - i. Five of the 6 mock-vaccinated negative control ducks became DHBV positive following challenge.

The Chi-square analysis was used to compare differences between treatments. Significantly more control ducks (mock vaccinated) became DHBV positive following challenge than the ducks vaccinated with delipidated serum (p<0.05).

Vaccination of ducklings with delipidated DHBV positive serum using the above protocol resulted in prevention of DHBV infection following challenge with DHBV positive serum in 5 of 6 ducklings. This suggests that the delipidated serum vaccine is capable of inducing a positive immunogenic response in vaccinated ducks. It is further believed that the delipidation process exposed patient-specific antigens that were previously unexposed and/or caused a structural change in the viral particle structure to enable the positive immunogenic response. In comparison 5 of 6 mock vaccinated and 4 of 4 sham-vaccinated ducks became DHBV positive following vaccination suggesting no induction of immunity in these ducks due to lack of immune response.

EXAMPLE 2

A. Delipidation of Cattle Pestivirus (bovineviral diarrhea virus, BVDV), as a Model for Hepatitis C

A standard cattle pestivirus isolate (BVDV) was used in these experiments. This isolate, “Numerella” BVD virus, was isolated in 1987 from a diagnostic specimen submitted from a typical case of ‘Mucosal Disease’ on a farm in the Bega district of New South Wales (NSW), Australia. This virus is non-cytopathogenic, and reacts with all 12 of a panel of monoclonal antibodies raised at the Elizabeth Macarthur Agricultural Institute (EMAI), NSW, Australia, as typing reagents. Therefore, this virus represents a ‘standard strain’ of Australian BVD viruses.

The Numerella virus was grown in bovine MDBK cells tested free of adventitious viral agents, including BVDV. The medium used for viral growth contained 10% adult bovine serum derived from EMAI cattle, all of which tested free of BVDV virus and BVDV antibodies. This serum supplement has been employed for years to exclude the possibility of adventitious BVDV contamination of test systems, a common failing in laboratories worldwide that do not take precautions to ensure the test virus is the only one in the culture system. Using these tested culture systems ensured high-level replication of the virus and a high yield of infectious virus. Titration of the final viral yield after 5 days growth in MDBK cells showed a titer of 10^6.8infectious viral particles per ml of clarified (centrifuged) culture medium.

1. Treating Infectious BVDV

100 ml of tissue-culture supernatant, containing 10^6.8viral particles/ml, was harvested from a 150 cm²tissue-culture flask. The supernatant was clarified by centrifugation (cell debris pelleted at 3000 rpm, 10 min, 4° C.) and 10 ml set aside as a positive control for animal inoculation (non-treated virus). The remaining 90 ml, containing 10^7.75infectious virus, was treated using the following protocol: 180 ml of a solvent mixture butanol:diisopropyl ether (DIPE) (2:1) was added to a 500 ml conical flask and mixed by swirling. The mixture was then shaken for 60 min at 30 rpm at room temperature on an orbital shaker. It was then centrifuged for 10 min at 400×g at 4° C., after which the organic solvent phase was removed and discarded. In subsequent steps, the bottom layer (aqueous phase) was removed from beneath the organic phase, improving yields considerably.

The aqueous phase, after the butanol:DIPE treatment, was washed four times with an equal volume of fresh diethyl ether (DEE) to remove all contaminating traces of butanol. After each washing, the contents of the flask was swirled to ensure even mixing of both aqueous and solvent phases before centrifugation as above (400×g, 10 min, 4° C.). After four washes, the aqueous phase was placed in a sterile beaker covered with a sterile tissue fixed to the top of the beaker with a rubber band to prevent contamination and placed in a fume hood running continuously overnight (16 hr) to remove all remaining volatile ether residue from the inactivated viral preparation. Subsequent culture of the treated material demonstrated no contamination. The treated viral preparation was then stored at 4° C. under sterile conditions until inoculation into tissue culture or animals to test for any remaining infectious virus.

2. Testing of Treated BVDV Preparation

a. Tissue-Culture Inoculation

2 ml of the solvent-treated virus preparation, expected to contain about 10^7.1viral equivalents, was mixed with 8 ml tissue-culture medium Minimal Eagles Medium (MEM) containing 10% tested-free adult bovine serum and adsorbed for 60 min onto a monolayer of MDBK cells in a 25 cm²tissue-culture flask. As a positive control, 2 ml of non-treated or substantially lipid-containing infectious virus (also containing about 10^7.1viral equivalents) was similarly adsorbed on MDBK cells in a 25 cm²tissue-culture flask. After 60 min, the supernatant was removed from both flasks and replaced with normal growth medium (+10% ABS). The cells were then grown for 5 days under standard conditions before the MDBK cells were fixed and stained using a standard immunoperoxidase protocol with a mixture of 6 BVDV-specific monoclonal antibodies (EMAI panel, reactive with 2 different BVD viral proteins).

There were no infected cells in the monolayer of MDBK cells that was inoculated with the organic solvent treated virus. In contrast, approximately 90% of the cells in the control flask (that was inoculated with non-inactivated BVDV) were positive for virus as shown by heavy, specific, immunoperoxidase staining. These results showed that, under in vitro testing conditions, no infectious virus remained in the treated, at least partially delipidated BVDV preparation.

b. Animal Inoculation

An even more sensitive in vivo test is to inoculate naive (antibody negative) cattle with the at least partially delipidated virus preparation. As little as one infectious viral particle injected subcutaneously in such animals is considered to be an infectious cow dose, given that entry into cells and replication of the virus is extremely efficient for BVDV. A group of 10 antibody-negative steers (10-12 months of age) were randomly allocated to 3 groups.

The first group of 6 steers was used to test whether BVDV had reduced infectivity. The same at least partially delipidated preparation of BVDV described above was used in this example. Two steers were inoculated with a vaccine having at least partially delipidated viral particles to act as a positive control for the vaccine group. These two positive control animals were run under separate, quarantined conditions to prevent them from infecting other animals when they developed a transient viraemia after infection (normally at 4-7 days after receiving live BVDV virus). The two remaining steers acted as negative “sentinel” animals to ensure there was no naturally-occurring pestivirus transmission within the vaccinated group of animals. Antibody levels were measured in all 10 animals using a validated, competitive ELISA developed at EMAI. This test has been independently validated by CSL Ltd and is marketed by IDEXX Scandinavia in Europe.

The six animals in the first group each received a subcutaneous injection of 4.5 ml of the at least partially delipidated BVDV preparation, incorporated in a commercial adjuvant. Since each ml of the at least partially delipidated preparation contained 10^6.8viral equivalents, the total viral load before the delipidation process was 10^7.4tissue culture infectious doses (TCID)₅₀. The positive-control animals received 5 ml each of the non-delipidated preparation, that is, 10^7.5TCID₅₀injected subcutaneously in the same way as for the first group. The remaining two ‘sentinel’ animals were not given any viral antigens, having been grazed with the first group of animals throughout the trial to ensure there was no natural pestivirus activity occurring in the group while the trial took place.

There was no antibody development in any of the vaccinated steers receiving the at least partially delipidated BVD virus preparation until a second dose of vaccine was given. Thus, at 2 and 4 weeks after a single dose, none of the 6 steers seroconverted showing that there was no infectious virus left in a total volume of 27 ml of the at least partially delipidated virus preparation. This is the equivalent of a total inactivation of 10^8.2TCID₅₀. In contrast, there were high levels of both anti-E2 antibodies (neutralizing antibodies) and anti-NS3 antibodies at both 2 and 4 weeks after inoculation in the two steers receiving 5 ml each of the viral preparation prior to delipidation. This confirmed the infectious nature of the virus prior to delipidation. These in vivo results confirm the findings of the in vitro tissue-culture test. The two ‘sentinel’ animals remained seronegative throughout, showing the herd remained free of natural pestivirus infections.

The panel of monoclonal antibodies used detected host antibodies directed against the major envelope glycoprotein (E2), which is a glycoprotein incorporated in the lipid envelope of the intact virus. The test systems also detected antibodies directed against the non-structural protein, NS3 that is made within cells infected by the virus. This protein has major regulatory roles in viral replication and is not present within the infectious virus. There was no evidence in the vaccinated cattle that infectious virus was present, indicating all infectious viral particles had been destroyed. All pestiviruses are RNA viruses. Therefore, there was no viral DNA present in the delipidated preparation. These results demonstrate the efficacy of the present method to at least partially delipidate virus such that substantially no infectious virus is found in animals receiving the delipidated virus.

B. Delipidated BVDV Preparation as a Vaccine in Steers

All six steers that had received an initial dose of 4.5 ml of the at least partially delipidated BVDV preparation described in above in Section A were again injected subcutaneously with a similar dose at 4 weeks after the first priming dose. At this time there were no antibody responses after the initial dose. It is normal for an animal to react after the second dose. Strong secondary immune responses for anti-E2 antibody levels (equivalent to serum neutralizing antibodies SNT) were observed in 3 of the 6 steers at 2 weeks after the second dose of the at least partially delipidated virus. This response was more than 70% inhibition in a competitive ELISA. The remaining 3 animals showed weak antibody responses (23-31% inhibition).

In contrast to the anti-E2 antibody responses, only one animal developed a strong anti-NS3 antibody response (93% inhibition) at 2 weeks after the second dose of at least partially delipidated BVDV. A second animal had a weak anti-NS3 response (29% inhibition) and four animals showed no antibody following administration of 2 doses. This was not unexpected since similar responses following administration of at least partially delipidated BVDV vaccines have been observed previously. The antibody levels in steers following 2 doses of the at least partially delipidated BVDV preparation demonstrate its potential as a vaccine since antiE2 antibody levels were measurable in all 6 vaccinated steers at 2 weeks after the second dose.

EXAMPLE 3Use of Delipidated SIV to Induce or Augment SIV Specific Humoral and CD4+ T Cell Memory Responses in Mice—A Model for a New Auto-Vaccination Strategy Against Lentiviral Infection

The following studies focused on the simian equivalent of human HIV, termed SIV. The purpose was to utilize delipidated SIVmac251 (an uncloned highly pathogenic isolate of SIV) to carry out studies to determine the relative immunogenicity of the delipidated virus in mice. The complete nucleotide sequence of an infectious clone of simian immunodeficiency virus of macaques, SfVmac239, has been determined. Virus produced from this molecular clone causes AIDS in rhesus monkeys in a time frame suitable for laboratory investigation. The proviral genome including both long terminal repeats is 10,279 base pairs in length and contains open reading frames for gag, pol, vif, vpr, vpx, tat, rev, and env. The nef gene contains an in-frame premature stop after the 92nd codon. At the nucleotide level, SIVmac239 is closely related to SIVmac251 (98%) and SIVmac142 (96%). (Regier D A, Desrosiers Annual Review Immunology. 1990;8:557-78.)

Experiments were performed to determine the minimal dose of delipidated simian immunodeficiency virus (SIV) that would produce a readily recognizable boosting of the virus specific humoral and/or cellular immune response in previously primed Balb/c mice. All experiments were carried out in a BSL3 facility.

The immunogenicity of the delipidated virus preparation was compared with an aliquot of the same virus in its native form. The quality (titer of antibody, the conformational and linear epitope specificity of the antibody, the isotype content of the antibody and the function of the antibody) and quantity of antibody induced by immunization of mice with equivalent protein amounts of the non-delipidated and delipidated virus preparation were ascertained as described below. Total protein from an aliquot of wild type virus and total protein recovered following delipidation of the same aliquot of virus were determined using standard quantitative protein assay (Biorad, BCA kit assay, Rockford, Ill.). The total protein profile was determined using SDS-PAGE analysis of the wild type virus and the delipidated virus preparation and the relative epitope preservation was ascertained by Western Blot comparison of wild type with delipidated virus.

Equivalent protein amounts of the chemically treated wild type and the delipidated virus were analyzed for their ability to boost virus specific immune response in groups of mice. The sera from these immunized mice were assayed by ELISA and Western Blot analysis for reactivity against native wild type and for comparison the delipidated virus preparation. Spleen cells were assayed for their CD4 and CD8 SIV virus env and gag specific immune response enhancing capacity as outlined below. Standard statistical analyses were performed for the analysis of the data.

Four to six week old healthy female Balb/c mice from the Jackson labs, Bar Harbor, Me. were purchased and housed in the BSL2/3 mouse housing facility at Emory University. Twenty Balb/c mice were each immunized subcutaneously with 25 ug of protein of 2-2 dithiopyridine-inactivated SIVmac251 incorporated in an equal volume of Freunds incomplete adjuvant.

A sufficient quantity of SIVmac251 was delipidated to provide the amount needed for boosting these mice per schedule. Delipidation consisted of incubating SIVmac251 with 10% DIPE in phosphate buffered saline (PBS). 1.0 ml of a 10% DIPE solution in PBS was prepared and mixed on a vortexer until it appeared cloudy.

The virus preparation: A 1 ml tube from Advanced Biotechnologies SIVmac251 was used as seed stock (Sucrose Gradient Purified Virus 1 mg/ml). The supplier reported a titer of 10^6.7with total protein of 1.074 mg/mL (Pierce BCA protein method) and virus particle count of 6.95¹⁰/ml (EM). It was confirmed that the virus had a titer of 10^7.0using CEMx174, the first time as a rapid assay, and the second time in quadruplicate cultures/dilution. A measurement of p27 in this preparation revealed a value of 106 ug/ml. Next, 25 μl of the undiluted viral stock was introduced into 0.6 ml clear snap-cap polypropylene Eppendorf tube.1 Then, 2.5 μl of 10% DIPE solution was added into the Eppendorf tube containing virus and vortexed for 15 seconds. The tube was spun (using an Eppendorf 5810R centrifuge) at room temp at 1000×g for 2 minutes. No bulk solvent was removed. The solvent was removed by vacuum centrifugation (Speedvac Concentrator Model SVC200H) at 2000 rpm with no heat for 30 minutes. The volume in the tube was adjusted to 25 μl with PBS. Total protein recovery was measured using a Pierce BCA protocol. Gels (12% SDS-PAGE) were employed for specific protein recoveries (env protein, pol protein, gp41, p27 and gag protein) and stained with Coomasie Blue and provided semi-quantitative results using OD. Western blots were run using serum from SIV-infected monkeys to measure envelope protein, gp66, gp41, p27, gag, and p6 gag. The viral infectivity of the preparation was determined using a luciferase assay and CEM-174 cells. The virus titer was 10^4.5, a 2.5 log reduction from that measured in undelipidated stock. This delipidated SIV preparation appears to retain greater than 90% of the major protein constituents of SIVmac251 such as the gag and env proteins.

Next, the immunogenicity of the modified viral preparation was determined in the twenty adult female Balb/c mice described above that were each immunized subcutaneously with 25 μg of protein of 2-2 dithiopyridine-inactivated SIVmac251 . On day 14, groups 3-6 were boosted with 10 ug to 0.01 ug (based on total protein of stock) of delipidated virus in 0.5 ml normal saline. The estimated actual virus protein content was equal to 1/10 that of total protein based on the ratio of total protein/p27 protein in stock. The mice were injected with the delipidated vaccine composition as follows:

TABLE 3

	Initial Immunization
	s.c. 2-2
	dithiopyridine-
Groups (containing 4	inactivated	Day 14 - Booster
mice each)	SIVmac251	Injections i.v.

GROUP 1 - Control	Non-immunized	Administered-saline
		without delipidated
		virus
GROUP 2	Immunized	Not administered
GROUP 3	Immunized	0.5 ml saline + 10 ug
		of delipidated virus
GROUP 4	Immunized	0.5 ml saline + 1.0 ug
		of delipidated virus
GROUP 5	Immunized	0.5 ml saline + 0.1 ug
		of delipidated virus
GROUP 6	Immunized	0.5 ml saline + 0.01 ug
		of delipidated virus

Four days after the booster injection, the mice were anesthetized and blood was collected via retro-orbital puncture and intra-cardiac puncture. About 0.5 ml of blood was collected from each mouse, primarily from intra-cardiac puncture. The blood was permitted to clot at room temperature. The spleen of each mouse was aseptically removed and transported to the lab under double bag containment. The clotted blood from each mouse was centrifuged at about 450×g at room temperature, and serum was collected from tube, transferred to a sterile tube, and stored at −70° C. until use. ELISA was performed to determine antibody titers against SIV for each serum sample.

SIV ELISA Protocol

Stocks of positive and negative serum and fluids to be tested were frozen in aliquots to be used on every plate to standardize each run.

Coated Corning Easy-Plates were washed with 100 ul per well of poly-1-lysine at a concentration of 10 ug per ml of PBS, pH 7.2-7.4. Plates were covered and incubated overnight at 4° C. Several plates were coated at one time and stored for subsequent use. Next, excess polylysine was removed and the plate dried for a few minutes. About 100 ul of 2% Triton-X was added to 100 ul of the stock ABI SIVmac251 the samples sat for 5 minutes. Next, 50 ul of coating buffer of pH 9.6 was added. Next, 100 ul of the viral antigen was added to each well of 5 plates, which were covered and incubated at 4° C. overnight.

After the overnight incubation, wells were washed 3 times with PBS-T. The wells then received 200 ul per well of 2% nonfat dry milk in PBS for one hour at room temperature to block non-specific binding. Excess fluid was removed. About 100 ul of test or control serum diluted at 1/100 in 10% RPMI 1640 or PBS with 10% calf serum was added to duplicate wells and incubated for 2 hours at 37° C. Wells were washed 4 times with PBS-T. Next 100 ul of Southern Biotech (from Fisher) alkaline phosphatase anti Mouse IgG (diluted 1/800 in media or PBS with 10% calf serum) was added and incubated 1 hour at 37° C. Wells were washed 4 times with PBS-T.

The BIORAD Alkaline Phosphatase Substrate kit was used to develop a reaction product. One substrate tablet was added for each 5 ml of 1× buffer and mixed. Next 100 ul was added per well and evaluated at about 5, 10, 15, 30 and then at 1 hour intervals for color development.

Blank readings were obtained from the media controls when the positive control was above 1.500 and the negative control was 0.100 to 0.200 for the serum. The results were then recorded and the means and the standard deviations of the negative control, positive control and the experimental samples were calculated. The negative cutoff value was the mean of the negative control plus 0.150.

Immunogenicity Results

The immunogenicity of the delipidated SIV virus preparation in mice was examined with an ELISA assay. The mean optical density (O.D.) was examined at 405 nm at various dilutions of serum. Table 4 provides the results of the ELISA test on serum samples.

1/100	2.541	3.663	3.289	2.846	2.627
1/500	1.035	2.86	2.055	1.458	1.257
1/2500	0.449	1.239	0.855	0.601	0.445
1/12500	0.194	0.463	0.304	0.229	0.181
1/62500	0.127	0.151	0.153	0.129	0.123
1/312500	0.11	0.116	0.108	0.108	0.107

Analysis of Responses of Dissociated Spleen Cells Obtained from Immunized Mice

A single cell suspension of spleen cells was prepared from each individual mouse by gently teasing the splenic capsule and passing the cells through a 25 gauge needle. Spleen cells were dissociated into a single cell suspension in medium (RPMI 1640 supplemented with 100 ug/ml penicillin, 100 ug/ml streptomycin, 2 mM L-glutamine), washed twice in medium and subsequently adjusted to 10 million cells/ml. 0.1 ml of this cell suspension from each mouse was dispensed into each well of a 96 well round bottom microtiter plate containing medium. Remaining cells were cryopreserved. These spleen cell cultures were then assessed for the ability of CD4+ and CD8+ T cells to synthesize IFN-gamma by standard intracellular cytokine staining (ICC) and flow cytometry.

Two individual wells containing the duplicate cell cultures from an individual mouse received either a) 0.1 ml of medium containing 2 ug/ml of each of a pool of 9 SIV envelope (SE) peptides (n=14 pools), or b) 0.1 ml of medium containing a pool of 7 SIV gag (SG) peptides (n=17 pools). Each pool contained 2 ug/ml of 7 peptides each for SIV env and SIV gag. Controls consisted of spleen cell cultures that received media alone (background control) or a previously determined optimum concentration of phorbol myristic acetate (PMA 1 ug/ml)+ionomycin (0.25 ug/ml) for maximal IFN-gamma staining (positive control). The SIVenv peptides (n=49 individual peptides) were mixed in a grid fashion of a 7×7 matrix and the SIV gag peptides (n=72 peptides) were mixed in a grid fashion of a 9×8 matrix which permitted identification of individual peptide specific immune responses. The SIV env and gag peptides were synthetic 20 mer peptides that overlapped each other by 12 amino acids and encompassed the entire SIV env and gag sequence. Peptide pools were made to contain 2.0 ug/ml of each peptide. For each spleen cell preparation there were 36 wells of culture. The components of the 9 pools and 7 pools of env and gag overlapping peptides are described below. Shown are the peptides that compose the pools with their respective position within SfVmac239gag (SG) and env (SE).

TABLE 5

Pool arrangement of individual SIVmac239 env peptides.
7 SG Peptide pools

SG18	1	2	3	4	5	6	7
SG19	8	9	10	11	12	13	14
SG20	15	16	17	G-6*	G-5*	20	21
SG21	22	23	24	25	26	27	28
SG22	29	30	31	32	33	34	35
SG23	36	37	38	39	40	41	42
SG24	43	44	45	46	47	48	49

TABLE 6

Pool arrangement of individual SIVmac239 env peptides.
9 SE Peptide pools

SE9	1	2	3	4	5	6	7	8
SE10	9	10	11	12	13	14	15	16
SE11	17	18	19	20	21	22	23	24
SE12	25	26	27	28	29	30	31	32
SE13	33	34	35	36	37	38	39	40
SE14	41	42	43	44	45	46	47	48
SE15	49	50	51	52	53	54	55	56
SE16	57	58	59	60	61	62	63	64
SE17	65	66	67	68	69	70	71	72

TABLE 7

SIVmac239 gag overlapping peptides for epitope
mapping

SEQ ID NO: 1	MGVRNSVLSGKKADELEKIRLR	SG1	1-22

SEQ ID NO: 2	KKADELEKIRLRPNGKKKYMLK	SG2	11-32

SEQ ID NO: 3	LRPNGKKKYMLKHVVWAANELD	SG3	21-42

SEQ ID NO: 4	LKHVVWAANELDRFGLAESLLE	SG4	31-52

SEQ ID NO: 5	LDRFGLAESLLENKEGCQKILS	SG5	41-62

SEQ ID NO: 6	LENKEGCQKILSVLAPLVPTGS	SG6	51-72

SEQ ID NO: 7	LSVLAPLVPTGSENLKSLYNTV	SG7	61-82

SEQ ID NO: 8	GSENLKSLYNTVCVIWCIHAEE	SG8	71-92

SEQ ID NO: 9	TVCVIWCIHAEEKVKHTEEAKQ	SG9	81-
			102

SEQ ID NO: 10	EEKVKHTEEAKQIVQRHLVVET	SG10	91-
			112

SEQ ID NO: 11	KQIVQRHLVVETGTTETMPKTS	SG11	101-
			122

SEQ ID NO: 12	ETGTTETMPKTSRPTAPSSGRG	SG12	111-
			132

SEQ ID NO: 13	TSRPTAPSSGRGGNYPVQQIGG	SG13	121-
			142

SEQ ID NO: 14	RGGNYPVQQIGGNYVHLPLSPR	SG14	131-
			152

SEQ ID NO: 15	GGNYVHLPLSPRTLNAWVKLIE	SG15	141-
			162

SEQ ID NO: 16	PRTLNAWVKLIEEKKFGAEVVP	SG16	151-
			172

SEQ ID NO: 17	IEEKKFGAEVVPGFQALSEGCT	SG17	161-
			182

SEQ ID NO: 18	VPGFQALSEGCTPYDINQMLNCVGD	G-6	171-
			195*

SEQ ID NO: 19	GCTPYDINQMLNCVGDHQAA	G-5	180-
			199*

SEQ ID NO: 20	NCVGDHQAAMQIIRDIINEEAAD	SG20	191-
			213

SEQ ID NO: 21	IIRDIINEEAADWDLQHPQPAP	SG21	202-
			223

SEQ ID NO: 22	ADWDLQHPQPAPQQGQLREPSG	SG22	212-
			233

SEQ ID NO: 23	APQQGQLREPSGSDIAGTTSSV	SG23	222-
			243

SEQ ID NO: 24	SGSDIAGTTSSVDEQIQWMYRQ	SG24	232-
			253

SEQ ID NO: 25	SVDEQIQWMYRQQNPIPVGNIY	SG25	242-
			263*
			(*)

SEQ ID NO: 26	RQQNPIPVGNIYRRWIQLGLQK	SG26	252-
			273(*)

SEQ ID NO: 27	IYRRWIQLGLQKCVRMYNPTNIL	SG27	262-
			284(*)

SEQ ID NO: 28	KCVRMYNPTNILDVKQGPKEPF	SG28	273-
			294

SEQ ID NO: 29	ILDVKQGPKEPFQSYVDRFYKS	SG29	283-
			304

SEQ ID NO: 30	PFQSYVDRFYKSLRAEQTDAAV	SG30	293-
			314

SEQ ID NO: 31	KSLRAEQTDAAVKNWMTQTLLI	SG31	303-
			324

SEQ ID NO: 32	AVKNWMTQTLLIQNANPDCKLV	SG32	313-
			334

SEQ ID NO: 33	LIQNANPDCKLVLKGLGVNPTL	SG33	323-
			344

SEQ ID NO: 34	LVLKGLGVNPTLEEMLTACQGV	SG34	333-
			354

SEQ ID NO: 35	TLEEMLTACQGVGGPGQKARLM	SG35	343-
			364

SEQ ID NO: 36	GVGGPGQKARLMAEALKEALAP	SG36	353-
			374

SEQ ID NO: 37	LMAEALKEALAPVPIPFAAAQQ	SG37	363-
			384

SEQ ID NO: 38	APVPIPFAAAQQRGPRKPIKCW	SG38	373-
			394

SEQ ID NO: 39	AQQRGPRKPIKCWNCGKEGHSA	SG39	382-
			403

SEQ ID NO: 40	KCWNCGKEGHSARQCRAPRRQG	SG40	392-
			413

SEQ ID NO: 41	SARQCRAPRRQGCWKCGKMDHV	SG41	402-
			423

SEQ ID NO: 42	RQGCWKCGKMDHVMAKCPDRQAG	SG42	411-
			433

SEQ ID NO: 43	HVMAKCPDRQAGFLGLGPWGKK	SG43	422-
			443

SEQ ID NO: 44	AGFLGLGPWGKKPRNFPMAQVH	SG44	432-
			453

SEQ ID NO: 45	KKPRNFPMAQVHQGLMPTAPPE	SG45	442-
			463

SEQ ID NO: 46	VHQGLMPTAPPEDPAVDLLKNY	SG46	452-
			473

SEQ ID NO: 47	PEDPAVDLLKNYMQLGKQQREK	SG47	462-
			483

SEQ ID NO: 48	NYMQLGKQQREKQRESREKPYK	SG48	472-
			493

SEQ ID NO: 49	EKQRESREKPYKEVTEDLLHLN	SG49	482-
			503

SEQ ID NO: 50	YKEVTEDLLHLNSLFGGDQ	SG50	492-
			510

denotes peptides containing defined or ()semi defined gag epitopes (156-158)

TABLE 8

Overlapping peptides in Env of SIVmac239
(25-mer with 13-mer overlapping)

SEQ ID	MGCLGNQLLIAILLLSVYGIYCTLY	SE1	1-25
NO: 51

SEQ ID	LLLSVYGIYCTLYVTVFYGVPAWRN	SE2	13-37
NO: 52

SEQ ID	YVTVFYGVPAWRNATIPLFCATKNR	SE3	25-49
NO: 53

SEQ ID	NATIPLFCATKNRDTWGTTQCLPDN	SE4	37-61
NO: 54

SEQ ID	RDTWGTTQCLPDNGDYSEVALNVTE	SE5	49-73
NO: 55

SEQ ID	NGDYSEVALNVTESFDAWNNTVTEQ	SE6	61-85
NO: 56

SEQ ID	ESFDAWNNTVTEQAIEDVWQLFETS	SE7	73-97
NO: 57

SEQ ID	QAIEDVWQLFETSIKPCVKLSPLCI	SE8	85-109
NO: 58

SEQ ID	SIKPCVKLSPLCITMRCNKSETDRW	SE9	97-121
NO: 59

SEQ ID	TMRCNKSETDRWGLTKSITTTAST	SE10	109-133
NO: 60

SEQ ID	WGLTKSITTTASTTSTTASAKVDMV	SE11	121-145
NO: 61

SEQ ID	TTSTTASAKVDMVNETSSCIAQDNC	SE12	133-157
NO: 62

SEQ ID	VNETSSCIAQDNCTGLEQEQMISCK	SE13	145-169
NO: 63

SEQ ID	CTGLEQEQMISCKFNMTGLKRDKKK	SE14	157-181
NO: 64

SEQ ID	KFNMTGLKRDKKKEYNETWYSADLV	SE15	169-193
NO: 65

SEQ ID	KEYNETWYSADLVCEQGNNTGNESR	SE16	181-205
NO: 66

SEQ ID	VCEQGNNTGNESRCYMNHCNTSVIQ	SE17	193-217
NO: 67

SEQ ID	RCYMNHCNTSVIQESCDKHYWDAIR	SE18	205-229
NO: 68

SEQ ID	QESCDKHYWDAIRFRYCAPPGYALL	SE19	217-241
NO: 69

SEQ ID	RFRYCAPPGYALLRCNDTNYSGFMP	SE20	229-253
NO: 70

SEQ ID	LRCNDTNYSGFMPKCSKVVVSSCTR	SE21	241-265
NO: 71

SEQ ID	PKCSKVVVSSCTRMMETQTSTWFGF	SE22	253-277
NO: 72

SEQ ID	RMMETQTSTWFGFNGTRAENRTYIY	SE23	265-289
NO: 73

SEQ ID	FNGTRAENRTYIYWHGRDNRTIISL	SE24	277-301
NO: 74

SEQ ID	YWHGRDNRTIISLNKYYNLTMKCRR	SE25	289-313
NO: 75

SEQ ID	LNKYYNLTMKCRRPGNKTVLPVTIM	SE26	301-325
NO: 76

SEQ ID	RPGNKTVLPVTIMSGLVFHSQPIND	SE27	313-337
NO: 77

SEQ ID	MSGLVFHSQPINDRPKQAWCWFGGK	SE28	325-349
NO: 78

SEQ ID	DRPKQAWCWFGGKWKDAIKEVKQTI	SE29	337-361
NO: 79

SEQ ID	KWKDAIKEVKQTIVKHPRYTGTNNT	SE30	349-373
NO: 80

SEQ ID	IVKHPRYTGTNNTDKINLTAPGGGD	SE31	361-385
NO: 81

SEQ ID	TDKINLTAPGGGDPEVTFMWTNCRG	SE32	373-397
NO: 82

SEQ ID	DPEVTFMWTNCRGEFLYCKMNWFLN	SE33	385-409
NO: 83

SEQ ID	GEFLYCKMNWFLNWVEDRNTANQKP	SE34	397-421
NO: 84

SEQ ID	NWVEDRNTANQKPKEQHKRNYVPCH	SE35	409-433
NO: 85

SEQ ID	PKEQHKRNYVPCHIRQIINTWHKVG	SE36	421-445
NO: 86

SEQ ID	HIRQIINTWHKVGKNVYLPPREGDL	SE37	433-457
NO: 87

SEQ ID	GKNVYLPPREGDLTCNSTVTSLIAN	SE38	445-469
NO: 88

SEQ ID	LTCNSTVTSLIANIDWIDGNQTNIT	SE39	457-481
NO: 89

SEQ ID	NIDWIDGNQTNITMSAEVAELYRLE	SE40	469-493
NO: 90

SEQ ID	TMSAEVAELYRLELGDYKLVEITPI	SE41	481-505
NO: 91

SEQ ID	ELGDYKLVEITPIGLAPTDVKRYTT	SE42	493-517
NO: 92

SEQ ID	IGLAPTDVKRYTTGGTSRNKRGVFV	SE43	505-529
NO: 93

SEQ ID	TGGTSRNKRGVFVLGFLGFLATAGS	SE44	517-541
NO: 94

SEQ ID	VLGFLGFLATAGSAMGAASLTLTAQ	SE45	529-553
NO: 95

SEQ ID	SAMGAASLTLTAQSRTLLAGIVQQQ	SE46	541-565
NO: 96

SEQ ID	QSRTLLAGIVQQQQQLLDVVKRQQE	SE47	553-577
NO: 97

SEQ ID	QQQLLDVVKRQQELLRLTVWGTKNL	SE48	565-589
NO: 98

SEQ ID	ELLRLTVWGTKNLQTRVTAIEKYLK	SE49	577-601
NO: 99

SEQ ID	LQTRVTAIEKYLKDQAQLNAWGCAF	SE50	589-613
NO: 100

SEQ ID	KDQAQLNAWGCAFRQVCHTTVPWPN	SE51	601-625
NO: 101

SEQ ID	FRQVCHTTVPWPNASLTPKWNNETW	SE52	613-637
NO: 102

SEQ ID	NASLTPKWNNETWQEWERKVDFLEE	SE53	625-649
NO: 103

SEQ ID	WQEWERKVDFLEENITALLEEAQIQ	SE54	637-661
NO: 104

SEQ ID	ENITALLEEAQIQQEKNMYELQKLN	SE55	649-673
NO: 105

SEQ ID	QQEKNMYELQKLNSWDVFGNWFDLA	SE56	661-685
NO: 106

SEQ ID	NSWDVFGNWFDLASWIKYIQYGVYI	SE57	673-697
NO: 107

SEQ ID	ASWIKYIQYGVYIVVGVILLRIVIY	SE58	685-709
NO: 108

SEQ ID	IVVGVILLRIVIYIVQMLAKLRQGY	SE59	697-721
NO: 109

SEQ ID	YIVQMLAKLRQGYRPVFSSPPSYFQ	SE60	709-733
NO: 110

SEQ ID	YRPVFSSPPSYFQQTHIQQDPALPT	SE61	721-745
NO: 111

SEQ ID	QQTHIQQDPALPTREGKERDGGEGG	SE62	733-757
NO: 112

SEQ ID	TREGKERDGGEGGGNSSWPWQIEYI	SE63	745-769
NO: 113

SEQ ID	GGNSSWPWQIEYIHFLIRQLIRLLT	SE64	757-781
NO: 114

SEQ ID	IHFLIRQLIRLLTWLFSNCRTLLSR	SE65	769-793
NO: 115

SEQ ID	TWLFSNCRTLLSRVYQILQPILQRL	SE66	781-805
NO: 116

SEQ ID	RVYQILQPILQRLSATLQRIREVLR	SE67	793-817
NO: 117

SEQ ID	LSATLQRIREVLRTELTYLQYGWSY	SE68	805-829
NO: 118

SEQ ID	RTELTYLQYGWSYFHEAVQAVWRSA	SE69	817-841
NO: 119

SEQ ID	YFHEAVQAVWRSATETLAGAWGDLW	SE70	829-853
NO: 120

SEQ ID	ATETLAGAWGDLWETLRRGGRWILA	SE71	841-865
NO: 121

SEQ ID	WETLRRGGRWILAIPRRIRQGLELTLL	SE72	853-877
NO: 122

The cultures were incubated overnight at 37° C. in a 7% CO₂humidified atmosphere. Cells from each well were gently removed, transferred to 5.0 ml FACS test tubes and washed. One set of cells was stained with anti-CD3+ anti-CD4+. The other duplicate set was stained with anti-CD3+ anti-CD8+ (see below). These cell surface stained cells were then permeabilized and stained for intracellular content of IFN-gamma using an anti-IFN-gamma staining antibody using standard intracellular staining protocols. Each stained cell population (about 10,000 cells from each tube) was then analyzed using a FACS flow cytometer and the frequency of CD3+ CD4+ and CD3+ CD8+ T cells synthesizing IFN-gamma was determined. The negative and positive controls were utilized for background control and for positive control references. About 1000 analyses were performed in this manner during this experiment.

The frequency of CD4+ T cells (y axis) that expressed IFN-gamma by spleen cells from the six groups of mice in response to pools of SIV env peptide (9 pools) and SIV gag peptides (7 pools) were determined. Also determined was the frequency of CD8+ T cells (y axis) that express IFN-gamma by spleen cells from the same six groups of mice in response to pools of SIV env peptide (9 pools) and SIV gag peptides (7 pools). Data were the mean value from 4 mice/group. Results of these initial studies indicated that delipidated SIVmac251 at a dose of 10 ug or 1.0 ug led to marked augmentation of the SIV specific humoral responses in previously primed BALB/c mice. Even a dose of 0.1 ug (5×10⁶viral particles) led to detectable enhancement of the SIV specific humoral responses in these mice. A dose of 1.0 ug, but not 10 ug, led to markedly broad breadth of SIV env and SIV gag peptide specific CD4+ T cell responses as measured by IFN-g synthesis in previously primed BALB/c mice.

EXAMPLE 4

Charcoal Removal of Solvents after Plasma Delipidation

A charcoal column was generated by loading 2 ml of PBS-washed Hemasorba charcoal into 3-ml BD LuerLock syringe containing a Whatman filter frit. The column was washed with 5% glucose/PBS (5 to 10 column volumes). The column was incubated in 5% glucose/PBS for 30 min. This column was used to remove solvents from treated plasma.

About 2 ml of freshly isolated human plasma (ACD) was mixed with 1 ml of one the following solvents: 1% DIPE; 10% DIPE; or butanol/DIPE (25:75). The mixture was vortexed for 15 seconds and then centrifuged 5 min at 3000 rpm (˜1000×g). The solvent layer was aspirated. The plasma was passed through the charcoal column described in the preceding paragraph. About 0.5 ml of PBS was used to wash the column. Washing may occur several times as needed. The results are shown in Table 9. Total cholesterol (TC), triglycerides (TG), phospholipid (PL), apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and HDL were measured. The results show good recoveries of ApoA1, ApoB and HDL compared to controls

TABLE 9

Analysis of Plasma Delipidated and Passed Through Charcoal Syringe
Columns

Sample	TC	TG	PL	ApoA1	ApoB	HDL

Assay CSI	132.8	83.5	154.2	106.0	60.5	17.3
Assay CSII	197.4	164.7	224.0	87.2	107.7	21.3
Control 1	66.2	76.0	57.4	47.8	31.5	10.5
Control 2	103.6	113.2	98.7	79.6	48.8	15.6
1% DIPE (1 pass)	48.1	59.2	38.6	39.4	15.6	10.1
1% DIPE (2 pass)	40.1	46.5	29.3	26.0	15.7	6.8
10% DIPE (1 pass)	56.9	60.3	45.0	37.0	20.1	8.9
10% DIPE (2 pass)	58.9	61.7	52.4	42.2	25.5	8.5
But/DIPE (1 pass)	57.4	65.0	54.1	47.7	24.8	9.1
But/DIPE (2 pass)	81.7	84.4	73.8	53.8	34.2	9.2

Plasma Virus Recovery after Passage through Charcoal Column

Freshly isolated human plasma (ACD) was combined with HIV-1 to 1 ug/ml p24. HIV was added to the plasma such that the final concentration or particle content was 1 ug/ml of virus p24 antigen. Next, 1 ml of this plasma was passed through the column followed by 1 ml of PBS wash. The flow through and wash were combined. This procedure was repeated twice on fresh columns using 1 ml of the plasma. The flow through and wash from each of these three runs were analyzed separately. The results showed excellent recovery of p24 from the columns. P24 was measured by a standard capture ELISA protocol with a monoclonal antibody coated plate (for capture) and a polyclonal antibody for detection. Standard curves with known amounts of p24 are used to determined the p24 content of unknowns.

Direct Delipidation of HIV-1 and Removal of Solvents with Charcoal Column and Retention of HIV Proteins

About 25 ul of 1000× HIV-1 IIIB was mixed with 1) nothing; 2) 12.5 ul butanol/DIPE (25:75); 3) 2.5ul 100% DIPE; or 4) 12.5 ul 1% DIPE in PBS and the samples were vortexed for 15 seconds. Charcoal columns (0.5-ml) were prepared as described above. The virus-solvent mixtures were loaded individually onto separate columns. The columns were eluted with 1 ml of PBS. The elution volumes were measured and samples assayed for p24 by ELISA, protein, and subjected to Western blotting.

The samples treated with 1% DIPE showed excellent p24 recovery compared to controls. The samples treated with 10% DIPE or butanol/DIPE showed slightly less p24 recovery. The total protein recovery was similar in terms of percentage relative to control, to the p24 results obtained 1% DIPE, 10% DIPE or butanol/DIPE.

Western blot analysis, performed in a similar manner to the protocol provided below in this example, revealed numerous immunoreactive bands when probed with human anti-HIV IgG with butanol/DIPE, 10% DIPE or 1% DIPE solvent treatments. Western blot analysis also revealed positive immunoreactive bands corresponding to p24 with butanol/DIPE, 10% DIPE or 1% DIPE. Positive immunoreactive bands were observed for gp41 using 10% DIPE or 1% DIPE. Additional positive immunoreactive bands were observed for gp120 with butanol/DIPE, 10% DIPE or 1% DIPE, although the intensity of staining was higher with 10% DIPE or 1% DIPE.

SIV and HIV Western Blot Analysis

Reagents for comparison included delipidated SIVmac251, heat inactivated SIV mac251 and a rabbit polyclonal antibody against whole SIV (available through the AIDS reagent repository, Rockville, Md.). About 1 ug of protein was required to visualize most of the SIV bands in the Western blot. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the viral lysates (lysate buffer: 50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 ug/ml each of aprotinin, leupeptin and pepstatin; 1 mM sodium vanadate; 1 mM NaF).

A silver stain was used to visualize the bands which reveal the various viral proteins present following delipidation with respect to molecular weight standards. The heat inactivated SIVmac251 proteins were compared with the delipidated SIVmac251 proteins on the gels. A similar SDS-PAGE was run and the proteins are transferred to nitrocellulose. The blotted nitrocellulose was washed twice with water. A minimum of three blots each for the delipidated SIVmac251 and the heat inactivated SIVmac251 were run.

The blotted nitrocellulose was blocked in freshly prepared PBS containing 3% nonfat dry milk (MLK) for 20 min at 20-25° C. with constant agitation. The nitrocellulose strips were incubated with a freshly prepared pre-determined optimum concentration of the rabbit polyclonal anti-SIV antiserum (about 5 ml of a 1:1000 dilution of the antiserum in PBS-MLK) overnight with agitation. The nitrocellulose strips were washed twice with water. The strips were incubated with horseradish peroxidases (HRP)-conjugated goat anti-rabbit IgG 1:3000 dilution in PBS-MLK for 90 min at room temperature with agitation. The nitrocellulose was washed with water twice and then with PBS-0.05% Tween 20 for 3-5 min. The nitrocellulose strips were washed with 4-5 changes of water. Detection of the developed bands was achieved via detection of the developed bands. The bands developed using the heat inactivated SIV with the delipidated SIV were compared.

EXAMPLE 5Use of a Kit for Delipidation of a Plasma Sample Containing HIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected to an opening in the bag, is obtained from blood drawn from a 22 year old patient afflicted with the human immunodeficiency virus (HIV) and showing symptoms of acquired immunodeficiency syndrome (AIDS). The patient requires a reduction in the viral load in the blood. The plasma sample is exposed to a first extraction solvent to remove lipid from the viral envelope of the HIV virus.

A first container with a 500 ml capacity is removed from the kit. The first container, which is graduated, contains a known volume (about 200 ml) of a first extraction solvent. The entire plasma sample is added to the first container through a removable screw cap. The first container is agitated through repeated inversion, thereby mixing the first extraction solvent and the plasma sample sufficiently to create a mixture. The first container is placed on a counter and the mixture settles into two phases.

The delipidated plasma phase is removed with a manual pipette or a pipette connected to a vacuum, and placed in a second container from the kit. The volatile components of the first extraction solvent evaporate. Mild heating may be employed at this step. A tube, obtained from the kit, is inserted into the second container. The tube serves as, or is connected to, an intravascular line leading to a needle introduced into the antecubital vein of the patient. The delipidated fluid containing delipidated plasma and delipidated HIV viral particles with reduced infectivity is introduced into the vascular system through the force of gravity by elevating the second container above the patient. The needle is optionally obtained from the kit. Administration of the delipidated HIV viral particles into the vascular system induces an immune response in the patient to epitopes on the delipidated HIV viral particles.

EXAMPLE 6Use of a Kit for Delipidation of a Plasma Sample Containing HIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected to an opening in the bag, is obtained from blood drawn from a 22; year old patient afflicted with the human immunodeficiency virus (HIV) and showing symptoms of AIDS. The patient requires a reduction in the viral load in the blood. The plasma sample is exposed to a first extraction solvent to remove lipid from the viral envelope of the HIV virus.

A first container with a 500 ml capacity is removed from the kit. The first container, which is graduated, contains a known volume (about 200 ml) of a first extraction solvent. The entire plasma sample is added to a second container through a removable screw cap. The contents of the first container and the second container are added to a third container obtained from the kit. The third container is agitated through repeated inversion, thereby mixing the first extraction solvent and the plasma sample sufficiently to create a mixture. The third container is placed on a counter and the mixture settles into two phases.

The delipidated plasma phase is removed with a manual pipette or a pipette connected to a vacuum, and placed in a fourth container from the kit. The volatile components of the first extraction solvent evaporate. Mild heating may be employed at this step. A tube, obtained from the kit, is inserted into the fourth container. The tube serves as, or is connected to, an intravascular line leading to a needle introduced into the antecubital vein of the patient. The delipidated fluid containing delipidated plasma and delipidated HIV viral particles with reduced infectivity is introduced into the vascular system through the force of gravity by elevating the fourth container above the patient. The needle is optionally obtained from the kit. Administration of the delipidated HIV viral particles into the vascular system induces an immune response in the patient to epitopes on the delipidated HIV viral particles.

EXAMPLE 7Use of a Kit for Delipidation of a Plasma Sample Containing HIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected to an opening in the bag, is obtained from blood drawn from a 22 year old patient afflicted with the human immunodeficiency virus (HIV) and showing symptoms of AIDS. The patient requires a reduction in the viral load in the blood. The plasma sample is exposed to a first extraction solvent to remove lipid from the viral envelope of the HIV virus.

The delipidated plasma phase is removed with a manual pipette or a pipette connected to a vacuum, and placed in a fourth container from the kit. The volatile components of the first extraction solvent evaporate. Mild heating may be employed at this step. A second extraction solvent, contained in a graduated fifth container, is poured into the fourth container in order to remove residual first extraction solvent. The fourth container is agitated through repeated inversion, thereby mixing residual first extraction solvent, the partially delipidated plasma sample and the second extraction solvent sufficiently to create a mixture. The fourth container is allowed to sit and the mixture separates into a delipidated plasma layer and a solvent layer containing the second extraction solvent and residual first extraction solvent. The delipidated plasma layer is removed and placed in a sixth container obtained from the kit. A tube, obtained from the kit, is inserted into the sixth container. The tube serves as or is connected to an intravascular line leading to a needle introduced into the antecubital vein of the patient. The delipidated fluid containing delipidated plasma and delipidated HIV viral particles with reduced infectivity is introduced into the vascular system through the force of gravity by elevating the sixth container above the patient. The needle is optionally obtained from the kit. Administration of the delipidated HIV viral particles into the vascular system induces an immune response in the patient to epitopes on the delipidated HIV viral particles.

All patents, publications and abstracts cited above are incorporated herein by reference in their entirety. It should be understood, of course, that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims.

0.1 ug boost

0.01 ug boost