Incorporation of soluble antigens into ISCOMs: HIV gpl20 ISCOMs induce virus neutralizing antibodies Michael Browning*, G e o r g e Reid, R o b e r t O s b o r n e and O s w a l d Jarrett
Through a process of covalent attachment of palmitic acid, we have incorporated recombinant gp120 of H I V strain IIIB into ISCOMs. Rabbits immunized with 1SCOMs incorporating 10 #g gp120 produced high levels of gp120-specific antibody, comparable to the response to ten times as much antigen in complete Freund's adjuvant. The ISCOM-induced antisera showed virus neutralizing activity against the homologous strain, but failed to neutralize two heterologous strains of HIV-1. The antisera recognized non-conformationally determined epitopes on gp120, and antibody binding to gp120 was affected by glycosylation of the viral glycoprotein. Keywords:ISCOM; Human ImmunodeficiencyVirus type 1; viral glycoprotein;virus neutralizing antibody
INTRODUCTION Immunostimulating complexes (ISCOMs) are stable particulate complexes of protein antigens incorporated into cage-like structures consisting of the adjuvant glycoside Quil A and lipid 1. ISCOMs have been used as a delivery system for amphipathic glycoproteins from a variety of enveloped viruses, and have been shown to induce protective immune responses to several of these, including measles virus, influenza virus and feline leukaemia virus 2-4. Presentation of viral antigens in ISCOMs greatly enhances their immunogenicity for the induction of antibody responses as compared with antigen presented in micellar form or in the virus particle x. In addition, recent work s'6 has shown that ISCOMs elicit antigen-specific cytotoxic T-lymphocyte (CTL) responses in immunized animals. The ability of ISCOMs to stimulate both the humoral and cellular limbs of the anti-viral immune response in a naive host may be of relevance to the selection of antigen delivery systems in the design of anti-viral vaccines. One of the limitations of ISCOMs as a delivery system for viral antigens has been the requirement for a hydrophobic or membrane-spanning region in the protein of interest to permit incorporation into ISCOMs. This has limited both the range of antigens which may be incorporated and also the antigenic purity of the resulting complex. We have developed a process where, through covalent attachment of fatty acids, we are able MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow G61 1QH, UK. *To whom correspondence should be addressed at: ICRF Cancer Immunology Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. (Received 9 September 1991; revised 19 December 1991; accepted 21 January 1992) 0264-410X/92/090585-06 © 1992Butterworth-Heinemann Ltd
to incorporate soluble proteins into ISCOMs. By this method, ISCOMs have been prepared incorporating ovalbumin 6, cytochrome C and Tamm-HorsfaU glycoprotein 7. In this study we have used this method to incorporate the external viral glycoprotein (gpl20) of HIV-1, expressed as a recombinant protein in mammalian cells, into ISCOMs. Immunization of rabbits with gpl20-ISCOMs induced high levels of gpl20-specific antibody. Sera from these animals showed virusneutralizing activity when tested against the homologous strain (IIIB) of HIV-1. MATERIALS AND METHODS
Materials Recombinant gpl20 (rgpl20) of HIV-1 strain IIIB produced by mammalian cells (Celltech, UK ) or by insect cells (American Biotechnology, USA) was obtained through the MRC AIDS Reagent Project. These preparations were > 90% pure and < 20% cleaved when examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Palmitic acid was used as the N(palmitoyloxy) succinimide derivative (Sigma). I SCO Ms were prepared with Quil A (Coopers Animal Health Ltd, U K ) and lipid mix consisting of an equal concentration (10mgm1-1) of cholesterol and phosphatidyl choline in 20% Mega-10 (Sigma).
Preparation of gpl20 ISCOMs The method for incorporation of soluble antigens into ISCOMs is described in detail elsewhere 7. The preparation of rgpl20 ISCOMs is illustrated in Figure /. Briefly, mammalian cell-derived rgpl20 was mixed with palmitic acid at a molar ratio of 1:40 in the presence of the detergent deoxycholate. The mixture was shaken
Vaccine, Vol. 10, Issue g, 1992 585
HIV gp120 ISCOMs: M. Browning et al. gp120
~
4
N- (palmitoyloxy)succinimide
:
(20g d e o x y c h o l a t e )
[
Shake ON; 16°C
I
0.1~ d e o x y c h o l a t e
pH 8.6
Quil A (1 mg/ml) lipid mix
J
Dialy~"
Anti-gpl20 antibody enzyme immunoassay
I
Tris-HCI pH 8.6 I
I0-40~ sucrose density gradient 35K, 20°C, 16 h
~- F r a c t i o n s a s s a y e d - Q u i l A - gp120
J
Pooled f r a c t i o n s - EM
Figure 1 Preparation of rgp120 ISCOMs.The inset shows an electron micrograph of ISCOMs prepared using soluble protein antigen
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200
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Through a process of covalent attachment of palmitic acid, we have incorporated recombinant gp120 of H I V strain IIIB into ISCOMs. Rabbits immunized with 1SCOMs incorporating 10 #g gp120 produced high levels of gp120-specific antibody, comparable to the response to ten times as much antigen in complete Freund's adjuvant. The ISCOM-induced antisera showed virus neutralizing activity against the homologous strain, but failed to neutralize two heterologous strains of HIV-1. The antisera recognized non-conformationally determined epitopes on gp120, and antibody binding to gp120 was affected by glycosylation of the viral glycoprotein. Keywords:ISCOM; Human ImmunodeficiencyVirus type 1; viral glycoprotein;virus neutralizing antibody
INTRODUCTION Immunostimulating complexes (ISCOMs) are stable particulate complexes of protein antigens incorporated into cage-like structures consisting of the adjuvant glycoside Quil A and lipid 1. ISCOMs have been used as a delivery system for amphipathic glycoproteins from a variety of enveloped viruses, and have been shown to induce protective immune responses to several of these, including measles virus, influenza virus and feline leukaemia virus 2-4. Presentation of viral antigens in ISCOMs greatly enhances their immunogenicity for the induction of antibody responses as compared with antigen presented in micellar form or in the virus particle x. In addition, recent work s'6 has shown that ISCOMs elicit antigen-specific cytotoxic T-lymphocyte (CTL) responses in immunized animals. The ability of ISCOMs to stimulate both the humoral and cellular limbs of the anti-viral immune response in a naive host may be of relevance to the selection of antigen delivery systems in the design of anti-viral vaccines. One of the limitations of ISCOMs as a delivery system for viral antigens has been the requirement for a hydrophobic or membrane-spanning region in the protein of interest to permit incorporation into ISCOMs. This has limited both the range of antigens which may be incorporated and also the antigenic purity of the resulting complex. We have developed a process where, through covalent attachment of fatty acids, we are able MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Bearsden, Glasgow G61 1QH, UK. *To whom correspondence should be addressed at: ICRF Cancer Immunology Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. (Received 9 September 1991; revised 19 December 1991; accepted 21 January 1992) 0264-410X/92/090585-06 © 1992Butterworth-Heinemann Ltd
to incorporate soluble proteins into ISCOMs. By this method, ISCOMs have been prepared incorporating ovalbumin 6, cytochrome C and Tamm-HorsfaU glycoprotein 7. In this study we have used this method to incorporate the external viral glycoprotein (gpl20) of HIV-1, expressed as a recombinant protein in mammalian cells, into ISCOMs. Immunization of rabbits with gpl20-ISCOMs induced high levels of gpl20-specific antibody. Sera from these animals showed virusneutralizing activity when tested against the homologous strain (IIIB) of HIV-1. MATERIALS AND METHODS
Materials Recombinant gpl20 (rgpl20) of HIV-1 strain IIIB produced by mammalian cells (Celltech, UK ) or by insect cells (American Biotechnology, USA) was obtained through the MRC AIDS Reagent Project. These preparations were > 90% pure and < 20% cleaved when examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Palmitic acid was used as the N(palmitoyloxy) succinimide derivative (Sigma). I SCO Ms were prepared with Quil A (Coopers Animal Health Ltd, U K ) and lipid mix consisting of an equal concentration (10mgm1-1) of cholesterol and phosphatidyl choline in 20% Mega-10 (Sigma).
Preparation of gpl20 ISCOMs The method for incorporation of soluble antigens into ISCOMs is described in detail elsewhere 7. The preparation of rgpl20 ISCOMs is illustrated in Figure /. Briefly, mammalian cell-derived rgpl20 was mixed with palmitic acid at a molar ratio of 1:40 in the presence of the detergent deoxycholate. The mixture was shaken
Vaccine, Vol. 10, Issue g, 1992 585
HIV gp120 ISCOMs: M. Browning et al. gp120
~
4
N- (palmitoyloxy)succinimide
:
(20g d e o x y c h o l a t e )
[
Shake ON; 16°C
I
0.1~ d e o x y c h o l a t e
pH 8.6
Quil A (1 mg/ml) lipid mix
J
Dialy~"
Anti-gpl20 antibody enzyme immunoassay
I
Tris-HCI pH 8.6 I
I0-40~ sucrose density gradient 35K, 20°C, 16 h
~- F r a c t i o n s a s s a y e d - Q u i l A - gp120
J
Pooled f r a c t i o n s - EM
Figure 1 Preparation of rgp120 ISCOMs.The inset shows an electron micrograph of ISCOMs prepared using soluble protein antigen
0.8 ,.~
200
i "~
100
