Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity

European Journal of Pharmaceutical Sciences 22 (2004) 459–468

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity Atthachai Homhuan a , Sompol Prakongpan a , Prachak Poomvises b , Riks A. Maas c , Daan J.A. Crommelin d , Gideon F.A. Kersten e , Wim Jiskoot d,∗ a

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand Department of Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand c Central Institute of Animal Disease Control Lelystad (CIDC-Lelystad), P.O. Box 2004, Lelystad 8203 AA, The Netherlands d Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, Utrecht 3508 TB, The Netherlands e Research and Development Unit, Netherlands Vaccine Institute, P.O. Box 457, Bilthoven 3720 AL, The Netherlands b

Received 26 January 2004; received in revised form 26 April 2004; accepted 4 May 2004 Available online 2 July 2004

Abstract The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl glucoside (OG) followed by detergent removal. Biochemical analysis revealed that these virosomes contained both the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), with preserved biological activity. Acidic environment triggered the fusion between virosomes and chicken erythrocyte ghosts. Formation of ISCOMs was achieved by solubilizing phospholipids, cholesterol, envelope protein antigen and Quil A in Triton X-100. The ISCOM particles were formed by removal of the detergent. In each formulation the relative HN content correlated with the capability to agglutinate red blood cells. The immunogenicity of these lipid-based subunit vaccines was determined in chickens after subcutaneous immunization. The relative HN content of the subunit vaccines correlated with the haemagglutination-inhibition (HI) antibody titres. Virosomes prepared with Triton X-100 and ISCOMs offered high clinical protection (> 80%) upon challenge with virulent NDV. Virosomes prepared with OG yielded lower clinical protection despite high HI antibody titres. Virosomes with reduced antigen density showed poor immunogenicity and protection. In conclusion, ND virosomes and ISCOMs were found to be immunogenic and provided good protection. © 2004 Elsevier B.V. All rights reserved. Keywords: ISCOMs; Newcastle disease virus; Subunit vaccine; Virosomes

1. Introduction

Abbreviations: Chol, cholesterol; CMC, critical micelle concentration; DLS, dynamic light scattering; EPC, egg phosphatidylcholine; F protein, fusion protein; HBS, hepes-buffered saline; ISCOMs, immunostimulating complexes; HA, haemagglutination activity; HAU, haemagglutinating units; HI, haemagglutination-inhibition; HN, haemagglutininneuraminidase protein; ND, Newcastle disease; NDV, Newcastle disease virus; OG, n-octyl ␤-d-glucopyranoside; PBS, phosphate-buffered saline; PE, phosphatidyl ethanolamine; PyrPC, 1-hexadecanoyl-2-(1pyrenedecanoyl)-sn-glycero-3-phosphocholine ∗ Corresponding author. Tel.: +31 30 253 6970; fax: +31 30 251 7839. E-mail address: [email protected] (W. Jiskoot). 0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2004.05.005

Newcastle disease (ND) is one of the most serious contagious diseases affecting chickens worldwide. It causes great economic loss in developing countries and acts as a trade barrier (Westbury, 2001; King, 1996). The causative agent, Newcastle disease virus (NDV), is an enveloped RNA virus, belonging to the family of Paramyxoviridae. The viral envelope is composed of a lipid bilayer and two surface glycoproteins, namely HN (haemagglutinin-neuraminidase) and F (fusion) protein. HN is responsible for the attachment of the virus to receptor molecules on host cells. F protein plays a major role in fusion of the viral membrane with the cellular plasma membrane (Yusoff and Tan, 2001). These

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two membrane glycoproteins are the antigenic components that can elicit a protective immune response (Seal et al., 2000). Vaccination is the most effective method to control and prevent ND in poultry. A single immunization with live NDV vaccine at early age induces antibody titres which last for 2–6 months. When these titres become too low, immunization with inactivated NDV vaccine is carried out to ensure sufficient protection. Although live attenuated and inactivated whole virus vaccines have been used successfully, both types of vaccine have shown serious drawbacks. Live NDV vaccines have the disadvantage of causing respiratory distress under certain field condition and there is a potential for reversion to virulent strains with passage from bird to bird (Alexander, 2001). On the other hand, inactivated oil–emulsion vaccines generally result in local inflammation at the site of injection. Mineral oil also persists in tissues and there is a possibility of contamination by carcinogenic aromatic hydrocarbons (Yamanaka et al., 1993; Droual et al., 1990). Therefore, replacement of existing vaccines with a formulation that avoids all of these disadvantages – but still is sufficiently immunogenic – is desired. Lipid-based delivery systems such as virosomes and ISCOMs have been used in human and veterinary vaccines successfully (Glück and Metcalfe, 2003; Kersten and Crommelin, 2003). Virosomes or reconstituted viral envelopes antigenically resemble the intact virus, but do not contain the virus replication machinery. They therefore represent a useful system for presentation of antigens. Virosomes have a comparable dimension to the viral pathogen that the immune system evolved to combat (Morein and Simons, 1985). Reconstitution of viral membranes is usually based on solubilization of the viral envelope with a detergent, removal of the viral nucleocapsid by ultracentrifugation, and subsequent removal of the detergent from the supernatant. Two non-ionic detergents, Triton X-100 and octyl glucoside (OG), are commonly used in the reconstitution of viral membrane proteins (Bron et al., 1993). Triton X-100 has a low critical micelle concentration (CMC of about 0.24 mM), and is therefore difficult to remove by dialysis. OG has a relatively high CMC (23 mM) and is therefore much easier to remove. Virosomes reconstituted with these two detergents were reported to have different properties, such as protein incorporation efficiency and fusion activity (Bron et al., 1993; Metsikko et al., 1986). Immunostimulating complexes (ISCOMs) are generally prepared by solubilization of viral protein antigen in detergent, mixed together with Quil A and lipids (Barr and Mitchell, 1996). The detergent is removed and subsequently ISCOMs form spontaneously. A typical feature of ISCOMs is their spherical cage-like structure having a size of about 30–40 nm. Apart from the multimeric presentation of antigens in ISCOM particles, the built-in adjuvant effect contributed from Quil A gives ISCOMs strong capacity to induce high antibody and cell-mediated immune responses (Kersten and Crommelin, 2003).

In this study, we have examined the influence of different detergents, Triton X-100 and OG, on the physicobiological properties of virosomes. The effect of decreasing protein density in the virosomal membrane on the physicochemical and immunological characteristics was also studied. In addition, ISCOMs were produced by using Triton X-100 as detergent and they were characterized in the same way as virosomes. Chickens were immunized with lipid-based subunit vaccines. The immune response was assessed by measuring serum antibody levels and clinical protection after NDV challenge. It is demonstrated that virosomes and ISCOMs are immunogenic and provide good protection.

2. Materials and methods 2.1. Materials Egg phosphatidylcholine (EPC) and phosphatidylethanolamine (PE) were from Avanti Polar Lipids Inc. (Alabaster, USA). 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3phosphocholine (PyrPC) was from Molecular Probes (Eugene, USA). Cholesterol (Chol) and n-octyl ␤-d-glucopyranoside was purchased from Sigma (Zwijndrecht, The Netherlands). Triton X-100 was purchased from BDH Laboratory Supplies (Poole, UK). The hydrophobic resin Bio-beads SM-2 and the DC Protein Assay kit were obtained from Bio-Rad (Hercules, USA). N-[2-hydroxyethyl]piperazine-N -[2-ethanesulfonic acid (Hepes) was obtained from ACROS ORGANICS (NJ, USA). Quil A was supplied by Iscotec (Lulea, Sweden). All other chemicals were of analytical grade. 2.2. Chickens Male BABCOCK chickens (Kerd-Charoen Hatcheries, Bangkok, Thailand) were maintained in conventional cages, with feed and water ad libitum. The chickens were found to be free from maternal immunity to NDV. Guidelines and legislative regulations on the use of animals for scientific purposes of Mahidol University, Thailand were followed. 2.3. Virus NDV Clone-30 grown in the allantoic cavity of chicken embryos was kindly provided by Intervet International B.V. (Boxmeer, The Netherlands). The virus-infected allantoic fluid was concentrated by cross-flow ultrafiltration with a polyethersulfone filter with a pore size of 300 kDa. The virus was further purified by layering 8 vol. of concentrated virus on top of 4 vol. of 20% sucrose (w/w) in HBS (5.0 mM Hepes, 0.15 M NaCl, pH 7.4) and pelleting by ultracentrifugation (80,000 × g, 1 h, 4 ◦ C).

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2.4. Preparation of virosomes from Triton X-100 and octylglucoside To compare the effect of different detergents on the biophysical and immunological characteristics of virosomes, Triton X-100 and octyl glucoside (OG) were used to solubilize the NDV membrane as previously described for influenza virus with minor modifications (Stegmann et al., 1987; Bron et al., 1993). Sucrose-purified NDV was resuspended in HBS at a protein concentration of 5 mg/ml. Triton X-100 was added to a final concentration of 2% (w/v). In the same way, OG was added to a final concentration of 150 mM. The mixtures were incubated at room temperature for 1 h with gentle shaking. The suspension was then layered on a cushion of 20% sucrose (w/v) in HBS and ultracentrifuged for 90 min at 100,000 × g to remove nucleocapsid complexes. The optically clear supernatant above the sucrose solution was collected. Triton X-100 was removed by stepwise addition of methanol-washed SM2 Bio-Beads. Briefly, 80 mg of SM2 Bio-Beads was added to 1 ml of supernatant and incubated for 2 h on a shaking-device (1400 per min) at room temperature. Subsequently, two batches of 35 mg of Bio-Beads were added to the mixture within a time interval of 30 min while shaking was continued. The formed ND virosomes were collected. The remaining Bio-Beads were washed with 0.5 ml of HBS and the liquid was pooled with the virosomes. To remove OG, the supernatant was dialyzed overnight against HBS at 4 ◦ C. The thus formed virosomes were collected. High molecular weight aggregates present in virosome suspensions were removed by a gradient centrifugation. A discontinuous sucrose gradient of 1 ml 50% sucrose, 1 ml 40% sucrose, 2 ml 10% sucrose, 1 ml 5% sucrose and 4 ml HBS was prepared. Fifty ␮l of 50% (w/v) sucrose in HBS was added into 1 ml of virosomes in HBS, yielding a virosomal suspension in a 2.5% sucrose solution. The virosomes in 2.5% (w/v) sucrose were applied to the discontinuous sucrose gradient on top of interface between 5% sucrose solution and HBS. The gradient was then ultracentrifuged at 100,000 × g for 2 h at 4 ◦ C. The virosomes located at about 40% sucrose, were isolated and dialyzed overnight against HBS at 4 ◦ C. 2.5. Preparation of diluted virosomes Diluted virosomes were made of EPC, PE, Chol and viral membrane lipids in a 4:2:4:1 molar ratio by detergent dilution method as previously described (Jiskoot et al., 1986). In short, appropriate amounts of each lipid were dissolved in chloroform/methanol (2:1, (v/v)) in a round bottom flask and a lipid film was obtained by solvent evaporation with a rotavapor under reduced pressure. The film was solubilized in the viral membrane solution containing 150 mM OG. The resulting mixed micelles were rapidly diluted 11-fold in HBS, allowing the formation of lipid vesicles (diluted virosomes). Subsequently, diluted virosomes were pelleted by

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ultracentrifugation (370,000 × g, 1 h) and resuspended in HBS. The diluted virosomes were then dialyzed overnight against HBS at 4 ◦ C.

2.6. Preparation of ISCOMs ISCOMs were prepared by using classical centrifugation method with some modifications (Barr and Mitchell, 1996). Lipid film composed of PE and Chol at a weight ratio of 1:1 was prepared by evaporating the chloroform and methanol from the lipid solution. Then the lipid film was solubilized in viral membrane solution containing 2% (w/v) Triton X-100. Quil A was added as 10% w/v solution to give a ratio between lipid and Quil A of 1:2 by weight. The mixture was incubated at room temperature for 1 h. Triton X-100 was removed by vigorously shaking with SM2 Bio-Beads as described above. Thereafter the formed ISCOMs were collected with a needle and syringe. To remove free compounds from ISCOM particles, sucrose gradient centrifugation was applied. Six blocks of sucrose layers, composed each 1.5 ml of 60-50-40-30-20-10% sucrose (w/v) in HBS were prepared. The ISCOMs sample was layered onto the top of the discontinuous sucrose gradient and ultracentrifuged at 50,000 × g for 18 h at 4 ◦ C. ISCOMs, positioned between 30 and 40% sucrose, were isolated and dialyzed overnight against HBS at 4 ◦ C.

2.7. Characterization of virosome, diluted virosome and ISCOM formulations The mean hydrodynamic diameter of virosomes, diluted virosomes, and ISCOMs was measured by dynamic light scattering (DLS) at 25 ◦ C with a Malvern 4700 system equipped with a 75 mW Argon ion laser (488 nm, Uniphase, San José, CA, USA), a remote interface controller and PCS software, version 1.35 (Malvern Ltd., Malvern, UK). The particle size distribution was reflected by a polydispersity index ranging from 0.0 for entirely monodisperse particles, up to 1.0 for heterodisperse particles. Each preparation was measured after production and after storage at 4 ◦ C for 3 months. The morphology of virosomes, diluted virosomes, and ISCOMs structure was analyzed by transmission electron microscopy. The formulations were adsorbed to carbon-stabilized formvar-coated grids, negatively stained using 2.0% potassium phosphotungstate (pH 5.2) and analyzed using a Philips TEM 400 electron microscope at an operating voltage of 80 kV. The protein content of virosomes and ISCOMs was determined according to Bio-Rad DC protein assay with bovine serum albumin (Pierce, Rockford, IL) as a relative standard. The phospholipid content was determined according to Rouser et al. (1970) with sodium phosphate (Merck, Darmstadt, Germany) as standard.

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2.8. SDS–PAGE and Western immunoblotting

as:

Protein composition was determined by SDS–PAGE. A 12.5% acrylamide gel was used and the protein was visualized by Coomassie Brilliant Blue staining. For Western blotting, following SDS–PAGE the separated proteins were electrophoretically transferred onto nitrocellulose membranes at 100 V for 90 min. Then the membrane was blocked with 0.5% protifar (Nutricia, Zoetermeer, The Netherlands) in PBS (10 mM Na2 HPO4 , 0.15 M NaCl) containing 0.05% Tween 20. The membrane was then incubated with IDNDV 134.1 (CIDC, Lelystad, The Netherlands), a monoclonal antibody directed against HN-protein of NDV, diluted 1:35 (v/v) in PBS-Tween 20 for 1 h. The membrane was washed, and incubated in diluted 1:2,500 (v/v) goat anti-mouse horseradish peroxidase conjugate (GAMPO, BioRad) in PBS-Tween 20 for 1 h. After washing, n-chloronaphtol (Sigma, Zwijndrecht, The Netherlands), a substrate solution was used as colour reagent.

f = 100

2.9. Haemagglutination activity (HA) Haemagglutination assay was performed using 1% v/v chicken erythrocytes in PBS. Serial five-fold dilution of virosomes, diluted virosomes, and ISCOMs in PBS was set up in a U-shaped 96-wells microtitre plate. An equal volume of 1% red blood cells was added to each well. The plate was gently shaken to mix the contents of each well and incubated for 60 min at 4 ◦ C. Sedimentation of the cells was visually assessed in each well. The HA was expressed as haemagglutinating units (HAU)/␮g protein, where HAU was defined as the lowest sample dilution able to inhibit the sedimentation of red blood cells. 2.10. Virosome fusion assay The extent of membrane fusion was carried out as described before (Stegmann et al., 1993). Briefly, virosomes labelled with the fluorescent probe pyrPC (10 mol% relative to the total amount of lipid) at a concentration of 5 ␮M of phospholipid were mixed with chicken erythrocyte ghosts (100 ␮M of phospholipid). The virosome mixture was placed into a cuvette and stirred at a temperature of 37 ◦ C. After 2 min, the pH of the medium in the cuvette was lowered to 5.5 and 4.0 by addition of 70 ␮l of 0.1 M Mes (morpholinoethane sulfonic acid)-0.2 M acetic acid, pretitrated with NaOH and HCl, respectively. Fusion was continuously monitored at 37 ◦ C by measuring the decrease in pyrPC excimer fluorescence with a LS50B fluorescence spectrophotometer (Perkin-Elmer, Beaconsfield, UK) set at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The decrease of fluorescence was expressed relative to the difference in the initial fluorescence and the excimer fluorescence at ‘infinite’ dilution, which was obtained by adding 70 ␮l of 10% (w/v) Triton X-100 in HBS. Fusion extents (f) were calculated

(E0 − Et ) (E0 − E∞ )

where Et represents the excimer fluorescence intensity at time (t), and E0 and E∞ represent the intensities at time zero and after addition of Triton X-100, respectively, corrected for dilution. 2.11. ELISAs for NDV-HN and NDV-F Antigenicity of ND-virosomes, diluted virosomes, and ISCOMs was tested by ELISA as previously described (Maas et al., 2000). In short, samples were serially diluted in 96-well flat-bottom plates that were coated with 1 ␮g/ml anti-HN monoclonal antibody (IDNDV134.1) or 1 ␮g/ml anti-F monoclonal antibody (IDNDV133.1, CDIC, Lelystad, The Netherlands). After incubation for 2 h at room temperature, plates were washed and incubated for 1 h at room temperature with horseradish peroxidase conjugated-IDNDV134.1 or IDNDV133.1. Plates were washed and the colour reaction was performed. The reference ND antigen, formalin-inactivated NDV strain Ulster (CIDC, Lelystad, The Netherlands), was also included in each plate. The results are shown as the ratio between relative ELISA titre and the protein concentration determined by Bio-Rad DC protein assay. 2.12. Immunization and virus challenge Six groups each of 15 chickens were used. The five vaccinated groups received a single dose of 0.5 ml of vaccine injected subcutaneously when 4 weeks of age. Birds in group 1 received commercial oil emulsion vaccine (INACTIVAC® CHICK-ND, Maine Biological Laboratory, USA). Birds in group 2, 3, 4, and 5 received virosomes prepared with Triton X-100, virosomes prepared with OG, diluted virosomes, and ISCOMs, respectively. The dose of subunit vaccines tested was maintained at 30 ␮g viral protein. Chickens in the sixth group received no vaccine. Four weeks after vaccination, all birds were challenged by oral administration of a virulent strain of NDV with an approximate dose of 104 EID50 per bird. After challenge, birds were observed daily for clinical signs of ND. The protective efficacy was calculated by dividing the number of chickens that survived without showing any clinical evidence of ND during 14 days by the total number of challenged chickens. Blood samples were collected for serological analysis 1, 2, 3, and 4 weeks after vaccination and from the survivor chickens 2 weeks after challenge. After heat treatment at 56 ◦ C for 30 min, sera samples were stored at −20 ◦ C and subjected to the HI test for assessment of antibody level against ND.

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2.13. Haemagglutination inhibition (HI) assay The HI tests were performed by standard microtitre plate method as essentially described by Alexander (1988). In brief, dilution series of sera were incubated with four HAU of NDV La Sota strain at room temperature for 30 min. The HAU was titrated before each assay. Thereafter, chicken erythrocytes were added and agglutination was monitored after incubation at room temperature for 45 min. The HI titre was defined as the reciprocal of the highest serum dilution completely inhibiting agglutination. 2.14. Statistical methods HI antibody titres of the groups were expressed as geometric mean titre. Analysis of variance was used for statistical evaluation of the data. The significance of the differences between geometric mean antibody titres was determined by the Fisher’s least-significant-difference test at a confidence level of 95%.

3. Results 3.1. Preparation and characterization of virosomes and ISCOMs Following the classical procedure for reconstitution of viral envelopes (Bron et al., 1993), ND virosomes from viral extracts containing surface proteins and endogenous viral lipids were prepared. Two non-ionic detergents, Triton X-100 and OG, were used as membrane solubilizer to determine the effect of different detergents on structural, biophysical, and immunological properties of virosomes. The particle size of the virosomes prepared with both detergents as measured by DLS was comparable. However, virosomes reconstituted from OG-solubilized viral membrane showed a wider size distribution as reflected

Fig. 1. SDS–PAGE of NDV (lane 1), solubilized viral membrane proteins (lane 2), nucleocapsid and matrix protein (lane 3), virosome formulation (lane 4), and ISCOMs formulation (lane 5). In panel is indicated the presence of two membrane proteins, HN and F, at molecular weight of about 70 and 60 kDa. The molecular weight (kDa) of reference proteins is indicated at the right (M).

by a higher polydispersity index (as shown in Table 1). The formation of virosomes was confirmed by electron microscopy, which showed images of spherical particles with external spikes protruding from their membranes (not shown). ND virosomes reconstituted with OG yielded a lower protein:phospholipid ratio (ca. 1.5 (w/w)) than virosomes prepared with Triton X-100 (ca. 2.1), as shown in Table 1. Decreasing protein density in virosomal membrane by addition of exogenous lipids (diluted virosomes), as expected, exhibited a lower protein:phospholipid ratio (ca. 0.3) than non-diluted virosomes. The data obtained from DLS revealed the formation of particles with an average size of 120 nm, slightly smaller than non-diluted virosomes (see Table 1). ISCOMs prepared with Triton X-100 solubilized viral membranes bore all the classic characteristics of ISCOMs. Electron microscopy showed typical cage-like structures

Table 1 Physicochemical characteristics and haemagglutination activity of the formulations used in this study Formulation

Virosomes prepared with Triton X-100 Virosomes prepared with OG Diluted virosomes ISCOMs a

Particle size (nm), PDa

Protein/phospholipid ratio (w/w)b

Relative content/ ␮g protein

Haemagglutination activity (HAU/␮g)d

HNc

Fc

2.05 ± 0.33

2.1 ± 0.3

2.6 ± 0.0

3311

176, 0.41

1.49 ± 0.59

2.7 ± 0.3

0.4 ± 0.0

2289

125, 0.28 44, 0.26

0.32 ± 0.02 0.55 ± 0.33

0.3 ± 0.0 0.6 ± 0.3

0.0 ± 0.0 1.3 ± 0.3

211 1366

After preparation

After storage for 3 months at 4 ◦ C

155, 0.19

165, 0.20

157, 0.36 117, 0.26 40, 0.27

Polydispersity index (see Section 2). Calculated using an approximate molecular weight of phospholipid of 750. Data represent mean ± S.D. of three different samples for each preparation. c Relative content (relative to that of reference antigen, inactivated NDV strain Ulster) as determined with IDNDV 134.1 and 133.1 monoclonal antibody in an ELISA divided by protein content. The results are shown as mean value (±upper/lower value of duplicate means). d Haemagglutination activity was defined as the highest dilution of samples able to inhibit the sedimentation of red blood cells; the haemagglutination titre was expressed as HAU/␮g protein. b

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with a mean diameter of 35 nm. DLS confirmed the presence of particles having an average size of 37 nm with a polydispersity index of 0.25 (Table 1). Because of the addition of lipids, Chol and Quil A necessary for the formation of ISCOMs, the ISCOMs had a lower protein:phospholipid ratio (ca. 0.6) compared to that of virosomes. The physical stability of the virosomes and ISCOMs after storage at 4 ◦ C for 3 months was studied by DLS. The size and polydispersity of these lipid-based subunit vaccines had not changed significantly (Table 1). This finding implies good physical stability of these subunit vaccines. SDS–PAGE of the virosomes and ISCOMs revealed protein composition as shown in Fig. 1. Unlike the parent NDV, virosomes and ISCOMs contain only two prominent

protein bands corresponding to HN and F protein. To examine the identity of HN, the formulations were analyzed by Western-immunoblotting with the use of a monoclonal antibody against HN protein. The results confirmed that the protein band visible on SDS–PAGE at about 70 kDa (Fig. 1) truly represents glycoprotein HN (results not shown). To ensure the presence of HN and F protein in the formulations and to compare their relative quantity, an HN-specific as well as an F protein-specific ELISA was done. The relative ELISA titre divided by the protein concentration was used as an arbitrary unit for the relative antigen contents of the formulations. Virosomes prepared with either Triton X-100 or OG showed a relatively high HN content (2.1 and 2.7, respectively) (Table 1), whereas diluted virosomes as well

40 35

% Fusion

30

pH 4.0

25 20

pH 5.5

15

pH 7.4

Acidification of buffer

10 5 0 0

1

2

3

4

5

6

7

8

Time (min)

(a) 40 35

% Fusion

30

pH 4.0 25 20

Acidification of buffer

15

pH 5.5

10

pH 7.4

5 0 0

(b)

1

2

3

4

5

6

7

8

Time (min)

Fig. 2. Extent of fusion between pyrPC labelled virosomes and erythrocyte ghosts. The pyrPC labelled virosomes prepared with Triton X-100 (panel a) or OG (panel b) were incubated with excess chicken erythrocyte ghosts at 37 ◦ C under different pH conditions with continuous stirring. Data are representative for three repeated assays.

<1 (0/15) 1.20 ± 0.20 (5/15) <1 (0/15) <1 (0/15) 1.0 (1/15) <1 (0/15)

30 ␮g protein/dose for virosomal and ISCOM vaccines; 0.5 ml dose for W/O emulsion vaccine. Antibody response measured by HI assay represented as geometric mean titre, log2 ± S.E.M. Groups, which are not statistically different at the level of α = 0.05 are indicated by the same letter for each separate column. c Challenge was done by oral inoculation of virulent NDV with an approximate dose of 104 EID . 50 b

a

43 80 20 9.30 ± 0.33 (6) 8.04 ± 0.63 (12) 2.00 ± 0.88 (3) <1 (0/15) 3.73 ± 0.38B (15/15) <1 (0/15)

1.44 ± 0.24 (9/15) 1.14 ± 0.14 (7/15)

<1 (0/15) 3.27 ± 0.32B (15/15) <1 (0/15)

8.76 ± 0.63 (10) 4.40 ± 0.34A,B (15/15)

3.40 ± 0.41 (15/15) 1.20 ± 0.20 (5/15) 1.25 ± 0.25 (4/15) 1.0 (1/15)

4.20 ± 0.17A (15/15)

100 93 4.93 ± 0.30A (15/15) 4.47 ± 0.13A,B (15/15) 5.33 ± (15/15) 4.27 ± 0.23A (15/15)

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W/O emulsion vaccine Virosomes prepared with Triton X-100 Virosomes prepared with OG Diluted virosomes ISCOMs Negative control

2

3

0.25C

4

9.47 ± 0.29 (15) 9.01 ± 0.48 (14)

Antibody response 2 weeks post challengeb (number of survivors) 1

The immunogenicity of virosomes and ISCOMs was studied in chickens. All subunit vaccines (virosomes, diluted virosomes, and ISCOMs) containing 30 ␮g protein were tested, together with 0.5 ml dose of commercial W/O emulsion vaccine as positive control. The choice of 30 ␮g viral protein for subunit vaccines was based on a preliminary study, where the protein content was variable (10, 30, and 50 ␮g). We found that 30 ␮g protein was the optimal dose for the virosomal vaccine (unpublished data). One and 2 weeks after immunization with subunit vaccines, low antibody levels had developed in chickens’ sera (See Table 2). However, 3 and 4 weeks after vaccination the groups having received virosomes prepared with Triton X-100 and virosomes prepared with OG showed similarly high HI titres (see Table 2). Serum HI antibody titres in these groups showed no tendency to decline four weeks after vaccination. Chickens injected with diluted virosomes, in contrast, did not develop HI antibodies throughout the experiment (HI titre < 1). ISCOMs elicited lower HI antibody responses than virosomes did. The commercial W/O emulsion vaccine induced the highest HI titres, although titres four

Antibody response at weeks after vaccinationb (Number of responders/total)

3.3. Immunogenicity of virosomes and ISCOMs

Formulation

A lipid-mixing assay was performed to determine whether virosomes prepared from different detergents have similar fusogenic properties. The fluorescence reporter molecules, pyrPC, were co-reconstituted in the virosomal membrane during reconstitution of viral envelopes. Chicken erythrocyte ghosts were used as a model biological target membrane. Lipid mixing between labelled virosomes and an excess of erythrocyte ghosts was monitored continuously at different pH values, i.e. 7.4, 5.5 and 4.0. The results are shown in Fig. 2. Virosomes prepared with either Triton X-100 or OG displayed a similar fusion activity being little at pH 7.4 (cf. panels a and b of Fig. 3). Decreasing pH resulted in an increase of the fusogenic activity of virosomes.

Table 2 Serum antibody responses and protection after challenge in chickens vaccinated subcutaneously with various subunit ND vaccine formulationsa

3.2. Fusogenic activity of virosomes

Challengec percentage protected

as ISCOMs (to which exogenous lipids were added during preparation) showed much lower values (0.3 and 0.6, respectively). Virosomes prepared with Triton X-100 and ISCOMs had a relatively high F protein content (2.6 and 1.3, respectively). In contrast, virosomes prepared with OG-solubilized viral membrane showed a relatively low F protein content (0.4) and the F protein content of diluted virosomes was undetectable. We characterized the functional feature of HN incorporated in virosomes, diluted virosomes, and ISCOMs by an HA assay. The HA of virosomes prepared with Triton X-100 ranked the best, followed by virosomes prepared with OG (see Table 1). Decreasing protein density in virosomal membrane (diluted virosomes) caused a considerable decrease of HA. ISCOMs exhibited an HA between that of virosomes and diluted virosomes.

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weeks after vaccination with W/O emulsion vaccine were not significantly higher (P < 0.05) as compared with virosomes. All unvaccinated chickens were negative for NDV antibody (HI titre < 1) throughout the experiment. In the challenge experiment, the protection level in chickens vaccinated with W/O emulsion vaccine was 100%. Most of the control (unvaccinated) birds did not survive the challenge and exhibited 80% mortality (Table 2). Virosomes prepared with Triton X-100 gave 93% protection, whereas virosomes prepared with OG yielded lower protection (67%). Although chickens receiving diluted virosomes were found to be free of HI antibody, six out of fourteen birds tolerated the challenge. The ISCOM formulation offered a high protection (80%), even though the HI antibody titres were not as high as those induced by virosomes. The antibody levels in challenged and protected chicken are very high. Even the 43% surviving chicken receiving ‘diluted’ virosomes respond strongly, although they had no detectable antibodies when challenged.

4. Discussion Developing subunit vaccines containing only antigens relevant for protective immunity has been extensively investigated (Singh and O’Hagan, 2003; Bowersock and Martin, 1999). Antigens in monomeric form are usually poorly immunogenic and therefore require efficient antigen delivery systems (Morein and Simons, 1985). Virosomes and ISCOMs have been reported as excellent vaccine delivery vehicles for improving the immunogenicity of purified viral surface proteins (Huckriede et al., 2003; Kersten and Crommelin, 2003; Glück and Metcalfe, 2003). In the present work we describe a systematic approach for the reconstitution of HN and F protein derived from NDV in virosomes and ISCOMs. Previous reports have detailed different functional characteristics of virosomes reconstituted with Triton X-100 and OG. As for vesicular stomatitis virus, reconstituted virosomes based on OG removal by dialysis did not display fusion activity (Metsikko et al., 1986). Our data revealed similar structural characteristics of virosomes prepared with either Triton X-100 or OG. Functionally, these virosomes retained both HA and fusogenic properties to a comparable degree. The degree of membrane fusion between virosomes and chicken erythrocyte ghosts was pH dependent: the more acidic the environment, the more fusion was observed. This finding is similar to results from previous studies reporting acidic pH triggered fusion between NDV and target cell membrane (Román et al., 1999; Trybala, 1987). However, the optimum pH for NDV fusion with erythrocyte ghosts has been found to be 7.4 (Cobaleda et al., 1994), whereas in the present study we observed the fusion to be highest at pH 4.0. It is assumed that the entry into target cells of paramyxovirus like NDV is a pH-independent mechanism that occurs only by fusion of the viral membrane with the host cell plasma membrane

(White et al., 1983; Nagai et al., 1983). On the other hand, viruses that use the endocytic pathway like influenza virus show a significant enhancement of fusion at low pH (Glück and Metcalfe, 2003; Bungener et al., 2002). Taken together, these contradictory findings may suggest that NDV is able to enter the cell by an endocytic route, although direct fusion of the viral NDV envelope with the host cell plasma membrane would be the main pathway for viral entry. Although F protein-specific ELISA determination indicated that virosomes prepared with Triton X-100 had relatively higher F protein contents than virosomes prepared with OG, the degree of fusion between these virosomes was similar. It is possible that the arrangement of F protein in virosomes prepared with OG is such that it is less accessible for binding with specific F-antibody in ELISA, but the F protein can still undergo fusion with the target membrane. Another possible explanation is that a high F protein density in the virosomal membrane is not critical for membrane fusion. It remains to be seen whether one F protein is enough to induce fusion or the concerted action of several F proteins is needed for inducing membrane fusion. ISCOMs, in which the antigens are presented in multimeric form together with a built-in adjuvant, were also characterized and their immunogenicity was determined. In this study, we have used Triton X-100 to solubilize the viral membrane. By using ultracentrifugation the ISCOM particles were formed. SDS–PAGE analysis revealed two major protein bands corresponding to the HN and F proteins on the basis of molecular weight as similar as virosomes. These results are in agreement with recent studies, showing that the HN and F proteins were incorporated in reconstituted ND liposomes (Cobaleda et al., 2001; Kapczynski and Tumpey, 2003). ISCOMs and diluted virosomes elicited lower HA as compared to that of undiluted virosomes. This is likely to be a consequence of the lower protein density resulting from the addition of exogenous lipids. Interestingly, we found a correlation between relative HN content as determined by ELISA and HA (Table 1). Diluted virosomes displayed a low HA, in parallel with their low relative HN content. The dilution effect of larger amount of lipids may cause more surface protein to be oriented inward in the bilayer of virosomes, which would result in less receptor binding and lower ELISA titres. A correlation between mean serum HI antibody levels and the relative HN content in the formulation was found in this study. ELISA of virosomes prepared with either Triton X-100 or OG revealed relatively high HN contents, which corresponded with high serum HI antibody titres. Reduced antigen density by addition of extra lipids in ISCOMs and diluted virosomes accounted for much lower relative HN contents. Consequently, the HI antibody titres after immunization with ISCOMs was lower than that of virosomes whereas no serum HI antibodies were detected after immunization with diluted virosomes. This finding is in close agreement with a previous study in that the serological response of

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vaccinated chickens correlated with the HN content of inactivated NDV vaccines (Maas et al., 2003). Four weeks after vaccination HI antibody titres elicited by virosomes were comparable with those raised by the commercial W/O emulsion vaccine. Geometric mean HI titres ranging from 2 log2 to 5 log2 have been proposed as a good predictor for clinical protection of birds immunized with inactivated vaccine (Allan et al., 1978; Goddard et al., 1988). Indeed, the mean serum HI antibody titres of groups receiving the W/O emulsion vaccine (4.93 log2 ) and virosomes prepared with Triton X-100 (4.47 log2 ) correlated with a high level of protection against virulent NDV challenge. However, virosomes prepared with OG yielded only 67% protection though the mean serum HI titres were comparable to virosomes prepared with Triton X-100. This might be explained by the much lower relative F protein content in the virosomal vaccine prepared with OG and indicates that the presence of both HN and F protein in the vaccine is important for vaccine efficacy. The results of the present study support the notion that antibodies against F protein contribute significantly to effective protection and that the design of effective paramyxovirus vaccines requires the presentation of not only HN but also the F protein (Merz et al., 1980). Diluted virosomes offered the lowest clinical protection. It has been postulated that viral epitope organization is a major attribute for inducing B-cell response (Bachmann et al., 1993). The repetitive antigenic determinants at a spacing of 5–10 nm are unique to viral antigens and therefore the immune system has evolved to respond strongly to this arrangement of epitopes (Chackerian et al., 2002; Huckriede et al., 2003; Bachmann and Zinkernagel, 1996; Bachmann et al., 1993). Decreasing protein density in the virosomal membrane (diluted virosomes) might result in loss of the highly organized viral surface structure, which in turn makes the virosomes fail to induce serum HI antibodies and provide clinical protection. On the other hand, virosomes in which the protein was more densely packed did induce a strong HI antibody response and offered better protection. The chickens that had received ‘diluted’ virosomes and survived had comparable titres, although they showed no detectable antibody titres prior to challenge (see Table 2). This seems surprising but it may be that these 43% of the group had a very fast booster response. The absence of antibodies during the booster prevents virus neutralization. Therefore, the effective dose may be higher in this group, resulting in a more pronounced booster effect during infection (from non-detectable to 9.30 log2 ). In the other groups the challenge virus will be (partly or completely) neutralized and as a result the booster effect, although still very considerable, is much smaller: from between 4 and 5 to about 9 log2 (see Table 2). ISCOMs induced lower serum HI antibody levels than virosomes prepared with OG, but a high protection (80%) was achieved after challenge with virulent NDV. The relatively high F protein content together with the presence of HN

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found in the ISCOM formulation, together with the presence of Quil A, may be responsible for this. Moreover, the capacity of ISCOMs to induce cell-mediated immunity is well documented (Kersten and Crommelin, 2003). Accordingly, the induction of cell-mediated immunity in addition to antibody responses may contribute to the high clinical protection in vaccinated birds. In conclusion, we have characterized biophysical properties of virosomes and ISCOMs and have shown that they are immunogenic in the target species. Immunization with virosomes or ISCOMs provides protection from lethal NDV challenge. The detergent chosen for isolation and reconstitution of ND viral membrane had a considerable impact on the properties of the ND virosomes. The use of Triton X-100, rather than OG, to solubilize ND viral membrane, followed by controlled detergent removal using polymer beads adsorption is the preferred method for obtaining ND virosomes containing a high content of HN and F protein. Moreover, a high density of functional antigens on the virosomal membrane appears to enhance their immunogenicity. The outcome of this study may be useful for further development of other enveloped veterinary or human virus vaccines.

Acknowledgements This study was supported by a grant from the Royal Golden Jubilee-Thailand Research Fund. We thank Intervet International B.V. (Boxmeer, The Netherlands) for providing the lentogenic ‘Clone-30’ strain. We are grateful to Dr. I.J.T.M. Claassen (CIDC-Lelystad) for his valuable guidance.

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