JP2001302541A - Adjuvant composed of hvj-charged liposome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an adjuvant composed of an HVJ-charged liposome capable of inducing humoral immunity and cellular immunity and inducing high immunoenhancing activity even on a peptide having low immunogenicity. SOLUTION: The present invention relates to an HVJ-charged liposome composed of a lipid having electric charge and Sendai virus (HVJ) as main constituent components, an immune composition produced by sealing an epitope peptide of human immunodeficiency virus in the HVJ-charged liposome and a method for the preparation of the liposome and the composition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a charged liposome capable of effectively enhancing humoral immunity and cellular immunity, and a method for producing the same. More specifically, Hemagglutinating Vir
us of Japan: sometimes referred to as HVJ) and a positively-charged liposome containing a negatively charged lipid, an immune composition containing the charged liposome containing an antigenic substance, and a method for producing the composition.

[0002]

2. Description of the Related Art In order to enhance the effectiveness of vaccines used for the prevention and treatment of infectious diseases, adjuvants for enhancing an immune response and immunostimulants added to adjuvants are used. A variety of adjuvants have been reported so far, but many of them cannot be used in humans, such as exhibiting toxicity or forming ulcers.

[0003] The only adjuvants that can be used by humans are adsorption-type adjuvants such as aluminum hydroxide and aluminum phosphide gels. However, although this aluminum gel effectively induces humoral immunity related to antibody production, its ability to induce cellular immunity is not strong. In addition to humoral immunity, cellular immunity is deeply involved in ecological defense. Control of cellular immunity is particularly important in the prevention and treatment of persistent infectious viral diseases such as immunodeficiency virus (HIV). The importance of is pointed out. In addition, aluminum gels have the disadvantage that they have low immunopotentiating activity against relatively weak immunogenic substances, such as small peptides.

[0004] In recent years, liposomes, which have been widely used as research materials for cell membrane structures and the like as biomembrane models, have attracted attention as one method of solving such problems. Liposomes not only analyze biological phenomena such as membrane fluidity and membrane permeation barrier, but also various protein antigens, DNA,
As a method of effectively administering substances such as drugs and drugs into cells, application to the treatment of various diseases is required, and the necessity and utilization techniques thereof have been pursued.

For example, a liposome vaccine in which antigen-presenting glycoprotein present on the surface of cancer cells is reconstituted into liposomes (Japanese Patent Application No. 2-188532), a hemagg of influenza
A virosome-type influenza vaccine incorporating lutinin (HA) into a liposome membrane (Vaccine, Vol.15, pp.1675
-1679, 1997), inactivated in influenza virosomes A
Vaccines conferring immunopotentiating activity, such as the hepatitis virus (HAV) -conjugated IRIV-HAV vaccine (J. Clin. Invest Vol. 90, 2491-2495, 1992), have been reported.

[0006] However, the immunogens used in these liposome vaccines are all high molecular weight protein molecules or virus particles, and it is clear whether or not they have the same effect on substances with low immunogenicity. It has not been. In addition, the use of influenza HA in liposomes may reduce the substantial effect by half, since most humans have antibodies to the influenza virus.

[0007] Attempts have also been made to increase the immunopotentiating activity by incorporating a substance having adjuvant activity into liposomes. For example, PCT Publication (WO 964024)
In 3), a lipopolysaccharide having immunopotentiating activity or interferon-inducing activity (eg, lipid A, an endotoxin present in the outer membrane of the cell wall of gram-negative bacteria) is reconstituted into liposomes, and the HIV gp120 epitope is added thereto. It has been described that the added liposome induces humoral immunity and cellular immunity against gp120. However, lipid A is a body of pyrogen and may cause fever or inflammation, so that it needs to be handled carefully when used in humans.

In addition, liposomes are greatly affected by the shape, size, stability, size and amount of a substance that can be held depending on the lipid used, and the affinity of the liposome for a cell membrane and the permeability of a substance due to the polarity of the liposome. It is known that there is a difference in
0 p784-798, 1985). Therefore, when preparing liposomes, it is considered important to properly use lipids according to the purpose.

HVJ has an envelope composed of sialic acid-attached glycolipids and glycoproteins (F: fusion, HN: two types of glycoproteins known as hemoglutinin-neuraminidase), and most of the proteins other than lymphocytes. Cells. Since the discovery of the cell fusion effect of HVJ, studies on the method of introducing substances into cells using HVJ have been actively conducted, and the introduction of enzymes into cells using HVJ, antibodies and nuclear proteins using erythrocyte ghosts and HVJ, etc. Numerous reports have been reported, such as the introduction of liposomes and the introduction of toxins by fusion liposomes in which HVJ fusion proteins are implanted on the liposome surface (hereinafter sometimes referred to as HVJ-liposomes). In particular, HVJ
-Since liposomes can efficiently introduce substances such as proteins and nucleic acid fragments into cells and have low cytotoxicity, they have recently attracted attention as a method for introducing genes into cells, and are expected to be applied to gene therapy. (Biogenic A
mines, Vol.14, pp.553-572, 1998, MOLECULAR MEDICIN
E TODAY, JULY 1999 (VOL.5), P298-303, Chemistry and Biology
Vol.36, No6. P376-384, 1988).

[0010]

As described above, the HVJ-
Although several efficient methods for introducing substances into cells using liposomes have already been attempted, there have been few reports on the research results relating to the adjuvant activity of HVJ-liposomes. At present, there are no other adjuvants that can withstand practical use from the viewpoint of efficacy and safety, and it is desired to develop a more practical and effective adjuvant that overcomes the disadvantages of aluminum gel that can be substituted for it. ing.

Under these circumstances, the present invention provides an HVJ-charge that can induce humoral immunity and cellular immunity, and can also induce high immunopotentiating activity against peptides with low immunogenicity. It is an object of the present invention to provide a liposome of the type and a method for producing the same.

[0012]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and as a result, HVJ-charged liposomes incorporating HIV epitope peptides have heretofore been low in antigenicity. The present inventors have found that HIV antigenicity, which has been considered difficult to use as a vaccine, is significantly improved, and that HVJ-charged liposomes have a strong booster effect. Reached.

Accordingly, the present invention encompasses HVJ-charged liposomes, vaccines containing HVJ-charged liposomes incorporating HIV epitope peptides as main components, and methods for producing these.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to an HVJ in which the envelope glycoprotein (F, HN) of HVJ is reconstituted into a charged liposome.
-Characterized by charged liposomes and compositions incorporating the protective antigen into the liposomes. By administering this composition to a subject animal or human, high immunity against a protective antigen can be imparted.

Positively charged liposomes have excellent local convection and are easily fused with local cells, and thus are excellent in local immunity as a booster. On the other hand, since negatively charged liposomes are likely to repel the cell membrane, they are scattered throughout the body and easily taken up by lymphatic tissues throughout the body. Therefore,
By selecting a positive charge type or a negative charge type in accordance with the purpose and the immunization schedule, it is possible to more effectively induce the immunity. For example, the use of positively charged forms is more effective in inducing local mucosal immunity, and delayed hypersensitivity (DT
The positive charge type is also effective for the induction of H). In contrast, b
When inducing systemic immunity by subcutaneous administration as an ooster,
The use of the negative charge type is effective.

The liposome particles can freely impart a liposome charge by changing the composition according to the above-mentioned purpose of enhancing immunity. The lipid component used for preparing the HVJ-charged liposome of the present invention includes phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (cholesterol: Chol), dimethylaminoethane / carbamyl cholesterol (dimethylaminoethane). -c
arbamoyl cholesterol: DC-chol, phosphadityl ethanolamine (dioleoylphosphatidylethanolamine: DOP)
E), sphingomyelin (Sphingomyelin: Sph), dioleyloxypropyltrimethylammonium chloride (N- {1- (2,3-dioleyloxy) propyl} -r, n, n-trimethyl
ammonium chloride: DOTMA), trimethylammonioacetyldidodecyl glutamate chloride (N- (α-tr
imethyl ammonioacetyl) didodecyl-D-glutamate chlori
de: TMAG), aminopropyldimethyldodecyloxypropanaminium bromide (N- (3-aminopropyl) -N, N-di
methyl-2,3-bis (dodecyloxy) -1-propanaminium bromid
e: GAPDLRIE). By combining these lipid components, various positively charged liposomes or negatively charged liposomes having different degrees of charge can be produced.

When producing positively charged liposomes, a composition comprising DC-Chol, PC, Sph, DOPE and Chol is preferably used. When preparing negatively charged liposomes, it is preferable to use a composition comprising PS, PC, Sph, DOPE and Chol. The amount ratio of each lipid component is changed according to the shape and size of the liposome and the purpose of use. For example, when preparing a positively charged liposome for encapsulating a low molecular weight substance, DC-Chol: PC: Chol = 6: 48: 24 (weight ratio) can be used. -Chol: PC: S
ph: DOPE: Chol = 6: 13: 12: 12: 24 (weight ratio) is used.
For the preparation of negatively charged liposomes, PS: PC: Chol = 10: 48: 24
(Weight ratio), but preferably PS: PC: Sph: DO
PE: Chol = 10: 13: 12: 12: 24 (weight ratio) is used.

For the preparation of HVJ-charged liposomes, purified HVJ virus particles and extraction and extraction from the virus particles are used.
Although any of the purified glycoproteins (F, HN) can be used, virus particles are preferably used. The HVJ is purified from the culture medium of the virus-infected cells by a general virus purification method, that is, a method such as centrifugation, membrane filtration and ion exchange chromatography, or a combination thereof.

For inactivating the virus, a method using ultraviolet irradiation or a method using a chemical substance such as beta-propyolactone is used. Extraction and purification of the HVJ envelope protein is performed using a surfactant such as NP-40, Triton X-
After extraction of the membrane protein with 100, octylglucoside, etc., this can be achieved by appropriately using a purification method usually used in protein chemistry, for example, ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like. .

HVJ-charged liposomes include viral particles, antigenic substances such as antigenic proteins and peptides, pharmaceuticals such as antibiotics and antisense oligonucleotides, and nucleic acid fragments constructed to be expressed in cells and living organisms. And various substances can be encapsulated. The HVJ-liposome of the present invention is particularly useful for enhancing the adjuvant activity of an antigenic substance. As the antigenic substance to be used at this time, any substance can be used as long as it is an immunological substance having a target epitope, and is not limited to the strength of antigenicity. Such antigenic substances include, for example, peptides having an epitope against immunodeficiency virus, and those produced by genetic recombination techniques or chemical synthesis can be used.

The amount of the substance to be encapsulated in the HIV-charged liposome is appropriately selected depending on the kind, shape, size and purpose of the substance. For example, the amount of the antigenic substance varies depending on the type of the antigen, the composition and structure of the liposome, etc., but is generally used in the range of 0 to 10 mg per 9.75 mg of the lipid constituting the liposome (for example, 200 to 500 μl of a solvent). If it can be dissolved, it can be encapsulated at a rate of 10 to 60%).

The HVJ-charged liposome of the present invention is prepared by the method of Kanada et al. (Saeki, Y., & Kaneda, Y .: Protein modifie).
d liposomes (HIV-liposomes) fro the delivery of ge
nes, oligonucleotides and proteins.Cell Biology; A
laboratory handbook (2 ^nd eds.) edited by JECelis
(Academic Press Inc., San Diego) vol4, 127-135, 1
998.). That is, after a predetermined lipid is dissolved in a solvent, the solvent is evaporated and removed by an evaporator or the like to form a lipid thin film. Examples of the solvent used at this time include chloroform, diethyl ether, tetrahydrofuran and the like, and they can be used alone or as a mixture of several kinds. Preferably, chloroform is used.

Next, an aqueous medium is added to the lipid thin film and vortexed vigorously to form charged liposomes. BBS (borate buffer), PBS as aqueous medium
(Phosphate buffer), physiological saline, water and the like can be used, but BBS and PBS are preferably used. The pH of the water-soluble medium can be used in the range of 7 to 8, but is preferably
7.5 is used.

By dissolving or suspending the target substance in a water-soluble medium, the target substance can be encapsulated in charged liposomes. The aseptic treatment of the thus obtained charged liposome is performed by passing through a 0.2 to 0.45 μm filter. The HVJ-charged liposome of the present invention may be any of a multi-layer type or a monolayer type, and these include vortex method, ultrasonic method, ethanol injection method, ether method and reverse phase evaporation method. Is appropriately selected or combined.

Next, HVJ particles or HVJ particles are added to the charged liposomes.
Of the envelope glycoprotein is introduced. For example, to the liposome solution, add HVJ inactivated by ultraviolet irradiation,
After leaving it on ice for 10 minutes, it was transferred to a 37 ° C constant temperature bath and shaken, so that HVJ in which HVJ was
It can form charged liposomes; HVJ−
Charged liposomes are separated and purified from unreacted components by performing density gradient centrifugation.

HVJ-charged liposomes encapsulating various infection-protecting antigens and other immunizing antigens are used as vaccines when administered alone or together with a suitable stabilizer that can be administered as a drug into a living body. In addition, the present invention
HVJ-charged liposomes are, like ordinary liposomes, auxiliary agents that promote the uptake of substances into cells by encapsulating target substances such as antibiotics and nucleic acid fragments in the HVJ-charged liposomes of the present invention. It can also be used as

[0027]

According to the present invention, HVJ-charged liposomes having adjuvant activity are provided.

The HVJ-charged liposome of the present invention encapsulating an immunogenic substance has a strong adjuvant activity, and thus can induce high immunity even to a peptide having low antigenicity. In addition, HVJ-charged liposomes have a booster effect of enhancing humoral and cellular immunity.
Furthermore, the charged liposomes having different polarities easily prepared by the method of the present invention can be effectively used as a drug delivery system, and have a cell fusion effect of HVJ, so that the target substance can be efficiently incorporated into cells. Can be made.

[0029]

EXAMPLES Example 1 HVJ-Positive Electron Containing HIV-V3 Peptide
Preparation of loaded liposome DC-Chol (provided by NOF) 6mg, DOPE (Sigma P5078)
12 mg, Sph (Sigma S0756) 12 mg, egg yolk PC (Sigma P
2772) 13 mg and 24 mg of Chol (Sigma C8667) were dissolved in 4 ml of chloroform (Nakarai), and 0.5 ml of each was dispensed into eight sharp glass tubes (13 cm long, Iwaki Glass). It was sealed by blowing and temporarily stored at -20 ° C. Take out the glass tubes one by one and rotate the evaporator (Tokyo
Rikakikai), and while evaporating chloroform, a thin film of the mixed lipid was formed on the inner wall of the glass tube. The mixture was sealed by blowing nitrogen gas and stored at -20 ° C. Glass tube 1
Add 300 μl of HIV-V3 peptide (1 mg) solution (PBS) to the book, vortex vigorously for 50 seconds, 37 ° C for 10 seconds
Was kept warm. This was repeated eight times. BSS (10 mM Tris-H
Cl pH7.5, 137 mM NaCl, 54 mM KCl)
Cellulose acetate filter (Iwaki Glass) 0.45
The liposome solution was passed through the same filter having a diameter of 0.20 μm, and the filter was washed again with 500 μl BBS.

HVJ was purified from allantoic fluid, and this virus solution was inactivated by irradiating 20,000 HAU (Hemagglutinating Unit) with ultraviolet light (198 mjoule / cm ² ), added to the liposome solution, and placed on ice for 10 minutes. Was transferred to a thermostat at 37 ° C. and shaken for 1 hour (twice per second). BBS containing 30% sucrose (W / V) 7 ml
Is placed in a centrifuge tube, and the HVJ-liposome solution is overlaid thereon.
Ultracentrifuged at 2,800 g for 90 minutes. HVJ- just above 30% sucrose solution
The liposome layer was collected with a Pasteur pipette and stored at 4 ° C.

Example 2: HVJ containing HIV-V3 peptide-negative electricity
Preparation of loaded liposome PS 10 mg, DOPE (Sigma P5078) 12 mg, Sph (Sigma S075)
6) 12 mg, egg yolk PC (Sigma P2772) 13 mg, Chol (Sig
ma C8667) (24 mg) was dissolved in chloroform (Nakarai) (4 ml), and 0.5 ml each was dissolved in a pointed glass tube (13 cm long, Iwa
ki Glass), dispensed into eight tubes, sealed with nitrogen gas, and stored temporarily at -20 ° C. Each glass tube was taken out one by one, attached to a rotary evaporator (Tokyo Rikakikai), a thin film of mixed lipid was formed on the inner wall of the glass tube while evaporating chloroform, sealed with nitrogen gas, and stored at -20 ° C. . 200 μl HIV-V per glass tube
3 Peptide (1 mg) solution (PBS) was added, and the mixture was vigorously vortexed for 30 seconds and kept at 37 ° C. for 30 seconds. This is 8
Repeated times. Sonicate for 3 seconds with a water bath type ultrasonic generator, vortex vigorously for 30 seconds, 300 μl BS
S was added, and the mixture was shaken (two rotations per second) at 37 ° C for 30 minutes.

HVJ was purified from allantoic fluid, 20,000 HAU (Hemagglutinating Unit) of this virus solution was inactivated by irradiation with ultraviolet light (198 mjoule / cm ² ), and this was added to the liposome solution and placed on ice for 10 minutes. Was transferred to a thermostat at 37 ° C. and shaken for 1 hour (twice per second). BBS containing 30% sucrose (W / V) 7 ml
Is placed in a centrifuge tube, and the HVJ-liposome solution is overlaid thereon.
Ultracentrifuged at 2,800 g for 90 minutes. HVJ- just above 30% sucrose solution
Collect the liposome layer with a Pasteur pipette,
Dilute with the same volume of BSS, place in a centrifuge tube, and centrifuge at 27,000 g for 30 minutes at 4 ° C to precipitate HVJ-liposome.
300 μl of PBS was added, suspended by vortexing, and stored at 4 ° C.

Example 3: Charge-type liposome booster
Effect (1) Induction of local skin reaction HIV-1 _HXB2 or HIV-1 _MN env V3-PND (major neutralizing region) gene is inserted into the hepatitis B virus core protein (HBc) gene as a carrier, and HIV -HBs chimera particles were prepared (the preparation method is described in Patent No. 2717799). HIV-HBc (HIV-HBc / CF) in which guinea pigs (Hartley species) are suspended in Freund's complete adjuvant (CFA)
A), HIV-HBc suspended in saline (HIV-HBc / PBS),
The mice were immunized twice with HBc (HBc / PBS) or HIV-1 PND peptide (HIV / PBS) suspended in physiological saline at intervals of 2 weeks. 7
A day later, guinea pigs of each group were injected subcutaneously with a conjugate of horseshoe crab hemagglutinin (KLH) and the peptide of the neutralizing region (PND) of the HIVenvV3 domain, or HBc particles, or PBS, and the skin reaction was observed. . Local skin reactions were measured 24 hours after challenge. The results are shown in Table 1 (the numerical values indicate the average value of each group (8 animals)). H
It showed specific skin reaction only to Bc antigen.

[0034]

[Table 1]

Next, guinea pigs were primed with HIV-HBc and two weeks later boosted with various HVJ-HIV-liposomes. According to the same method as above, the local skin reaction was measured. The results are shown in Table 2 (the numerical values indicate the average value of each group (8 animals)). Incorporation of HVJ into HIV liposomes induced an HIV-specific delayed skin reaction. Among them, in the local DTH reaction, cat
Anionic liposomes were more efficient than ionic liposomes.

[0036]

[Table 2]

(2) Induction of antibody production Guinea pigs (Hartley species) were prepared by suspending HIV-HBc in CFA.
(HIV-HBc / CFA), HIV-HBc (HIV-
HBc / PBS), HBc suspended in saline (HBc / PBS) or H
The mice were immunized twice with the IV-1 PND peptide (HIV / PBS) at two-week intervals. Blood was collected 6 weeks after the first immunization, and the HIV-1 antibody and HBc antibody in the serum were measured. Antibody titers were calculated by ELISA with limiting dilution of serum (values represent the average of four different assays). Specific antibodies to HBc antigen were detected.

[0038]

[Table 3]

Next, guinea pigs were primed with HIV-HBc and boosted with various HVJ-HIV-liposomes two weeks later. Blood was collected 6 weeks after the first immunization, and serum HI
V-1 antibody and HBc antibody were measured. FIG. 1 shows the results. Antibody titers were calculated in the same manner as above (numerical values represent the average of four different assays). Neutralizing activity was expressed as the inhibitory effect (% of control) by GHOST-cell assay (numerical values indicate the average of four measurements). IgG from guinea pigs boosted with HVJ-HIV-liposomes neutralized HIV-1LAI by 50% at 10-15 μl / ml.

(3) Induction of CTLs After immunization with the HIV-HBc chimer particles, HIV-HVJ-liposomes were boosted three weeks later. FIG. 2 shows the CTL activity in the first week of the booster immunization. CTL activity was measured by a conventional 51Cr chrome release assay. A is HIV in target cells
B shows the results when the PND peptide derived from the IIIB strain was pulse-labeled, and B shows the results when the target cells were pulse-labeled with the PND peptide derived from the HIV MN strain. HIV in any case
By incorporating HVJ into liposomes, CTL-inducing activity was dramatically improved. In the mice used for this immunization, no clinical abnormalities were observed, including the results of weight gain. This result indicates that the adjuvant activity of CTL induction is H
It is in IV-HVJ-liposomes, indicating that HVJ uptake as an active ingredient greatly contributes to its efficiency.

[Brief description of the drawings]

FIG. 1A is a view showing the results of measuring an ELISA antibody titer against a peptide in the PND region of HIV IIIB strain (same as LAI strain). B shows E to the peptide in the PND region of the HIV MN strain.
It is a figure which shows the result of having measured the LISA antibody titer. C is a diagram showing the results of measuring the neutralizing ability of the antibody.

FIG. 2A shows that mice immunized basal with HIV-HBc chimeric particles having the PND of the IIIB strain of HIV were given H-derived from the PND of the IIIB strain.
FIG. 9 shows the measurement results of HIV-1-specific CTL activity observed when booster immunization was performed with liposomes containing the cyclic V3 peptide of VJ-HIV-1HXB22. B shows that mice immunized with HIV-HBc chimera particles having PND of MN strain of HIV
FIG. 9 shows the measurement results of HIV-1-specific CTL activity observed when booster immunization was performed with liposomes containing the cyclic V3 peptide of HVJ-HIV-1HXB22 derived from the strain PND.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/115 A61K 37/02 F term (Reference) 4C076 AA19 BB11 CC06 EE41 EE51 EE56 4C084 AA02 AA03 BA44 CA01 NA05 NA14 ZB022 ZB092 ZB262 ZC552 4C085 AA02 AA38 BA69 CC08 CC21 CC32 FF19 4H045 AA11 AA30 BA53 CA01 CA05 DA86 EA31

Claims (10)

[Claims] 1. An adjuvant capable of inducing humoral immunity and cellular immunity to a low molecular substance, which comprises Sendai virus (sometimes referred to as HVJ) or an envelope glycoprotein of HVJ and a lipid component. The adjuvant, comprising HVJ-charged liposomes. 2. The adjuvant according to claim 1, wherein the HVJ-charged liposome is positively charged. 3. The adjuvant according to claim 1, wherein the HVJ-charged liposome is negatively charged. 4. The method according to claim 1, wherein the lipid component is phosphatidylcholine (P
C), cholesterol (Chol), dimethylaminoethane / carbamyl cholesterol (DC-Chol) phosphatidylserine (PS), sphingomyelin (Sph), phosphatidylethanolamine (DOPE), (DOTMA), (TMAG)
The adjuvant according to any one of claims 1 to 3, wherein the adjuvant is selected from the group consisting of: and (GAPDLRIE). 5. The method according to claim 5, wherein the envelope glycoprotein of HVJ is F or H.
The adjuvant according to any one of claims 1 to 4, which is an N protein. 6. An immunological composition comprising the adjuvant according to claim 1 and an optional antigenic substance. 7. The immune composition according to claim 6, wherein the antigenic substance is a protective antigen for HIV infection. 8. The immune composition according to claim 7, wherein the HIV infection protective antigen is an epitope peptide. 9. The immune composition according to claim 8, wherein the epitope peptide is a V3 peptide. 10. The method using any antigenic substance and lipid component,
A method for preparing an immune composition, comprising preparing a charged liposome encapsulating the antigenic substance, and incorporating HVJ or an envelope glycoprotein of the HVJ into the charged liposome.