Incorporation of soluble antigens into ISCOMs: HIV gpl20 ISCOMs induce virus neutralizing antibodies

M i c h a e l B r o w n i n g * , G e o r g e Reid , R o b e r t O s b o r n e a n d O s w a l d Ja r re t t

Through a process of covalent attachment of palmitic acid, we have incorporated recombinant gp120 of HIV 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 Immunodeficiency Virus type 1 ; viral glycoprotein; virus neutralizing antibody

I N T R O D U C T I O N

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)

to incorporate soluble proteins into ISCOMs. By this method, ISCOMs have been prepared incorporating ovalbumin 6, cytochrome C and Tamm-HorsfaU glyco- protein 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 virus- neutralizing activity when tested against the homologous strain (IIIB) of HIV-1.

M A T E R I A L S A N D M E T H O D S

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, UK) 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

0264-410X/92/090585-06 © 1992 Butterworth-Heinemann Ltd Vaccine, Vol. 10, Issue g, 1992 585

HIV gp120 ISCOMs: M. Browning et al.

gp120 ~ 4 N- (palmitoyloxy) succinimide

: (20g deoxycho la te )

[ Shake ON; 16°C

0.1~ I deoxycho la te pH Quil A (1 mg/ml) 8.6

J lipid mix

D i a l y ~ " I

Tris-HCI pH 8.6

I I0-40~ sucrose density gradient 35K, 20°C, 16 h

~- Fract ions a s s a y e d - Q u i l A - gp120

J Pooled f rac t ions - EM

Figure 1 Preparation of rgp120 ISCOMs. The inset shows an electron micrograph of ISCOMs prepared using soluble protein antigen

300 1.0

0.8 i ,.~ 200 "~

0.6

~_ 0.4 o~ 100 <

0.2 0

0 -- 0 3 5 7 9 11 13 15 17 1

Fract ion number

Figure 2 Content of gp120 and Quil A in sucrose density gradient fractions. A coincident peak of gp120 ( I ) and Quil A (/k) (fractions 9-13) correlated with the presence of ISCOMs on electron microscopic examination

overnight, and unbound palmitic acid was removed by dialysis against Tris (pH 8.6 )/0.1% (w/v) deoxycholate. The adjuvant Quil A (1 mg ml-1) was added together with the mixture of lipids (50 #g m1-1 final concentration) that forms the structural basis of the ISCOM. This mixture was dialysed extensively to remove detergent and allow the formation of ISCOMs. The crude ISCOM preparation was purified on 10-40% sucrose density gradients and the purified ISCOMs were assayed for antigen (by ELISA) and Quil A (by 3H-Quil A incorporation) content, and examined by electron microscopy. A coincident peak of gpl20 and Quil A in sucrose density gradient fractions (Figure 2) correlated with the presence of ISCOMs on electron microscopic examination.

Immunization of animals

Young adult male New Zealand white rabbits (Hylyne Farms) were immunized by intramuscular inoculation with mammalian cell-derived rgpl20 (10 #g) in ISCOMs at 0, 4 and 8 weeks, or with rgpl20 (100 #g) in Freund's

complete adjuvant (FCA; Difco) as a water-in-oil emulsion, followed by boosting at 4 weeks with rgpl20 in Freund's incomplete adjuvant, and at 8 weeks with soluble rgpl20 by the intravenous route.

Anti-gpl20 antibody enzyme immunoassay

Immulon 2 Microelisa plates (Dynatech) were coated overnight with monovalent sheep anti-gpl20 antibody D7324 (Aalto, Ireland) at 5/~g ml- 1 in carbonate coating buffer (pH 9.6). The plates were washed and blocked with 2% skimmed milk powder (Marvel, Premier Brands). Rgpl20 (10ng/well) in 2% Marvel was incubated for 1 h at room temperature, and unbound gpl20 was washed out. Rabbit antisera were added over a range of dilutions in 2% Marvel and 20% normal sheep serum (NSS; SAPU), and incubated for 1 h at room temperature. After washing, alkaline phosphatase- conjugated sheep anti-rabbit IgG (Sigma) was added at 1/1000 dilution in 2% Marvel and 20% NSS for 1 h. Bound conjugate was detected using the Ampak amplification system (Novo Biolabs), and was read at 492 nm in a Titertek Multiscan MCC/340 ELISA reader (Flow Laboratories).

Western blot analysis

Western blot analysis of anti-gp 120 antibody responses was carried out with HIV-1 antigen strips (DuPont) according to the manufacturer's instructions. Bound antibody was detected using protein A-biotin (Amersham)/ avidin-alkaline phosphatase (Bio-rad), and visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).

Virus neutralization assay

HIV-1 neutralization assays were carried out using a syncytium formation inhibition assay, modified from the method described by Clapham et al. 8. Briefly, using a quantal microtitre format, serial dilutions of cell-free virus (0.5 logl0-fold ) and antiserum (0.3 loglo-fold) were incubated for 90 min at 37°C prior to addition of C8166 indicator cells. The reactants were further incubated for 6 days at 37°C with the addition of fresh medium mid-term. End-point titres were determined from the Karber f o r m u l a 9

and subsequent regression analysis, and were recorded as the serum dilution which completely inhibited virus-induced syncytium formation.

RESULTS

Induction of anti-gpl20 antibody reslmnses

The ability of ISCOMs incorporating rgpl20 of HIV-1 to induce antigen-specific antibody responses in rabbits was compared with the response to the same antigen in FCA.

• Figure 3 shows the total gpl20-specific antibody responses of two rabbits immunized and subsequently boosted with ISCOMs incorporating 10 #g rgpl20, as compared with the response of a third rabbit hyperimmunized in parallel with 100 pg rgpl20 in Freund's complete adjuvant. High titres of gpl20-specific antibody were observed in all the animals. Western blot analysis against lysates of purified HIV-1 confirmed the specificity of these antisera for gpl20 (Figure 4). When tested by Western blotting against the homologous rgpl20, the ISCOM-induced antisera bound at dilutions of 1/1000 or greater (data not shown).

586 Vaccine, Vol. 10, Issue 9, 1992

2.0

1.6

1.2

L

~ o.8 .D <

0 i ~ - I .5 -2 .0 -2 .5 -3 .0 -3.5 -4 .0

Log10 ant ibody di lut ion

Flgu~ 3 Antibody responses of rabbits to immunization with gpILff)-ISCOMs or soluble gp120 in FCA. Rabbits were immunized and boosted with gp120-ISCOMs (/N and O) or with gp120 in Freund's complete adjuvant (m). GpIL~-specific serum antibody levels were estimated by ELISA 2 weeks after booster immunization. Pre-sera from the same animals are illustrated by o, + and ~ , respectively

The antisera were tested for their ability to neutralize different isolates of HIV-1 in vitro. All three antisera showed virus neutralizing activity against the homologous strain (IIIB) of HIV-1. Table 1 shows the neutralizing activity against HIV-IIIB of ISCOM-induced and FCA-induced antisera at a serum dilution of 1:5. At a dilution of 1:10, all the sera neutralized the lowest dose of virus, but at 1:20 no neutralizing activity was seen. None of the antisera neutralized strains RF or SF2.

Recognition of conformational or linear epitopes

The ability of the antisera to bind to gpl20 on Western blots indicated that the antisera recognized non- conformationally determined epitopes on gpl20. To examine whether the antisera also recognized conformational epitopes, we studied their ability to bind native or denatured rgpl20. Denaturation of rgpl20 was carried out by boiling in 1% SDS for 10 min prior to use in ELISA assays. Figure 5 shows that denaturation had no effect on the binding of the antisera to rgpl20. As the results were the same for both ISCOM-induced and FCA-induced antisera, this did not indicate a qualitative difference in antigen presentation by ISCOMs or FCA, but rather appears to reflect on the preparation of rgpl20 used in these experiments. However, these data indicate that strain-specific neutralizing antibodies to HIV may be raised to non-conformational epitopes on gpl20.

The role of glycosylation of rgpl20 on antibody binding

To determine whether glycosylation of rgpl20 played a role in antibody binding, we studied the inhibitory effect of soluble recombinant gpl20 on the binding of the antisera to solid phase rgpl20. Two preparations of

HIV gp120 ISCOMs: M. Browning et al.

rgpl20, derived from the same strain of HIV (IIIB) but expressed in either mammalian cells or in insect cells, were used. These preparations have identical amino acid sequences and differ only in the glycosylation of the proteins. As indicated in Figure 6, Chinese hamster ovary (CHO) cell-derived rgpl20 showed a dose-dependent inhibition of antibody binding, whilst insect cell-derived rgpl20 over the same concentration range showed little or no inhibition. This difference in inhibition of binding of the antisera by mammalian or insect cell-derived rgpl20 was reflected in comparatively poorer antibody binding to insect cell-derived rgpl20 in ELISA and Western blot assays (data not shown). The results indicate that glycosylation of rgp 120 played an important role in determining the ability of anti-gpl20 antibodies

Rgure 4 Western blot analysis: recognition of HIV antigens by gp120-specific antisera. Antisera as for Figure 2. A, C, E, pre-immune sera (1/10 dilution); B and D, ISCOM-induced and F, FCA-induced immune sera (1/10, 1/30, 11100 dilution, respectively); G, serum from HIV seropositive patient ( 1 / 100 )

Table I ISCOM-induced anti-gp120 antisera neutralize the homologous strain of HIV-1

Virus neutralization (TCIDso)

Serum Immunization 10 30 100

658/4 ISCOM + + - 659•4 ISCOM + + -t- 660 / 2 FC A + + +

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HIV gp120 ISCOMs: M. Browning et al.

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Log o ant ibody di lut ion Log o ant ibody di lut ion Log o ant ibody di lut ion

R g u r e 5 Anti-gp120 antisera bind native and denatured rgp120. Gp120 binding of antisera following third immunization with rgp120 in ISCOMs (a) and (b) or in FCA (c). ELISA estimation of binding to native ( • ) or denatured ( A ) mammalian cell-derived rgp120

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R g u r e 6 Glycosylation affects antibody binding to rgp120. Inhibition of gp120 binding by the addition of soluble rgp120 derived from mammalian cells ( i ) or from insect cells (A) . Antisera as for Figure 5

100000

~'~ 10000

looo

100

1 0

o Persistence of ISCOM-induced anti-gpl20 antibodies

We examined the persistence of anti-gpl20 antibodies for up to 8 weeks following a course of three immunizations with rgpl20-ISCOMs. Figure 7 shows the midpoint titres of anti-gpl20 antibodies in sera from rabbits immunized and boosted twice with rgpl20- ISCOMs or with rgpl20 in FCA. High levels of anti-gpl20 antibodies persisted in the sera of ISCOM- immunized animals following the course of immunization.

I I I I I I I I 2 4 6 8 10 12 14 16

f f DISCUSSION Time {weeks)

F i g u r e 7 Persistence of anti-gp120 antibodies. Rabbits were immunized at 0, 4 and 8 weeks (indicated by arrows). Anti-gp120 antibody r e s p o n s e s were estimated by ELISA, and are expressed as reciprocal half maximal titres. • and A, ISCOM-induced antisera; O, FCA-induced antiserum

to bind their ligand. The data do not distinguish, however, whether this effect was due to the induction of antibodies raised against sugar residues or.due to steric effects of glycosylation on the availability of peptide epitopes on the glycoprotein.

We have demonstrated a method whereby a soluble recombinant viral glycoprotein can be incorporated into ISCOMs. The method extends the range of antigens which may be incorporated into ISCOMs, and permits the formation of ISCOMs containing highly purified non-amphipathic recombinant viral proteins. The covalent attachment of fatty acids to a soluble protein allows its incorporation into ISCOMs under conditions that are unlikely to disrupt the native conformation of the protein. The method has been successfully employed in our laboratory to incorporate a range of soluble

588 V a c c i n e , Vol . 10, I ssue 9, 1992

proteins, including ovalbumin, cytochrome C and Tamm-Horsfall glycoprotein, into ISCOMs (Refs 6, 7). Other authors have reported incorporation of soluble antigens into ISCOMs following treatment of the antigen at low pH ~°'~ 1. This method results, however, in partial denaturation of the antigen 1°, with the potential loss of conformationally defined determinants which may be important in the induction of virus-neutralizing antibodies.

ISCOMs were prepared incorporating highly purified recombinant gp120 of HIV-1 strain IIIB expressed in the mammalian cell line CHO. Rabbits immunized with rgp120 ISCOMs produced high levels of antigen-specific antibodies, comparable with the response of a rabbit immunized with ten times as much rgpl20 in FCA. The antisera showed strain-specific virus neutralization following a course of three immunizations, but failed to neutralize two heterologous strains of HIV-1. T h e absence of cross-neutralizing antibodies in these antisera may reflect the fact that the external envelope protein of HIV-IIIB is poorly representative of HIV isolates in general 12. Alternatively, this may simply reflect the relatively low neutralizing antibody titres to the homologous strain.

Our results are similar to those reported by Pyle et al. 1~, who used acid treatment of virus-derived gpl20 (estimated purity 50%) to permit its incorporation into ISCOMs. These authors demonstrated the induction of strain-specific virus neutralizing antibodies in rhesus monkeys immunized on three occasions with gpl20- ISCOMs. Following multiple immunizations with gp120-ISCOMs, low levels of cross-neutralizing activity against heterologous strains of HIV were observed in the serum of one of these animals ~ x.

The data demonstrate the ability of antisera recognizing only non-conformational epitopes of HIV-1 gpl20 to neutralize the homologous strain of virus. As this was the case for both ISCOM-induced and FCA-induced antibodies, the data suggest that the preparation of rgp120 used in these experiments was partially denatured prior to its incorporation into ISCOMs. This observation was supported by studies of the ability of the native and denatured forms of rgp120 to bind recombinant CD4 in ELISAs (unpublished results). Much attention has focused on the role of the V3 loop of gpl20 as the principal neutralizing determinant for virus neutralizing antibody responses. Recent evidence 12 indicates that, in spite of high levels of sequence variability within this region, a common structural motif is conserved, suggesting that the conformation of this determinant may be important. As the antisera raised in our study recognized only non-conformational epitopes on gpl20, it would appear that either the virus neutralizing activity of the antisera was directed at epitopes outwith the V3 loop 13'~4, or linear sequences within the V3 region induce an antibody response capable of neutralizing at least the homologous strain of HIV. The ability of the antisera to bind overlapping synthetic 20-mer peptides derived from the V3 loop sequence (unpublished results) supports this latter case.

Glycosylation of rgp120 played an important role in determining the ability of experimentally induced anti-gp 120 antibodies to bind to the glycoprotein. Similar effects of differential glycosylation have been reported in studies of binding of antibodies from naturally infected

HIV gp120 ISCOMs: M. B r o w n i n g et al.

individuals to mammalian or insect cell-derived recom- binant gpl2015. It is not clear from these studies whether glycosylation modulates the binding of antibodies to peptide epitopes within the glycoprotein backbone or whether a proportion of the antibodies are directed against sugar residues. Davis and co-workers 16, however, have described the interference by glycosylation on binding to the native glycoprotein of peptide-induced antibodies to gp120.

It remains to be established which host immune responses and which viral antigens are critical for protection from lentivirus infection. Present studies indicate the requirement for the envelope glycoprotein for the induction of neutralizing antibody and protective immunity. A recent study of passive serum transfer in cynomolgous monkeys has shown that antibody alone may protect against infection with SIV ~. Other studies in chimpanzees, however, have failed to confer protection against HIV with antibody alone ~ a, suggesting that other limbs of the immune response may be involved also. The ability of ISCOMs to induce both virus-neutralizing antibody and virus-specific cellular responses suggests that ISCOMs represent a suitable antigen delivery system for study of potential vaccines against lentiviral infections. ISCOMs incorporating viral envelope glyco- protein have previously been shown to be effective in inducing protection against a type C retrovirus, FeLV (Ref. 4). The ability to incorporate non-amphipathic proteins into ISCOMs extends the range of viral antigens which may be investigated by this system.

R E F E R E N C E S

1 Morein, B., Lovgren, K., Hoglund, S. and Sundquist, B. The ISCOM: an immune stimulating complex. Immunol. Today 1987, 8,333-338

2 De Vries, P., Van Binnendijk, R.S., Van Der Marel, P., Van Wezel, A.L., Voorma, H.O., Sundquist, B. et al. Measles virus fusion protein presented in an immune-stimulating complex (ISCOM) induces haemolysis-inhibiting and fusion-inhibiting antibodies, virus-specific T cells and protection in mice. J. Gen. Virol. 1988, 69, 549

3 Sundquist, B., Lovgren, K., Hoglund, S. and Morein, B. Influenza virus ISCOMs: antibody response in animals. Vaccine 1988, 6, 49-53

4 Osterhaus, A., Weijer, K., Uytdehaag, F., Jarrett, O., Sundquist, B. and Morein, B. Induction of protective immune response in cats by vaccination with feline leukemia virus ISCOM. J. Immunol. 1985,135, 591-596

5 Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R.N. and Berzofsky, J.A. Induction of CD8+ T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature 1990, 344, 873-875

6 Mowat, A.M., Donachie, A.M., Reid, G. and Jarrett, O. Immune stimulating complexes containing Quil A and protein antigen prime class I-MHC restricted T lymphocytes in vivo and are immunogenic by the oral route. Immunology 1991, 72, 317-322

7 Reid, G. Soluble proteins incorporate into ISCOMs after covalent attachment of fatty acid. Vaccine 1992, 10, in press

8 Clapham, P.R., Weber, J.N., Whitby, D., Mclntosh, K., Dalgleish, A.G., Maddon, P.J. et al. Soluble CD4 blocks the infectivity of HIV and SIV for T cells and monocytes but not for brain and muscle cells. Nature 1989, 337, 368-370

9 Hawkes, R.A. In: Diagnostic Procedures for Viral, Rickettsial and Chlamidial Infections, 5th edition (Eds Lennette, E.H. and Schmidt, N.J.) American Public Health Association, Washington, DC, 1979, pp 34-35

10 Morein, B., Ekstrom, J. and Lovgren, K. Increased immunogenicity of a non-amphipathic protein (BSA) after inclusion into iscoms. J. Immunol. Methods 1990, 128, 177-181

11 Pyle, S.W., Morein, B., Bess, J.W., Akerblom, L., Nara, P.L. et al. Immune response to immunostimulatory complexes (ISCOMs)

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HIV gp120 ISCOMs: M. B rown ing et al.

prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 external envelope glycoprotein (gp120). Vaccine 1989, 7, 465-473

12 LaRosa, G.J., Davide, J.P., Weinhold, K. et al. Conserved sequence and structural elements in HIV-1 principal neutralizing determinant. Science 1990, 249, 932-935

13 Ho, D.D., Sarngadharan, M.G., Hirsch, M.S. et al. Human immunodeficiency virus neutralising antibodies recognize several conserved domains on the envelope glycoproteins. J. Virol. 1987, 61, 2024-2028

14 Chanh, T.C., Dreesman, G.R., Kanda, P., Linette, G.P., Sparrow, J.T., Ho, D.D. and Kennedy, R.C. Induction of anti-HIV neutralizing antibodies by synthetic peptides. EMBO J. 1986, 5, 3065-3071

15 Moore, J.P., McKeating, J.A., Jones, I.M., Stephens, P.E., Clements, G., Thomson, S. and Weiss, R.A. Characterisation of recombinant

gp120 and gp160 from HIV-I: binding to monoclonal antibodies and soluble CEN. AIDS 1990, 4, 307-315

16 Davis, D., Stephens, D.M., Willers, C. and Lachman, P.J. Glycosylation governs the binding of antipeptide antibodies to regions of hypervariable amino acid sequence within recombinant gp120 of human immunodeficiency virus type 1. J. Gen. Virol. 1990, 71, 2889-2898

17 Putkonen, P., Thorstensson, R., Ghavamzadeh, L, Albert, J., Hild, K., Biberfeld, G. and Norrby, E. Prevention of HIV-2 and SlVsm infection by passive immunization in cynomolgous monkeys. Nature 1991, 352, 436-438

18 Prince, A.M., Horowitz, B., Baker, L. et al. Failure of a human immunodeficiency virus (HIV) immune globulin to protect chimpanzees against experimental challenge with HIV. Proc. Natl Acad. Sci. USA 1988, 85, 6944-6948

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