WO1993019775A1

WO1993019775A1 - Administration of liposomes containing peptides or proteins including ctl eptitopes of hiv proteins

Info

Publication number: WO1993019775A1
Application number: PCT/US1993/002978
Authority: WO
Inventors: Carl R. Alving; David Cassatt; Scott Koenig; Nabila Wassef; Wendy White
Original assignee: Medimmune, Inc.; Department Of Army
Priority date: 1992-03-31
Filing date: 1993-03-25
Publication date: 1993-10-14

Abstract

A method of inducing a CTL response in an animal against HIV comprising administering to the animal lipid vesicles (e.g., liposomes) containing at least one peptide or protein including at least one CTL epitope of an HIV protein.

Description

ADMINISTRATION OF LIPOSOMES CONTAINING

PEPTIDES OR PROTEINS INCLUDING CTL EPITOPES OF HIV PROTEINS

This invention relates to the induction of a T-cell

response, in particular a cytotoxic T lymphocyte (CTL) response, against HIV proteins. More particularly, this invention relates to the induction of a CTL response against HIV by administering liposomes containing peptides or proteins including CTL epitopes of HIV proteins.

Cell-mediated immunity (or CMI) of infections is thought to be a major line of defense against certain infections, such as viral infections and certain bacterial infections. For example, CMI may be significant in the development of an effective vaccine against human immunodeficiency virus (HIV), or AIDS virus, because HIV vaccines and/or therapies based on the generation of passive transfer of HIV-specific antibody in the absence of cell-mediated immunity have not yielded consistent protection in primates challenged with the HIV virus. Also, immune responses to certain segments of the virus may be deleterious. Such problems associated with the development of vaccines against AIDS virus may be avoided by employing vaccines which include small regions of HIV proteins which are known to produce neutralizing antibody responses or cytotoxic T lymphocyte responses.

A potential target for use in a vaccine against HIV is the HIV envelope, or env protein (also referred to as gp 120 or gp 160). The HIV env protein contains several hypervariable regions (or V regions), of which one region, the V3 loop, contains a peptide sequence that is important for the generation of humoral and cellular immune responses. The V3 loop, which is defined by a disulfide bond, contains a principal neutralizing determinant (or PND), as antibodies specific for the V3 loop have been shown to neutralize HIV (Rusche, et al., PNAS, Vol. 85, pgs. 3198-3202 (1988); Goudsmit, et al., PNAS. Vol. 85, pgs. 4478-4482 (1988)) and to protect chimpanzees from viral challenge (Emini, et al., J. Virol. Vol. 64, pgs. 3674-3678 (1990); Girard, et al., PNAS, Vol. 88, pgs. 542-546 (1991). A fifteen amino acid peptide derived from the tip of the V3 loop, known as Peptide 18 or P18, is within the PND and contains the immunodominant region for CTL response. This epitope has also been found to provide T cell help for itself as shown by its ability to provide in vitro stimulation of cytotoxic T lymphocytes derived from mice

immunized with vaccinia virus containing a gp 160 construct.

(Takahashi, et al., J. Exp. Med.. Vol. 171, pgs. 571-576 (1990).)

It is an object of the present invention to provide an improved CTL response against HIV through the administration of peptides or proteins including CTL epitopes of HIV proteins.

In accordance with an aspect of the present invention, there is provided a method of inducing in an animal, a CTL response against HIV. The method comprises administering to an animal lipid vesicles, or liposomes including at least one peptide or protein which includes at least one CTL epitope of an HIV

protein. The lipid vesicles, or liposomes are administered in an amount effective to induce in an animal a CTL response against HIV.

The term "liposomes including at least one peptide or protein which includes at least one CTL epitope of an HIV

protein", as used herein means that the peptide or protein which includes a CTL eptiope of an HIV protein may be encapsulated within the liposome, ..or may form a portion of the liposome wall, or may be bound to the liposome wall. When the peptide or protein is bound to the liposome wall, the peptide or protein may be bound to the exterior or to the interior of the liposome wall.

Applicants have found that when lipid vesicles including a peptide or protein which includes a CTL epitope of an HIV protein are administered to an animal, an increased CTL response in the animal against HIV is obtained.

Although the scope of the present invention is not to be limited to any theoretical reasoning, it is believed that

liposomes are taken up by macrophages, and the peptide contained within them can be presented to T cells or can enter the

cytoplasm, thus making possible the peptide's presentation to cytotoxic T cells. Such ability of liposomes to direct the peptide to the proper antigen-presenting cells thus enhances the ability of the peptides to induce a CTL response.

Peptides or proteins which include CTL epitopes of HIV proteins, and which may be included in the liposomes, include, but are not limited to, HIV env (also known as gp 120 or gp 160); HIV-gp 41; HIV pol; HIV nef; HIV gag; HIV tat; HIV rev; HIV vif; HIV vpr; HIV vpu; and HIV vpx, or fragments or derivatives thereof. In one embodiment, the peptide or protein is an HIV env protein or fragment or derivative thereof. Preferably, the peptide or protein is the V3 loop of the HIV env protein gp 120, or a fragment or derivative thereof. In one embodiment, the peptide or protein is the P18 peptide of the V3 loop of HIV-III-B env protein.

P18 peptide has the following structural formula:

Arg Ile Gln Arg Gly Pro Gly Arg

5

Ala Phe Val Thr Ile Gly Lys

10 15

(SEQ ID NO:l)

The peptide or protein may, in one embodiment, be conjugated with a conjugate peptide or protein prior to being included in the liposome. The conjugate may be attached to the N-terminal or the C-terminal of the peptide or protein. Examples of conjugate peptides or proteins include, but are not limited to, ovalbumin. To facilitate attachment of the peptide to the conjugate peptide or protein, it is preferred that one or more amino acids, wherein at least one amino acid residue is a cysteine residue, be attached to the N-terminal or the C-terminal of the peptide or protein prior to attachment of the peptide or protein to the conjugate peptide or protein, and wherein the peptide or protein is attached to the conjugate peptide or protein via the cysteine residue. Thus, if the conjugate peptide or protein is to be attached to the N-terminal of the peptide or protein, a cysteine residue or an amino acid chain having a cysteine residue at its N-terminus is attached to the N-terminal of the peptide or protein. If the conjugate peptide or protein is to be attached to the C-terminal of the peptide or protein, a cysteine residue or an amino acid chain having a cysteine residue at its

C-terminus is attached to the C-terminal of the peptide or protein. In one embodiment, a Cys-Gly (CG) dipeptide is attached to the N-terminal of P18 peptide to form a peptide having the following structural formula:

Cys Gly Arg Ile Gln Arg Gly Pro Gly Arg

5 10

Ala Phe Val Thr Ile Gly Lys

15

(SEQ ID NO:2)

This peptide may then be attached to a conjugate peptide or protein, such as ovalbumin, to form a conjugate such as, for example, ovalbumin-Cys-Gly-P18 peptide. Alternatively, such peptides or proteins having a cysteine -containing amino acid chain at the N-terrainus or C-terminus may be included in a liposome alone (without being attached to a conjugate peptide or protein) in order to induce a CTL response against HIV.

Applicants have found that when a Cys-Gly-P18 peptide contained in a liposome is administered to an animal, that, in addition to a CTL response, an improved humoral immune response against HIV is generated as well. It is to be understood, however, that the scope of the present invention is not to be limited to any particular conjugate peptides or proteins, or to any particular cystein-containing amino acid chains which may be attached to the peptides or proteins of the present invention.

The peptides or proteins employed in the present invention may be synthesized by means well-known in the art. For example, the peptides or proteins may be synthesized on an automatic peptide synthesizer. (Merrifield, J. Am. Chem. Soc., Vol. 85, pg. 2149 (1963)). It is also contemplated that the peptides or proteins may be produced by genetic engineering techniques. For example, there may be provided DNA encoding a peptide or protein which includes a CTL epitope of an HIV protein. The DNA is placed in an appropriate expression vector, which is transformed into an appropriate organism which expresses the peptide or protein.

The liposome which includes the peptide or protein may be formed by a variety of methods known to those skilled in the art. For example, the liposome may be formed according to the methods of Alving, et al., Liposome Technology, Gregoriadis, ed., (CRC Press, Boca Raton, Florida) or Fries, et al., PNAS, Vol. 89, pgs. 358-362 (1992). The liposomes may be formed, for example, from one or more lipids and one or more sterols. Lipids which may be employed include, but are not limited to, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, spingomyelin,

phosphatidylethanolaimine, phosphatidylinositol, phosphatidic acid, and cardiolysin. Sterols which may be employed include cholesterol and lanosterol.

In a preferred embodiment, in addition to the lipids and sterols hereinabove described, the liposomes may further include lipid A or a lipopolysaccharide which, along with the above-mentioned lipids and sterols, also becomes incorporated in the liposome wall.

The peptides or proteins, either alone or coupled with a conjugate, may, in one embodiment, be encapsulated within the liposomes by a variety of means known to those skilled in the art. For example, a lyophilized lipid film may be reconstituted with a buffer containing the peptides, or proteins, whereby a portion of the peptide or protein becomes encapsulated within the liposome during reconstitution.

Although the present invention has been described with respect to encapsulation of the peptides or proteins within liposomes, it is to be understood that within the scope of the present invention, the peptides or proteins may form part of the liposome wall or may be attached to the liposome wall such that the peptide or protein is attached to the exterior or the

interior of the wall.

In accordance with another aspect of the present invention, there is provided a composition for inducing in an animal a CTL response against HIV which comprises a lipid vesicle including least one peptide or protein including at least one CTL epitope of an HIV protein. The lipid vesicle, or liposome, and the peptide or protein may be those hereinabove described.

The liposomes which include the peptide or protein which includes a CTL epitope of an HIV protein, may be employed in a composition, such as a vaccine, for inducing in an animal a CTL response against HIV. The vaccine may be administered to a human or non-human animal.

To form such a vaccine, the liposomes may be administered in conjunction with a suitable pharmaceutical carrier. As

representative examples of suitable carriers there may be

mentioned: alum, mineral oil, synthetic polymers, etc. Vehicles for vaccines are well known in the art and the selection of a suitable vehicle is deemed to be within the scope of those skilled in the art from the teachings contained herein. The selection of a suitable vehicle is also dependent upon the manner in which the vaccine is to be administered. Alternatively, because liposomes possess adjuvant properties, the liposomes may be administered without a carrier and instead may be administered in water alone. The vaccine may be in the form of an injectable dose and may be administered intramuscularly, intravenously, orally, intradermally, or by subcutaneous administration.

The peptide or protein may be administered to an average human adult in an amount of from about 1 microgram to about 1,500 micrograms, preferably from about 10 micrograms to about 1,000 micrograms. The peptide may be administered for example at monthly intervals for a period of time of about three months. It is to be understood, however, that the scope of the present invention is not to be limited to any particular dosage levels or intervals of administration.

The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.

EXAMPLE 1

Synthetic P18 peptide (SEQ ID NO:1); or P18 peptide having Cys-Gly chain attached to its N-terminus (SEQ ID NO:2), also sometimes hereinafter referred to as CG-P18 was synthesized by using the stepwise solid phase approach of Merrifield, J. Am.

Chem. Soc., Vol. 85, pg. 2149 (1963) on an Applied Biosystems Model 430A peptide synthesizer. All synthetic peptides were assembled on an insoluble copolymer MBHA resin consisting of styrene and divinylbenzene (1%). The symmetric anhydride

derivatives were used as the acylating species for all amino acids except asparagine, glutamine, arginine, and histidine.

These four amino acids were coupled as the 1-hydroxybenzotriazole esters. The reactive side chains of amino acids were protected during the synthesis. The protecting groups used were O-benzyl for Asp and Glu; benzyl for Ser and Thr; p-methylbenzyl for Cys; tosyl for Arg; benzyloxymethyl for His; 2-chlorobenzyloxycarbonyl for Lys; 2-br.omobenzyloxycarbonyl for Tyr; and formyl for Trp. Following their syntheses, peptides were cleaved with anhydrous liquid HF in the presence of anisole, dimethylsulfide, and ethanedithol. Once cleaved, the peptides were precipitated in ethyl acetate, and then extracted from the resin with .30%

(vol./vol.) glacial acetic acid in water.

Once the peptides were cleaved from the resin, each was analyzed and subsequently purified to greater than 95%

homogeneity, using a Vydec C-18 reversed phase column, and

Beckman "System Gold" HPLC. The correct amino acid content of each peptide was verified by hydrolyzing purified materials with constant boiling 6N HC1 at 150°C for 2 hours. Once hydrolyzed, these samples were then subject to amino acid compositional analysis using the Beckman "System Gold" amino acid analyzer.

EXAMPLE 2

Preparation of peptide-protein conjugate.

Ovalbumin (OVA) (5 mg. or 100 nmole) was dissolved in 1 ml PBS, pH 6.8. To this solution was added 3 mg (10 μmole) m-male- imidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in 300 ml of synthesis grade dimethyiformamide (DMF). The mixture was allowed to react for one hour at room temperature with gentle mixing. At the end of one hour reaction time, the entire mixture was desalted over a 1cm x 20 cm column packed with Sephadex G-50 equilibrated with 0.1M phosphate buffer, pH 6.0. The eluant was monitored for absorbance at 280 mm, and the derivatized fraction of maleimidobenzyl ovalbumin (OVA-MB) was collected in the void volume (V ).

Cysteine-containing peptides (3 to 5 mg) were dissolved in 1 ml cold 0.1M sodium borate buffer, pH 9.1. To this was added 100 ml of a 0.1M sodium borohydride solution. The reduction was permitted to proceed for 15 min at 4ºC. After 15 min excess reducing agent was destroyed by acidifying with 6N hydrochloric acid. Following this the solution containing reduced peptide (either P18 or CG-P18) was returned to neutral pH (pH 6-7) with sodium hydroxide. The reduced peptide was slowly added with stirring to the derivatized protein fraction (OVA-MB) prepared above. This mixture was reacted overnight at room temperature. The conjugation reaction mixture was extensively dialyzed (MWCO 12,000-14,000 daltons) against water at 4°C to remove excess peptide and buffer salts.

The number of moles of peptide conjugated per mole OVA was determined by amino acid analysis. The OVA-MB-peptide conjugates and OVA were hydrolyzed separately with 100 ul constant boiling (6N) HCl for 2 hours at 150°C. The hydrolyzates were then dried in vacuo in the presence of potassium hydroxide. The dried-down samples were then resuspended and applied to a Beckman "System Gold" analyzer for determination of the amino acid content. By examining and comparing the ratio of amino acids determined for both the OVA and OVA-MB-peptide the extent of crosslinking was evaluated.

EXAMPLE 3

Preparation of liposome encapsulated peptide, or peptide

conjugate.

Liposomes were prepared as taught by Alving, et al. (in

Liposome Technology, ed., Gregoriadis, G. (CRC Press, Boca Raton, FL), Vol. 2, pgs. 157-175, 1984) and Fries et al. (Proc. Natl. Acad. Sci. USA, Vol. 89, pgs. 358-362, 1992). Chloroform

solutions of dimyristoyl phosphatidylcholine, dimyristoyl

phosphatidylglycerol, and cholesterol (recrystallized three times from ethanol), or chloroform solutions of the above components plus a chloroform solution of lipid A (phospholipid molar ratio of 9:1:7.5) were added together in an appropriately-sized

round-bottom flask. When added, lipid A was added in a

concentration of 10 to 20 μg lipid A per μ-mole of total

phospholipid.

The chloroform was removed with a rotary evaporator and the lipid film further dried in a vacuum dessicator. The resultant lipid film was hydrated with sterile water to a final phospholipid concentration of 50-200 mM and then lyophilized. After lyophilization, liposomes were reconstituted to a

phospholipid concentration of 100-200 mM with sterile

phosphate-buffered saline, pH 7.4, (PBS) containing peptide P18, CG-P18, or P18 peptide conjugate. The mixture was vortexed and then incubated for 24-72 hours at 4°C. Unencapsulated P18 peptide, CG-P18, or peptide conjugate was removed by washing the liposomes with PBS and washed pellets were resuspended in PBS to a phospholipid concentration of 100-200 mM. The encapsulated peptide or peptide conjugate was determined and the final

liposome preparation was diluted appropriately according to the concentration of peptide or peptide conjugate required per injection. Concentration of the peptide or peptide conjugate was measured by hydrolyzing a sample of the liposomes in 6N HCl for 2 hours and examining by amino acid analysis as described above.

EXAMPLE 4

Immunization of mice.

Preparations for immunization contained peptide (P18 or CG-P18) alone or conjugated to ovalbumin. Peptide was

administered at 10 ug/dose, while the concentration of the conjugate was adjusted to yield 10 ug of peptide per dose. The non-encapsulated peptide or peptide conjugate was administered in PBS with no adjuvant, in PBS emulsified in complete Freund's adjuvant (CFA) for priming, and in incomplete Freund's adjuvant (IFA) for boosting, or adsorbed onto 1.5 mg of aluminum hydroxide (alum). Peptide or peptide conjugate encapsulated in liposomes was administered either in liposomes alone or in liposomes adsorbed with 1.5 mg of alum. BALB/c mice (4 per group) were given primary immunizations of peptide or peptide conjugate intraperitoneally, and boosting intraperitoneal immunizations 3 weeks later (for the peptide-protein conjugate) or 3 and 11 weeks later (for the peptides).

EXAMPLE 5

Detection of anti-P18 activity. Sera from mice immunized with OVA-CG-P18, P18, or CG-P18 were tested for anti-P18 activity by ELISA.

Immulon^R flat-bottom plates were coated overnight with 250 ng/well of P18. After washing and blocking with PBS containing 3% dry milk and 0.1% Tween-20, various dilutions of sera diluted in PBS containing 1% goat serum and 0.1% Tween-20 were added and incubated for 1 hour. The plates were washed and goat anti-mouse IgG conjugated to horseradish peroxidase was added and incubated for 1 hour. After washing, substrate solution was added and allowed to develop for 30 min. Readings were taken at a

wavelength of 405 nm.

Sera from mice immunized with OVA-CG-P18 were tested at 3, 5, 7, and 17 weeks after the initial immunization. The anti-P18 titers of the sera mice immunized with OVA-CG-P18 without

adjuvant, in CFA/IFA, alum, in liposomes with lipid A, or in liposomes with lipid A in alum is given in Table I below.

TABLE I

Adjuvant week 3 week 5 week 7 week 17

none < 100 < 100 < 100 < 100

CFA/IFA 4,000 128,000 256,000 128,000

alum 200 32,000 32,000 16,000

liposomes

with lipid A < 100 8,000 8,000 4,000

liposomes

with lipid A

in alum 16,000 128,000 128,000 32,000

The above results show that an immune response to the P18 peptide was generated when the conjugate was encapsulated in liposomes with lipid A, with a_^higher response generated by adsorbing the conjugate with alum.

Sera from mice immunized with P18 peptide or CG-P18 were tested at 7 weeks after the initial immunization. The peptides had been administered without adjuvant, in CFA/IFA, in alum, in liposomes, in liposomes in alum, in liposomes with lipid A, or in liposomes with lipid A in alum. The anti-P18 titers are given in Table II below.

TABLE II

Peptide Adjuvant Titer

P18 none < 100

P18 CFA/IFA < 100

P18 alum < 100

P18 liposomes < 100

P18 liposomes

plus alum < 100

P18 liposomes

with lipid A 200

P18 liposomes

with lipid A

plus alum < 100

CG-P18 none < 100

CG-P18 CFA/IFA < 100

CG-P18 alum < 100

CG-P18 liposomes < 100

CG-P18 liposomes

plus alum < 100

CG-P18 liposomes

with lipid A 6,400

CG-P18 liposomes

with lipid A

plus alum 3,200

The above results indicate that an antibody response is generated against P18 through the administration of P18 or CG-P18 only when the peptide is encapsulated in liposomes with lipid A.

EXAMPLE 6

Detection of cytolytic (CTL) activity.

Spleens from mice immunized with OVA-CG-P18, P18, or CG-P18 were taken and disrupted to make a single cell suspension. Spleen cells were washed and cultured in 25 cm² flasks at 5×10⁷ cells/flask in a volume of 10 ml. Syngeneic P815 cells were pulsed with 50 ug of P18 for 40 min and then mitomycin C treated for 40 min and added to the flasks at 5×10⁶ cells/flask.

After six days of culture, effector cells were added to 96 well microtiter plates at 5×10⁵, 2.5×10⁵, and 6.25×10⁴ cells/well.

P815 cells that were pulsed with 35 ug P18 or left unpulsed were loaded with 200 uCi of ⁵¹Cr for 2 hrs. and added to the cultures at 5×10³ cells/well. P815 cells that were loaded with ⁵¹Cr were cultured alone (to determine spontaneous release) or with 10% sodium dodecyl sulfate (to determine total release). Cells were cultured for 4 hrs. and the supernatants were harvested and counted to determine ⁵¹Cr release. Percent lysis was determined by the following equation:

Figure 1 shows the CTL activity of spleens from mice immunize with OVA-CG-P18 conjugate, which was administered as described in Example 5. The spleens were taken 14 weeks after the initial immunization. As shown in Figure 1, maximal CTL response was obtained when the OVA-CG-F18 conjugate was encapsulated in

liposomes with lipid A, without adsorption of the liposomes to alum.

Figure 2 shows the CTL activity of spleens from mice immunize with Plθ or CG-P18, which was administered as described in Example 5. The spleens were taken 18 weeks after the initial immunization As shown in Figure 2, mice that received peptide encapsulated in lipsomes containing lipid A were able to mount a CTL response to P18.

It is to be understood, however, that the scope of the prese invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

PATAP693.A

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Alving, Carl R.

Cassatt, David

Koenig, Scott

Wassef, Nabila

White, Wendy

(ii) TITLE OF INVENTION: Administration of

Liposomes

Containing Peptides or Proteins Including CTL Epitopes of HIV Proteins

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Carella, Byrne, Bain,

Gilfillan, Cecchi & Stewart

(B) STREET: 6 Becker Farm Road

(C) CITY: Roseland

(D) STATE : New Jersey

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMP UTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 inch diskette

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC-DOS

(D) SOFTWARE: DW4.V2 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Olstein, Elliot M.

(B) REGISTRATION NUMBER: 24,025

(C) REFERENCE/DOCKET NUMBER: 469201-144 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: P18 peptide.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe

5 10

Val Thr Ile Gly Lys

15

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(ix) FEATURE:

(A) NAME/KEY: P18 peptide derivative (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Cys Gly Arg Ile Gln Arg Gly Pro Gly Arg

5 10

Ala PheVal Thr He Gly Lys

15

Claims

WHAT IS CLAIMED IS:

1. A method of inducing in an animal a CTL response against HIV, comprising:

administering to an animal lipid vesicles including at least one peptide or protein including at least one CTL epitope of an HIV protein, said lipid vesicles being administered in an amount effective to induce in an animal a CTL response against HIV.

2. The method of Claim 1 wherein said peptide or protein is the HIV env protein or a fragment or derivative thereof.

3. The method of Claim 2 wherein said peptide or protein is the V3 loop region of the HIV env protein or a fragment or a derivative thereof.

4. The method of Claim 3 wherein said peptide or protein is the P18 peptide of the V3 loop region of the HIV-III-B env protein.

5. A composition for inducing in an animal a CTL response against HIV comprising a lipid vesicle including at least one peptide or protein,

said at least one peptide or protein including at least one CTL epitope of an HIV protein.

6. The composition of Claim 5 wherein said peptide or protein is the HIV env protein or a fragment or derivative thereof.

7. The composition of Claim 6 wherein said peptide or protein is the V3 loop region of the HIV env protein or a fragment or a derivative thereof.

8. The composition of Claim 7 wherein said peptide or protein is the P18 peptide of the V3 loop region of the HIV-III-B env protein.