Life Sciences, Vol. 50, pp. 481-489 Printed in the USA

ARTIFICIAL HUMAN

VIRAL ENVELOPES IMMUNODEFICIENCY

Pergamon Press

CONTAINING RECOMBINANT VIRUS (HIV) g p l 6 0

Ramesh Chander a and Hans Schreier b Department of Pharmaceutics Drug Delivery Laboratory, University of Florida Progress Center, Alachua, FL 32615, U.S.A. (Received in final form December 12, 1991) Summary

An artificial viral envelope was constructed, resembling the human immunodeficiency virus (HIV) envelope with respect to ultrastructure, size, phospholipid profile and lipid:cholesterol ratio. Recombinant HIV surface protein gpl60 was anchored in the outer surface of the envelope membrane using a double detergent dialysis. The envelopes remained physically stable for several months. Immunolabeling with anti-gpl60/41 monoclonal antibody revealed surface insertion and availability of gpl60 for binding. Cell fusion and cytosolic transfer of the encapsulated fluorescent marker FITC-dextran was demonstrated. Flow cytometry indicated more efficient transfer of the fluorescent marker to cells which were =60% CD4 ÷ (REX-IB), relative to cells which were only =18% CD4 ÷ (KG-I). However, plain lipid envelopes without gpl60 fused very efficiently with both cell types, indicating their potential usefulness as "fusogenic liposomes". Complete artificial viral envelopes may serve as subunit vaccines, and receptor-tarqeted delivery systems for drugs, toxins and genetic constructs. The lipid envelope of the human immun0deficiency virus (HIV), like the lipid envelopes of other enveloped viruses, consists of approximately equimolar amounts of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin, with a characteristic approximately equimolar cholesterol:phospholipid ratio (i). Electron spin resonance studies indicated that HIV has a very rigid membrane, and that the high membrane fraction of cholesterol regulates the rigidity as well as its infectivity (i). The HIV

aPermanent Address: Department of Food Technology and Enzyme Engineering, Bhabha Atomic Research Center, Bombay 400085, India 5To whom correspondence should be sent at Drug Delivery Lab, UF Progress Center, One Progress Blvd #19, A!achua , FL 32615, U.S.A. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.

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envelope glycoprotein (gpl60) consists of two subunits which span the viral envelope (gp41) and extend beyond it (gpl20) (2). The latter has been identified as a major antigenic moiety (3), exhibiting tropism to the CD4 ÷ receptor (4), whereas the hydrophobic gp41 subunit is thought to be involved in cell fusion (5). Analogues of viral membranes have been reconstituted from lipid and protein mixtures for various purposes including investigation of viral fusion (6), use as cytoplasmic delivery vehicle (7) and as subunit vaccine against rabies (8,9), influenza (i0), herpes simplex (Ii), and HIV (12). Invariably, investigators reported improved immunogenicity (9,11,12) and antigen-specific lymphocyte stimulation (ii), despite the fact that in most cases arbitrary phospholipid mixtures were employed, Specifically, p h o s p h a t i d y l e t h a n o l a m i n e and p h o s p h a t i d y l s e r i n e were not normally included, and the rigidity was not mimicked by adding appropriately high fractions of cholesterol (except control liposomes in ref. i0). The aim of the present study was to assemble analogues of the HIV envelope from its major components so that the resulting artificial envelope would resemble the natural envelope as closely as possible with respect to its unilamellar structure, size, lipid composition and lipid:cholesterol ratio. A second goal was to selectively anchor recombinant gpl60 in the outer surface of the envelope membrane so as to reflect its original membrane distribution. These requirements are difficult to fulfill with conventional liposome preparation techniques due to the low solubility of cholesterol and the labile nature of the surface glycoproteins. Therefore, a double detergent dialysis was employed, separating membrane formation from protein insertion. Materials

and

Methods

Egg p h o s p h a t i d y l c h o l i n e (PC), p h o s p h a t i d y l s e r i n e (PS) from bovine brain, egg p h o s p h a t i d y l e t h a n o l a m i n e (PE), cholesterol from porcine liver, deoxycholic acid, sodium cholate, FITC-dextran (avg. M.W. 20,000), and Sepharose 300 were from Sigma Chemical Co., St. Louis, MO. Egg sphingomyelin (SM) was from Avanti Polar Lipids, Alabaster, AL. Phosphate buffered saline (PBS) was made from 137 mM NaCI, 2.7 mM KCI, 8.1 mM Na2HPO4, and 1.5 mM KH2PO 4. Tris buffer was made with i0 mM Tris, 150 mM NaCl, and 3 mM sodium azide, adjusted to pH 8 with HCI. HIV gpl60 envelope protein was from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (13-15). Samples were also purchased from MicroGeneSys, West Haven CT, and Repligen, Cambridge, MA. Viral enveloDe construction: double deterqent dialysis. The first step of the double detergent dialysis comprised the preparation of the p h o s p h o l i p i d envelopes without gpl60. The mixture of p h o s p h o l i p i d s employed was very similar to the one found in natural HIV, except that minor fractions of acidic phospholipids were added as p h o s p h a t i d y l s e r i n e (Table I). Phospholipids in the molar ratios shown in Table I were dissolved in chloroform, cholesterol was dissolved in isopropanol and sodium cholate was dissolved in methanol. Of every lipid stock solutions 500 ~i were combined with 500 ~I cholesterol stock solution and 1,000 ~i of sodium cholate stock solution to give an approximate I:I molar lipid:cholesterol and 45:1 d e t e r g e n t : l i p i d ratio. This unusually high d e t e r g e n t : l i p i d ratio was found to be necessary to achieve complete solubilization

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TABLE I Phospholipid

Composition

of Artificial

Mole% of Total Phospholipid: Phosphatidylcholine (PC) Phosphatidylethanolamine Sphingomyelin (SM) Phosphatidylserine (PS) Phosphatidylinositol Phosphatidic acid Other

(PE)

n.a. = not added to artificial

Artificial

HIV Envelopes Natural (Ref.3)

23.7 22.6 28.1 25.7 n.a. n.a. n.a.

23.8 24.6 28.3 15.1 2.1 0.9 5.0

lipid envelope mixture

of the cholesterol-rich lipid mixture. The organic solvent was removed under a stream of nitrogen. The lipid/detergent film was dispersed in 5.0 ml i0 mM PBS to give a total lipid concentration (including cholesterol) of 1.2 mg/ml, and sonicated for I0 minutes in a bath sonicator (Lab Supplies, Hicksville, NY) until solubilization of the lipids was completed. The clear liquid was dialyzed in a teflon dialysis cell equipped with a Spectra/Por 2 membrane (MW cut-off 12-14,000) against 2 liters of PBS under nitrogen with 5 buffer changes over 48 hours. Cholesterol was determined colorim e t r i c a l l y (16). For phospholipid analysis a sample was extracted by the method of Bligh and Dyer (17) and phospholipid quantitated colorimetrically (18). Recovery of both total phospholipid and cholesterol was typically in the 70-80% range. Preformed envelopes were filtered through 0.22 ~m Acrodiscs and stored at 4°C. For the preparation of large stock volumes (~ i00 ml) of lipid envelopes, teflon cells were replaced with a counter-flow-through dialysis system consisting of two glass tanks and a hollow-fiber hemodialysis cartridge as described by Schwendener (19). In order to label the artificial envelopes with the fluorescent marker for cell interaction studies, FITC-dextran was added to PBS at a concentration of 23.6 mg/ml and vesicles formed as described above. U n i n c o r p o r a t e d marker was removed by filtration over a short Sepharose 300B column. The encapsulated marker concentration was determined following solubilization of a sample with 10% Triton-X i00, and determination of the fluorescence intensity using a PerkinElmer fluorescence spectrophotometer. The encapsulated concentration of FITC-dextran was generally in the range of 60-70 ~g/ml. The second step of the double dialysis procedure consisted of partial resolubilization of the preformed lipid envelopes with deoxycholate. Retention of the vesicular structure was monitored via laser light scattering (NICOMP Model 370; Particle Sizing Systems, St. Barbara, CA). Loss of the light scattering signal was taken as indicator for the conversion of vesicles to mixed lipid-detergent micelles (20) which occurred when >i0 mg deoxycholate per 2.5 mg lipid, corresponding to a detergent:lipid molar ratio of >8, was added. Accordingly, preformed envelopes were mixed under aseptic conditions with sodium deoxycholate to give a detergent:lipid ratio of 7.8. The partially solubilized envelopes were gently mixed with gpl60 and incubated for 45 minutes at room temperature. Typically, 2.5 mg total lipid were mixed with i00 ~g gpl60, corresponding to a

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total l i p i d / p r o t e i n m o l a r ratio of =7,000. The m i x t u r e was d i a l y z e d against Tris b u f f e r at 4°C under continuous p u r g i n g with n i t r o g e n under the same c o n d i t i o n s as d e s c r i b e d above and stored at 4°C. Samples were d i a l y z e d until homogeneous, i.e. until the size d i s t r i b u t i o n was unimodal (Gaussian distribution), indicating both the a b s e n c e of a m i x e d m i c e l l e fraction (bimodal distribution) and of unincorporated aggregated glycoprotein (wide polymodal distribution). Physical stability were m o n i t o r e d during storage over several m o n t h s by laser light s c a t t e r i n g (NICOMP Model 370). E l e c t r o n microscopy: Freeze fracture. Specimens were p r e p a r e d as d e s c r i b e d by M U l l e r et al. (21). A Balzers BA 360 was used for freeze fracture and replication. Replicas were e x a m i n e d with a Joel i00 CX e l e c t r o n m i c r o s c o p e operated at 60 kV. N e q a t i v e staining. This was p e r f o r m e d a c c o r d i n g to standard p r o c e d u r e s with 1% uranyl acetate. The grids were observed as above. Scanning. Specimens were adsorbed to p o l y - L - l y s i n e coated grids, washed, rapidly d e h y d r a t e d and dried with liquid CO 2. Samples were lightly coated with carbon by e v a p o r a t i o n and observed with a Hitachi S-400 field emission electron microscope. I m m u n o l a b e l i n q with anti gp160/41 antibody. Samples were a d s o r b e d to Formvar coated nickel grids and incubated for 1 hour on a 1:250 d i l u t i o n of the m o n o c l o n a l a n t i - H I V gp160/41 (Cellular Products, Inc., Buffalo, NY) or an irrelevant m o n o c l o n a l antibody. The m o n o c l o n a l antibody employed has been shown by W e s t e r n blot a n a l y s i s to recognize both HIV gp41 and gpl60 (22). Grids were incubated on a 1:20 d i l u t i o n of goat anti-mouse IgG coupled to 15 nm colloidal gold for 1 hour, n e g a t i v e l y stained and observed as d e s c r i b e d above. Control lipid envelopes w i t h o u t p r o t e i n were treated in a identical manner. Cell fusion assay. In order to exemplify the fusogenic ability of the artificial viral envelopes, two types of cells with varying c o n c e n t r a t i o n of CD4 receptors were employed, R E X - I B cells (=60% CD4 r e c e p t o r p o s i t i v e as d e t e r m i n e d by flow cytometry) or KG-I cells (=18% CD4 r e c e p t o r positive). Artificial envelope samples (I ml) with and w i t h o u t gpl60, all labelled with FITC-dextran, at a total lipid c o n c e n t r a t i o n of 1.5 mg/ml, were incubated with =2x106 of either cell type in 2 ml medium. Cells were p r o v i d e d by Dr. A.M. Miller, D i v i s i o n of Medical Oncology, D e p a r t m e n t of Medicine, U n i v e r s i t y of Florida. Samples were either w a s h e d immediately following m i x i n g and stored on ice (time "0"), or incubated at 37°C for 1 hour. Cells were c e n t r i f u g e d at 1,000 rpm at 5°C for 7 min, w a s h e d twice with 1 ml PBS, and r e s u s p e n d e d in 1.5 ml ice-cold PBS for f l u o r e s c e n c e d e t e r m i n a t i o n by flow c y t o m e t r y (FACSTAR PLUS, Becton-Dickinson). FITC-dextran of approximately equivalent c o n c e n t r a t i o n in PBS served as control. Results

Physical c h a r a c t e r i s t i c s of artificial viral envelopes. The double d e t e r g e n t dialysis employed g e n e r a t e d homogeneous, unilamellar v e s i c l e s in a size range of 150-300 nm, c o m p a r a b l e to natural e n v e l o p e d viruses. Freeze-fracture electron m i c r o s c o p y c o n f i r m e d the u n i l a m e l l a r structure of the artificial envelopes (Fig. IA). Fig. IB d e m o n s t r a t e s the intermediate state following partial r e s o l u b i l i z a t i o n of the lipid envelopes w i t h deoxycholate. Notable is a c h a r a c t e r i s t i c dumb-bell shaped s t r u c t u r e s w i t h rounded edges. A f t e r e x h a u s t i v e dialysis the vesicles r e v e r s e d to a spherical shape as shown in the scanning electron m i c r o g r a p h in Fig. iC.

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FIG.

485

1

A_I: Freeze fracture electron m i c r o g r a p h of envelopes after first dialysis step p r i o r to protein insertion. B." N e g a t i v e - s t a i n electron m i c r o g r a p h of p a r t i a l l y resolubilized envelopes p r e p a r e d for gpl60 insertion; C." Scanning electron m i c r o g r a p h of complete envelopes following gpl60 insertion and second dialysis step.

TABLE II Physical Stability of Artificial HIV Envelopes

Time post Preparation

Average Size ±

Size D i s t r i b u t i o n

Envelopes -gpl60 (Days)

0 2 6 9 17 60 69 73 163 347

Envelopes +gpl60

Sample 1

Sample 2

267 264 253 239 252

234 ±

± 137 ± 123 ± iii ± 92 ± 95

99

197 ± 89 162 ± 61

283 ± 112 285 ±

42 193 ± 69 286 ± 300 ±

(nm)

91 48

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HIV Envelopes

FIG.

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2

N e g a t i v e - s t a i n electron micrographs of envelopes following immunolabeling. A: gpl60-containing envelopes incubated with anti-HIV gp160/41 antibody and goat anti-mouse IgG coupled to 15 nm colloidal gold; B." the same envelopes incubated with irrelevant monoclonal antibody; C_I: envelopes without gpl60 incubated as in A. M a g n i f i c a t i o n 45,000x (insert in A 200,000x).

The r e p r o d u c i b i l i t y of the technique was remarkable, with an average diameter of 250 ± 26 nm (S.D. of the mean size) calculated from 15 experiments. Samples were always dialyzed until the size d i s t r i b u t i o n was homogeneous (Gaussian distribution). The physical stability of the artificial envelopes with and without gpl60 at 4°C as analyzed p e r i o d i c a l l y by laser light scattering varied very little, indicating prolonged physical stability (Table II). Immunolabelinq of artificial viral envelopes. Sandwich-immunolabeling with anti-gpl60/41 monoclonal antibody and colloidal goldcarrying mouse anti-IgG (Fig 2A and insert) demonstrated that gpl60 was available for binding of the anti-gpl60/41 monoclonal antibody. Fig. 2B shows the corresponding control with plain lipid envelopes, and Fig. 2C the complete envelopes incubated with unrelated monoclonal antibody. Cell fusion of artificial viral envelopes. When gpl60containing, FITC-dextran-labeled artificial viral envelopes were incubated with REX-IB and KG-I cells, respectively, flow cytometry indicated a shift in fluorescence intensity. While the mean fluorescence channel increased from 3.62 to 9.17 for REX-IB cells,

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the fluorescence intensity shift was much less pronounced for KG-I cells (3.09 to 5.42) (Table III). Fluorescence m i c r o s c o p y showed a large fraction of REX-IB cells stained h o m o g e n e o u s l y with the fluorescent dye, indicating cytosolic transfer via a fusion process. In contrast, only few KG-I cells showed staining under the fluorescence microscope (photographs not shown). There was no shift in fluorescence or cytosolic staining detectable when either type of cells was incubated with FITC-dextran in solution. Cell fusion of plain lipid envelopes (without aDl601. An unexpected finding was the strong interaction of plain lipid envelopes with both REX-IB and KG-I cells, resulting in a mean fluorescence channel shift from 3.62 to 22.49 (REX-IB), and 3.09 to 15.2 (KG-I), respectively (Table III). This interaction did not appear to be related to the relative difference of CD4 receptor concentration of the respective cells.

TABLE III Shift in Fluorescence Intensity of REX-IB and KG-I Cells Following Incubation with Artificial HIV Envelopes Variable

Incubation Time (h)

Mean Fluorescence REX-IB

(~60% CD4 ÷)

Channel KG-I

(Peak)

(=18% CD4 ÷)

+ gpl60

0

3.62

3.09

+ gpl60

1

9.17

5.42

gpl60

1

22.49

15.20

-

Fluorescence intensity was determined by flow cytometry. Envelopes were labeled with FITC-dextran. +gpl60 indicates complete artificial viral envelopes; -gpl60 indicates plain lipid envelopes (without protein present). For more details see Materials & Methods section.

Discussion

We have constructed an artificial envelope containing HIV gpl60, nearly identical to the natural HIV envelope with respect to size and unilamellar structure, lipid composition and cholesterol: lipid ratio, and insertion of gpl60 in the outer envelope surface. Employing a double detergent dialysis technique, preparation of the lipid envelopes and insertion of surface glycoprotein were performed as two separate steps. An important aspect of this method is that envelopes without protein can be stored for later insertion of the desired protein(s) as their physical stability is excellent. The method was remarkably reproducible with respect to vesicle size and size distribution, and flexible for preparation of batch sizes of $5 ml to ZI00 ml using either a 5 ml teflon dialysis cell or a hollow fiber dialysis apparatus. The excellent l o n g - t e r m p h y s i c a l stability

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was unexpected since conventional liposomes of comparable high cholesterol content grow upon storage (23).

size and

It appears that the gpl60 remained c o n f o r m a t i o n a l l y intact as anti-gpl60/41 monoclonal antibody binds to the envelopes' surface. Furthermore, when incubated with cells with different CD4 receptor c o n c e n t r a t i o n (-60% REX-IB; -18% KG-I), the e f f i c i e n c y of transfer of fluorescent marker appeared to correlate with the concentration of CD4 receptors. The finding that lipid envelopes without gpl60 apparently are highly fusogenic was seredipitous and may be of great consequence for the design of inherently fusogenic liposomes. The efficient transfer of encapsulated material to tissues or cells may be an advantageous property for use in therapy or in vitro for cell transfection. Liposomes have been engineered such that the bilayer is d e s t a b i l i z e d in a pH-dependent fashion and the contents are released into the c y t o p l a s m of the cell upon change of pH, by using a fatty a c i d / p h o s p h a t i d y l e t h a n o l a m i n e combination (24), a pHsensitive amphipathic peptide (GALA) (25), or a pH-sensitive lipid mixture consisting of dioleoylphosphatidylethanolamine and cholesteryl-hemisuccinate (26). However, no "natural" fusogenic mixture such as the one employed here has been reported. The potential usefulness of artificial viral envelopes containing viral surface glycoproteins is manifold. There is a great need for a safe and effective vaccine against HIV (27) and other viral infections. Insertion of a variety of natural epitopes or highly immunogenic conserved peptide residues may represent a new efficient way of a more natural presentation of viral antigens to the immune system. Intriguing alternative applications include their use as vehicles for receptor-targeted delivery of drugs, diagnostic agents, toxins, or genetic constructs. Studies are currently underway assessing the o p t i m u m surface glycoprotein density, the exact nature of the observed specific and nonspecific cell interaction, and the application of such vehicles as vaccines, and carriers for toxins (ricin-A) and plasmids (PCAT). Acknowledqements We thank F.T. Crews who provided laboratory space and equipment for R. Ch., R.A. Schwendener (Dept. of Nuclear Medicine, University of Zurich, Switzerland), for the blue-prints of the dialysis equipment, the NIH AIDS Research & Reference Reagent P r o g r a m for gpl60, A.M. Miller for REX-IB and KG-I cells, and M. Ausborn and S. GUnther for performing cell incubation studies. G. Erdos and B. O'Brien (ICBR EM Core Lab) performed the electron microscopic preparations, and N. Benson (ICBR Flow Cytometry Core Lab) the fluorescence analysis. Partial funding was provided to H.S. by a Florida High T e c h n o l o g y & Industry Council grant. R.Ch. was on e x t r a o r d i n a r y academic leave from the Bhabha Atomic Research Center, Bombay, India. References i.

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