Immune responses in mice induced by HSV-1 glycoproteins presented with ISCOMs or NISV delivery systems

Elsevier PII: SO26441OX(96)00155-7

ELSEVIER

Vaccine, Vol. 14. No. 17118, pp. 1581-1589. 1996 Copyright 0 1996 Elsevier Science Ltd. All nghts reserved Printed in Great Britain 0264-410X/96 $15+0.00

Immune responses in mice induced by HSV-1 glycoproteins presented with ISCOMs or NISV delivery systems Y. Hassan*,

J.M. Brewer-f, J. Alexander?

and R. Jennings*1

The purpose of this study was to evaluate the immunogenicity of a herpes simplex virus type I (HSV-I) antigen preparation, obtained following zwitterionic detergent treatment of virus, and incorporation of the antigens into either immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) delivery systems. Using Balblc mice the ISCOM and NISV HSV-1 vaccines were assayed for their capacity to induce and enhance both the humoral and cellular immune responses, and to elicit protection against both homologous and heterologous virus challenge. The serum from animals vaccinated with either the NISV or the ISCOM HSV-1 antigen preparation, were found to contain high levels of total IgG and IgGl and IgG2a subclass antibodies. In addition, both preparations were found to induce high neutralizing (NT) antibody levels following a two immunization protocol and to provide some protection against homologous and heterologous HSV challenge infection. Lymphoproltferative responses were observed in cultures of splenocytes from mice immunized with both HSV-I NISV vaccine and HSV-I ISCOMs vaccine, following various antigenic stimuli in vitro. In general, these were most marked in animals immunized with the HSV-I NISV preparation, and particularly so when the splenocytes were stimulated in vitro with live HSV-I. Both the NISV and ISCOM HSV-I vaccines were found to have induced interleukin 2, interleukin 10 and interferon-y in spleen cell culture supernatants, although again, the highest responses in general were observed in supernatant fluids from spleen cell cultures from animals immunized with the HSV-1 NISV preparation. These results suggest that a wide range of immune activity can be elicited by HSV-1 antigens presented to the immune system of mice in these formulations. Copyright 0 1996 Elsevier Science Ltd. Keywords:

HSV-I;

ISCOMs;

NISV; adjuvants;

Thl,

Th2

There is currently considerable interest in the development of vaccines against herpes simplex viruses (HSV), the causative agents of a number of infections of mucocutaneous surfaces, including primary and recurrent genital herpes, primary and recurrent orofacial disease, keratoconjunctivitis and also encephalitislP5. A number of these infections are becoming increasingly common amongst certain population groups, and are particularly severe in the immunocompromised6.7. Foremost amongst the several different strategies for vaccination against HSV primary or recurrent disease, is the induction of host immune responses by the use of one or more of the HSV surface glycoproteins8-14. These antigens may be prepared either by recombinant DNA tech*Department of Medical Microbiology, University of Sheffield Medical School, Sheffield, SIO 2RX, UK. TDepartment of Immunology, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow, G4 ONR, UK. $To whom correspondence should be addressed. (Received 22 February 1996; revised 11 June 1996; accepted 16 July 1996)

nology or by detergent solubilization of in vitro cultured HSV and partial purification of the surface glycoproteins. Althou h the HSV envelope contains at least 11 glycoproteins f5 , glycoprotein vaccine strategies have centred on the two major glycoproteins, B and D9.‘o.“*‘3 or mixtures of several HSV glycoproteins, including glycoproteins B and D16.17. These glycoproteins have been shown to elicit protection in experimental animal models for HSV infections. A major problem associated with the use of surface glycoproteins in viral vaccines is the relatively low immunogenicity of these isolated antigens in corn arison with the live or inactivated intact virus17.’6). One approach to circumvent this problem would be to use immunological adjuvants which enhance the immunogenicity of proteins formulated with them. A number of potential adjuvants for viral glycoprotein antigens have been investigated with regard to both their immunopotentiating capabilities19*20 and their capacity for targeting antigens to the host immume system in such a way as to preferentially trigger certain responses. These

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HSV-1 glycoprotein immune responses: Y Hassan et al. responses include, stimulation of antibody production of certain protective subclasses or CD8+ cytotoxic T lymphocyte responses. Amongst such adjuvant systems are immunostimulating complexes (ISCOMs) first described in 1984”. ISCOMs are assembled from Quil A, cholesterol and virus glycoprotein configured in a particulate “cage-like” structure visible by electron microscopy. ISCOMs have been demonstrated to target antigen to macrophages” and can generate an appropriate protective immune response against many viruses including the HSVs’ ‘.13-36. These protective responses are associated with the production of the Thl type cytokines, interleukin 2 (IL-2) and interferon-gamma (IFN-y), IFN-y-dependent IgG2a production, and CD8+ cytotoxic T cell (CTL) induction’7m29. However, Quil A, an extract from the bark of the tree, Quill&z saponiuria molina, contains a number of relatively toxic glycosides and is therefore unlikely to be suitable for medical use without further refinementJo. Thus the identification of a suitable, safe adjuvant which also generates the appropriate antigen-specific response remains highly desirable. The adjuvant activity of non-ionic surfactant vesicles (NISV) have recently been described as enhancing the production of specific antibodies to entrapped antigens when inoculated into mice” while possessing extremely low toxicity”. Relative to Freund’s complete adjuvant (FCA), a potent stimulator of Thl-type responses”, NISV were potent stimulators of IFN-y-dependent antiand therefore effecgen specific IgG2a production- “x~’ tive stimulators of Thl type responses themselves36.37. In a Toxoplasma gondii vaccine model protection mediated by IFN-y’s, has been associated primarily with CD8+ and NISV containing enlymphocyte activity39.‘0, trapped soluble parasite antigen were demonstrated to produce protective immunity and increase production of IFN-y in antigen stimulated splenocytes to levels higher that NISV were than FCA"' . These results indicated potent stimulators of not only Thl lymphocytes, but perhaps also CD8+ CTLs. The present studies are therefore concerned with a comparative assessment of the immunopotentiating capabilities of ISCOMs and NISV formulated with a mixture of several HSV-1 antigens derived following zwitterionic detergent treatment of HSV-1 infected Vero cells”.

MATERIALS

AND METHODS

HSV strains, antigen preparations and cell cultures Challenge infection of mice to assess the protective effects of vaccine preparations was carried out using strain WAL (HSV-1) or strain 333 (HSV-2). HSV-1 antigen preparations for use as vaccines were obtained using HSV-1 (strain F). All three HSV strains were originally supplied by Dr F. Rapp, Milton Hershey Medical Centre, Pennsylvania State University, USA. All viruses were grown and assayed by plaque-forming titration in Vero cell cultures (Flow Laboratories, Irvine, Scotland). HSV-1 antigen preparations were obtained using zwitterionic detergent extraction procedures4’ and characterized using methods described previously”3. HSV-1 for use as a stimulating antigen in the lymphoproliferation assay was heat-inactivated at 56°C for 30 min.

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HSV-1 vaccine preparations HSV-1 ISCOM vaccines were obtained by mixing HSV-1 antigen preparation with Quil A (kindly donated by Dr Bror Morein, National Veterinary Institute, Division of Vaccine Research, Uppsala, Sweden) as described by Erturk and his co-workers’ ’ . The presence of the characteristic cage-like ISCOMs was determined by electron microscopy. NISV were prepared under aseptic conditions by methods described previously”. Briefly, all glassware was heated at 180°C for 5 h to inactivate endotoxin, and autoclaved. Elgastat Ultra High Purity water (Elga, Bucks., UK) was used to prepare solutions. One hundred and fifty micromoles of l-mono palmitoyl glycerol, cholesterol and diacetyl phosphate (Sigma, UK) were mixed in a 15 ml Pyrex test tube in the molar ratio 5:4: 1 and then heated at 130°C in a dry-block (Grant Instruments, Cambridge, UK) until melted. Vesicles were formed when 2.5 ml of aqueous buffer (PBS pH 7.4) was added and the resulting suspension vortexed vigorously for 1 min. After shaking the suspension at 60°C for 2 h, HSV-1 antigens were added and entrapped by freezing the antigen/vesicle mixture in liquid nitrogen and thawing to 60°C five times, as described previously44. After a further 2 h shaking at 6O”C, non-entrapped antigen was removed from the vesicle suspension by centrifuging at 1OOOOOgfor 45 min. Protein or peptide content of the vesicle suspensions were determined by a modification of the ninhydrin method45 as described elswhere32. Mock ISCOMs were prepared by procedures described above, except that the Vero cells were not infected with HSV-1. Incorporation efficiency of HSV glycoprotein antigen preparation into NISV is ca 65%, whilst incorporation into ISCOMs has been shown to be ca 50%“. Characterization of ISCOMs has shown them to have a size ranging from 3040 nm dia”%‘9.“, while NISV particle size is ca 2-3 nm in diameter. We have previously demonstrated the association of HSV antigens with ISCOMs and indicated the prescence of at least six HSV glycoproteins in such preparations”, while previous studies have also shown the incorporation of rotein I? antigen in NISV following the above procedures- ‘.46,47.

Serum antibody determinations Blood was obtained from each individual mouse prior to immunization, and again 3 weeks following the second of two immunizations with either HSV-1 ISCOMs, HSV-1 NISV or PBS as a control, given 3 weeks apart. Serum, separated from each blood was tested individually for total and subclass IgG antibody by ELISA and ,also for neutralizing antibody.

Enzyme-linked immunosorbent assays (ELISA) Total and subclass IgG antibodies to HSV-1 were sandwich technique carried out using an antibod Y previously described in detail 9.42, using polyclonal rabbit IgG against HSV-1 (Dako Laboratories Ltd, Copenhagen, Denmark) at a 1: 1000 dilution as coating antibody, and rabbit anti-mouse IgG linked to horseradish peroxidase (HRP), also obtained from Dako Laboratories, as conjugate and used at a dilution of 1: 1000. For determination of levels of IgGl, IgG2a, IgG2b and IgG3 subclass antibodies in mouse sera, the same coating antibody was used at the same dilution as

HSV-1 glycoprotein immune responses: Y Hassan et al.

described above, but the conjugates consisted of rabbit anti-mouse subclass antibody linked to HRP, used at dilutions of 1:2000 (IgGl), 1:4000 (IgG2a), 1:1000 (IgG2b) and 1:200 (IgG3). These conjugates were obtained from Nordic Laboratories Ltd, Maidenhead, and all conjugates and other reagents were titrated prior to their use in these tests to determine optimal concentrations. All mouse sera were tested for both total IgG and subclass IgG at a dilution of 1:lOO. In addition, all post-immunization mouse sera were also diluted from 1:10’ to 1:1O6 and tested for total IgG antibody by endpoint titration using the same ELISA procedure as described above. tests (NT) These were carried out in 96-well sterile tissue culture microplates (Costar Ltd, Cambridge), and each mouse serum tested individually in duplicate. In brief, 50 ~1 of serum-free TC199 medium (Life Technologies Ltd, Paisley, Scotland) was placed in each well of the microplate, and 50,ul of each test serum added to the first well of each row to make a 1:2 dilution. Doubling dilutions were made from 1:4 to 1:2048 for each serum, and an equal volume of HSV-1 strain WAL at a concentration of 2 x 10’ p.f.u. ml-’ added to each well. The virusserum mixtures were gently agitated and the microplates incubated at 37°C for 2 h in 5% CO, in air. One hundred microlitres of Vero cells (2 x lo5 cells ml-‘) in TC 199+ 10% foetal calf serum (FCS) obtained from Life Technologies, Ltd, was then added to each well and the plates incubated for a further 48 h at 37°C. At the end of this period the mixture in each well was decanted, the wells stained with naphthalene black and observed for virus cytopathic effect (CPE). Controls, consisting of known HSV-1 positive and HSV-1 negative mouse sera were included on each microplate; in addition, HSV-1 (strain WAL) added to a series of wells containing medium only, was also included. Endpoints were taken as the dilution of serum causing at least 75% reduction in CPE. Neutralization

Lymphoproliferation

assays

Immune splenocytes were obtained from mice 21-24 days after the second of two immunizations with HSV-1 ISCOMs, HSV-1 NISV or PBS as control. The animals received the first immunization (ISCOMs or NISV formulated to contain 3 lug ml-’ of HSV-1 antigens) at 6 weeks of age, with a booster at the same antigen dose given 2 weeks later. Each mouse received 200 yl of the respective vaccines, or PBS, by the subcutaneous route. The spleen in RPM1 1640 medium (Life Technologies, Ltd) + 10% FCS, were disrupted, washed, resuspended in RPM1 1640 with 10% heat-inactivated FCS, and placed in 96-well cell cultures plates at a concentration of 4 x 10’ viable cells in 100 ~1 per well. HSV antigens in different forms, HSV-1 subunit preparation, HSV-1 ISCOMs, live HSV-1 virus (strain WAL), heat-inactivated HSV-1 strain WAL or mock ISCOMs were added in triplicate to the appropriate wells. After 72 h incubation at 37°C in 5% COZ, each well was pulsed with 50 ~1, 5 FuCi per well of [3H]thymidine at 1 mCi mmol- ’ (Amersham International, Amersham, UK) and harvested using a cell harvester. The amount of label

incorporated into cellular DNA was determined by liquid scintillation counting. Cell proliferation was expressed as counts per minute (c.p.m.) in presence of antigen minus c.p.m. in absence of antigen. Controls, including mock ISCOMs, RPM1 1640 medium alone, and phytohaemagglutinin (Sigma, U.K.) were used ‘throughout. Cytokine

assays

IFN-y, IL-2 and interleukin 10 (IL-lo) production by cultured splenocytes obtained from immunized and control mice was determined by ELISA. For IFN-y, assays were set up in triplicate in 96-well, flat-bottomed tissue culture plates (Costar Ltd, Cambridge), coated with purified rat anti-mouse IFN-), (Pharmingen, Cambridge Bioscience Ltd, Cambridge), at a concentration of 100 pg per well. After overnight incubation at 4°C and subsequent washing, wells were blocked (1 h/37”C) using 10% heat-inactivated FCS in phosphate-buffered saline, pH 7.2. After further washing, 100 ~1 of standard recombinant mouse IFN-1, (Cambridge Bioscience Ltd) was added at concentrations ranging from 54 pg ml ~ ’ to 120 ng ml-’ to produce a standard curve, and then 100 ~1 of undiluted test or control spleen cell culture supernates to appropriate test wells. After incubation for 2 h at 37”C, wells were washed and biotin-labelled rat antimouse IFN-), neutralizing antibody (Cambridge Bioscience Ltd) added at a concentration of 100 pg per well, contained in a volume of 100 pl. Following 45 min incubation at 37°C and further washes, HRP conjugate (Southern Biotechnology Associates Inc., Birmingham, USA) used in accordance with the manufacturer’s instructions at a dilution of 1:4000, was added in volumes of 100 ~1 to each well and the plates incubated at 37°C for 30 min. Following the addition of buffered TMB/DMSO substrate (Sigma Chemicals Ltd, Poole, Dorset) in volumes of 100 ~1 per well, and incubation for 30-60 min at 37”C, the reaction was stopped using 10% sulphuric acid (50 ~1 per well), absorbance values read at 450 nm, and the results expressed as IFN-), units ml-l. For determination of IL-2 in undiluted splenocyte culture supernatants, assays were set up essentially as described for IFN-y. Plates were coated with purified rat anti-mouse IL-2 (Cambridge Bioscience Ltd) at a concentration of 200 pg per well, standard recombinant mouse IL-2 (Genzyme Diagnostics Ltd, Cambridge) was added at concentrations ranging from 54 pg to 120 ng and biotin-labelled rat anti-mouse IL-2 (Cambridge Bioscience Ltd) neutralizing antibody was used at a final concentration of 100 pg per well. HRP conjugate was used at 1:4000, and substrate solution prepared as described above. The reaction was stopped using sulphuric acid and the results read at a wavelength of 450 nm. Similar procedures were used for the IL-10 assays. Plates were coated with purified rat anti-mouse IL-10 (Cambridge Bioscience Ltd) at a final concentration of 200 pg per well, blocked with 10% heat-inactivated FCS in PBS (pH 7.2) followed by addition of standard recombinant mouse IL-10 (Cambridge Bioscience Ltd), used at concentrations ranging from 54 pg to 120 ng to produce a standard curve, and test or control sample, diluted in blocking buffer. Biotin-labelled rat anti-mouse IL-10 neutralizing antibody (Cambridge Bioscience Ltd) was then added at a final concentration of 100 pg per

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HSV-1 glycoprotein immune responses: Y Hassan et al.

well, followed by HRP conjugate at 1:2000 dilution, and substrate. After addition of sulphuric acid, the results were read at 450 nm. Mouse protection experiments

In order to assess the ability of the various HSV-1 vaccine preparations to protect mice against homologous (HSV-1) or heterologous (HSV-2) lethal challenge infection, groups of 40 mice received two immunizations, 21 days apart with HSV-1 ISCOMs, HSV-1 NISV or PBS (as control) by the subcutaneous route in a volume of 0.2 ml, each containing 3 pg mll ’ total protein. Blood samples were collected from each mouse prior to the first immunization, and again 3 weeks following the booster dose. Serum samples from each mouse were stored and individually tested by ELISA and neutralization for antibody levels to HSV-1. Twenty-two days following the booster immunization, two groups of eight mice from each immunized group and from the control group were established. One group was inoculated with 6.0 mouse lethal doses (MLD,,) of HSV-1 (strain WAL), and the other with 6.0 MLD,, of HSV-2 (strain 333) by the intraperitoneal route in a volume of 0.2 ml. The animals were then observed for severe illness over a period of 15 days. Previous studies from this laboratory have indicated that mock ISCOM preparations do not induce protection against HSV-1 challenge infections in mice4’. Statistical

analyses

The Mann-Whitney “U”, Student’s t-test and the x2 test were used to statistically analyse antibody levels in mouse sera, cytokine levels in splenocyte cultures, lymphoproliferation results and the mouse protection experiments.

RESULTS Serum antibody levels in mouse sera

All antibody levels obtained by ELISA, for total and subclass antibody, and for neutralizing antibody, in sera collected from control or immunized mice are summarized in Table I. No HSV-1 antibody was detected in pre-immunization mouse sera. Sera were tested for total IgG antibody levels at both a single 1: 100 dilution and by titration to give an endpoint titre, using ELISA. Mean total IgG ELISA antibody levels, as determined at a single serum dilution of l:lOO, were significantly greater (PCO.05) in animals receiving HSV-1 NISV, as compared to those animals given HSV-1 ISCOMs, but this difference was not apparent when endpoint ELISA titres were determined (Table I). However, sera from mice immunized with either HSV-1 NISV or HSV-1 ISCOMs had significantly greater ELISA antibody levels irrespective of the procedure used, than those observed in animals given PBS, which were essentially at or below baseline levels. Although IgGl and IgG2a subclass antibodies were induced to similar levels by both the HSV-1 ISCOM and HIV-l NISV vaccine preparations (Table I), there was a marginal, but not significant difference, between HSV-1 ISCOMs and HSV-1 NISV in the amount of IgG2b subclass antibody elicited. Neither preparation induced levels of IgG3 subclass antibody above baseline values.

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Both vaccine preparations induced high levels of neutralizing antibody in mouse sera following the two dose immunization schedule (Table 1). Thus, NT antibody titres ranging from 1:64 to 1:1024 (geometric mean 1:168) and 1:32 to 1:128 (geometric mean 1:73) were elicited by HSV-1 ISCOMs and HSV-1 NISV vaccines, respectively. The NT antibody titres induced by the former vaccine were significantly greater (PCO.01) than those induced by the latter. No NT antibodies were detected in sera from mice given PBS (Table I). Induction of lympboproiiferative

responses

The lymphoproliferative responses, expressed as counts per minute, in mice immunized with HSV-1 ISCOMs, HSV-1 NISV or PBS, were determined in spleen cell cultures obtained 3 weeks after the second immunization and stimulated in vitro with various forms of HSV antigens used at varying concentrations. Figure la-c shows that no significant proliferative responses were observed in spleen cell cultures established from mice given PBS irrespective of the nature or concentration of the in vitro antigenic stimulant. In contrast, cell cultures established from spleens of mice immunized with HSV-1 ISCOMs or HSV-1 NISV showed high in vitro proliferative responses; these were significantly greater with most of the in vitro stimulants for the HSV-1 NISV (Figure I), including subunit HSV-1 antigen, HSV- 1 ISCOMs and live HSV-1. Significantly higher levels of stimulation (P
Supernatants from spleen cell cultures were tested for levels of three cytokines, IL-2, IL-10 and IFN-y. The results for IL-2 levels (Figure 2a) show that this cytokine was detectable in spleen cell cultures from mice immunized with either HSV- 1 ISCOMs or HSV- 1 NISV, and stimulated in vitro with 0.5 pug ml-’ of HSV-1 antigen preparation, HSV-1 ISCOMs, or with lo6 p.f.u. ml-’ of live HSV-1 virus. Highest IL-2 levels, 6.64 and 3.88 U ml-‘, were detected in supernatants stimulated in vitro with the HSV-1 subunit antigen preparation.

aat 492 nm

14 14 14

HSV-1 ISCOMs HSV-1 NISV PBS

S.D., standard deviation;

No. of sera tested

1.21 (kO.28) 1.72 (kO.41) 0.14 (kO.05)

1.14 (iO.62) 1 .15 (*0.47) 0.06 (kO.02) 0.67 (k0.27) 0.67 (kO.66) 0.06 (iO.02)

0.14 (+0.17) 0.39 (eO.44) 0.02 (+O.Ol)

IgG2b

0.73-l .I5 0.85-l .91 0.09-0.16

(Mean+S.D.) IgG2a

IgGl

Mean (+S.D.)

Subclass IgG

values?

Range

ELISA absorbance

Total IgG

Total/subclass

0.06 (kO.01) 0.06 (kO.02) 0.05 (+O.Ol)

lgG3 1:7000 1:8000 1:200

Mean ELISA endpoint titre

1:64-1:1024 1:32-1:128 <1:4

Neutralizing range

ELISA absorbance values for total and subclass specific IgG antibody levels and neutralizing antibody levels in sera from mice immunized with HSV-1 antigen preparations

Immunizing agent

Table 1 or NISV

into ISCOMs

1:168 1:73 <1:4

Antibody titre geometric mean

formulated

HSV-1 glycoprotein immune responses: Y Hassan et al.

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Figure

responses (c.p.m.-background 1 Lymphoproliferative c.p.m.) of spleen cells obtained from mice following two immunizations with HSV-1 antigens formulated into NISV or ISCOMs, with mock ISCOMs or PBS as controls, and stimulated in vitro with (a) HSV-1 subunit antigen preparations, (b) HSV-1 ISCOMs or (c) live or heat-inactivated HSV-1 (strain WAL).(a) Open square, 1 pg HSV-1 subunit antigen preparation; hatched square, 0.5 pg HSV-1 subunit antigen preparation; shaded square, 5.0 pg phytohaemagglutinin; filled square, RPM1 1640 medium. (b) Open square, 1 ,ug HSV-1 ISCOM preparation; diagonally-hatched square, 0.5 ,ug HSV-1 ISCOM preparation: vertically-hatched square, mock ISCOMs; shaded square, 5.0 ,ug phytohaemagglutinin; filled square, RPM1 1640 medium. (c) Horizontally-hatched square, live virus 106; open square, live virus 105; diagonally-hatched square, heat-inactivated virus 106; vertically-hatched square, heat-inactivated virus 105; shaded square, 5.0 pg phytohaemagglutinin; filled square, RPM1 1640 medium

Levels of the cytokine IL-10 (Figure 2b) were also detected in spleen cell culture supernatants from mice immunized with HSV-ISCOMs or HSV-1 NISV, although significantly higher (PcO.001) levels of this cytokine were found in spleen cell supernatants from mice immunized with the HSV-1 NISV preparation. Similar findings were made with respect to IFN-), (Figure 2c), where spleen cell culture supernates from HSV-1 NISV immunized mice contained relatively high concentrations of IFN-), after stimulation in vitro with lo6 p.f.u. ml-’ live HSV-1 (321 U ml-‘), 0.5 pug ml-’ HSV-1 subunit antigen preparation (199 U ml-‘) or 0.5 pug ml-’ HSV-1 ISCOMs (199 U ml-‘). In contrast, only live HSV-1 when used as the in vitro antigen stimulus, was found to elicit any detectable level of IFN-y (123 U ml-‘) in spleen cell culture supernates from mice immunized with HSV-1 ISCOMs (Figure 2~). In these studies, relatively high levels of IFN-), were observed in spleen cell culture supernates from HSV-1 NISV immunized mice, stimulated in vitro with mock ISCOMs or indeed without any stimulant at all. However, there were no significant IL-2, IL-10 or IFN-y cytokine levels

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”

NISV

ISCOMs Immunising

PBS

agent

Figure 2 Levels of cytokines (a) IL-2, (b) IL-10 and (c) IFN-71 in spleen cell culture supernatants from mice immunized with HSV-1 antigens formulated into NISV or ISCOMs, or with mock ISCOMs or PBS as controls, and stimulated in vitro with HSV-1 subunit antigen preparation, HSV-1 ISCOMs, live HSV-1, mock ISCOMs or RPM1 1640 medium. Vertically-hatched square, 1 pg HSV-1 subunit antigen preparation; open square, 1 pg HSV-1 ISCOMs; shaded square, lo6 p.f.u. live HSV-1 (strain WAL); vertically-hatched square, mock ISCOMs; filled square, RPM1 1640 medium

present, irrespective of the in vitro stimulant used, in spleen cell culture supernatants from the control mice inoculated with PBS. of mice immunized with HSV-ISCOMs or HSV-1 NISV following challenge with live HSV-1 or HSV-2 The survival of mice immunized with two doses of HSV-1 ISCOMs or HSV-1 NISV, or given PBS by the subcutaneous route, and challenged with 6LD,, of HSV-1 (strain WAL) or live HSV-2 (strain 333) is shown in Figure 3. It can be seen that following HSV-1 challenge infection (Figure 3a), five of eight mice (63%) immunized with HSV-1 ISCOMs survived, while only two of eight animals (25%) given HSV-1 NISV did not succumb to this lethal challenge. Following challenge with the heterologous HSV-2, just two of eight (25%) and three of eight (38%) of mice immunized with HSV-1 ISCOMs or HSV-1 NISV, respectively, survived. None of these differences were significant. None of the mice Protection

HSV-1 glycoprotein immune responses: Y Hassan et al.

lb)

0 HSV-IorHSV-2lSCOMs 0 HSV-IorHSV-2NlSV 0 PBS

'O"

75

0

5

10

15

20

Days Post Challenge

Days Post Challenge

Figure 3

Protection of mice against, (a) live HSV-1 (strain WAL) or, (b) HSV-2 (strain 333) challenge infection, following immunization with two doses of HSV-1 antioens formulated into NISV or ISCOMs, or with PBS as control. 0, HSV-1 or HSVQ ISCOMs; 0, HSV-1 or HSV-2 HISV; ‘9, PBS

inoculated with PBS survived challenge infection with either HSV-1 or HSV-2 (Figure 3).

DISCUSSION The studies described in this report show that a mixture of HSV surface glycoprotein antigens including major glycoproteins B, C, D and E’ ‘, formulated into either NISV or ISCOMs, induce high levels of both ELISA and NT antibodies in mice. However, the levels of NT antibody elicited were significantly greater following immunization with the ISCOM-formulated than NISV formulated HSV antigen preparations. It is likely that this difference relates to the effects of incorporation of the HSV antigens within different adjuvant active structures. It has been demonstrated previously that adjuvants influence the three dimensional structure of an antigen48. It is therefore possible that the processes used to formulate and entrap HSV antigens within NISV or ISCOMs may differentially affect the antigen structure and thus the antigenic determinants recognized by the immune system, although incorporation of antigen into vesicles can re-nature, rather than cause their denaturation4*. Some of the determinants recognized by the immune system may be non-neutralizing, and such epitopes have been previously identified within several HSV glycoproteins by monoclonal antibodies49. Differences in the protection levels afforded by the two adjuvants, although not significant, further suggest that the increased total antibody levels produced by HSV-1 NISV antigen preparation may not be directed against neutralizing epitopes. The circulating antibody response to HSV antigens incorporated into either NISV or ISCOMs was observed to be in both the IgGl and IgG2a subclasses; promotion of IgG2a antibody is especially important in viral infection as this subclass fixes complement5’ and has been reported to have the greatest neutralizing capacity51,52. In addition, in a recent study concerned with the prevention of herpes keratitis in a murine ocular infection model, it was reported that of seven monoclonal antibodies against HSV-1 glycoprotein D, only four were

protective and all were of the IgG2a subclass; the three non-protective monoclonals had a different isotype. Production of IgG2b antibodies as promoted by the use of NISV as adjuvant has been associated5’ with the generation of transforming growth factor /? (TGF-P), a cytokine with numerous effects on B cell antibody production53. TGF-/3 has also been associated with down regulation of Th2 type responses and induction of Thl type cells54. In contrast to the NT antibody responses, significantly higher levels of lymphoproliferative responses were observed in cultures of splenocytes from mice immunized with two doses of the HSV-1 NISV vaccine than those vaccinated with HSV-1 ISCOMs. This difference was particularly apparent when live or heatinactivated HSV-1 was employed as the in vitro stimulant. However, no assessment of the biological activity of the cell-mediated immune response, as shown by the activity levels of CDS+ CTLs, or delayed-type hypersensitivity (DTH) was undertaken in this study. There is some evidence that, although various cellular and humoral host immune mechanisms may contribute to protection against HSV infection’8,55-57, the level of neutralizing antibody plays a central ro1e’4,58~60.Thus, Martin and Rouse show that immunization of mice with a vaccinia virus recombinant expressing gD-1 will induce several T cell responses, including DTH and CTL, but that T-cell dependent induction of neutralizing antibodies plays a major role in protection58. The detection of cytokines IL-2, IL-10 and IFN-7 in spleen cell culture supernatants from mice immunized with HSV-1 NISV or HSV-1 ISCOMs suggests that a wide range of immune activity can be elicited by HSV-1 antigens presented to the immune system of mice in these formulations. It has already been reported that the protein antigen, bovine serum albumin (BSA) when formulated into NISV can promote high levels of serum IgG2a, indirectly indicating their potential for stimulation of the Th- 1 subset of T-cell lymphocyte$ . It has also been reported from this laboratory and elsewhere that ISCOMs incorporating viral or nonviral antigens can induce IgG2a, and IgGl subclass

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HSV-1 glycoprotein immune responses: Y Hassan et al. antibodies in mice, allow access of antigens into the class I MHC-processing pathway, enhance DTH responses and stimulate T cells to secrete cytokines with a Thl profile2S,29,61.6’. Other workers have reported that viral antigens presented as ISCOMs can elicit serum cytokine profiles characteristic of both Thl and Th2 T lymphocyte responses63. In the current study, induction of the signficantly higher levels of IFN-7 by HSV-1 antigens in NISV compared with HSV-1 ISCOMs supports earlier studies”’ suggesting that the former vaccine preparations may preferentially induce Thl lymphocyte responses, while antigens in ISCOMs may be less selective in the immune cell subset responses promoted. However, the present studies also show that NISV formulated with HSV-1 antigens induce relatively high levels of IL-10 which has been demonstrated to inhibit antigen-specific proliferation of Thl cell clones64. It has been suggested that induction of Th2 T cell activity may be less than optimum for the control of some viral infections, including HIV6’, but the relative importance of cellular and humoral immune responses in HSV infections remains unresolved. In the present study, there was little difference in the protection afforded to mice by the two vaccine preparations against lethal homologous or heterologous HSV challenge infection. Nevertheless, the study provides considerable support for the potential of viral vaccines prepared from a mixture of viral glycoproteins incorporated, together with lipids, into a particulate formulation for presentation to the immune system to promote a wide range of immune responses’ I and a consequent optimization of protective capability. Indeed, ISCOM type vaccines are already successful and commercially available for use against a number of veterinary infections”.66.67.

ACKNOWLEDGEMENTS The authors wish to thank Joan McCall, Maggie Conacher and June Irvine for their excellent technical assistance, and Proteus Molecular Design Ltd for their financial support during the duration of these studies.

REFERENCES Nahmias, A., Vistine, A., Caldwell, A. and Wilson, C. Eye infections. Sure. Oohfhamol. 1976. 21. 100-l 09 Hazen, P.G. and ippes, R.B. Eczema herpeticum caused by herpes virus type 2. Arch. Dermat. 1977, 113, 1085-1086 Easty, D.L. Clinical aspects of ocular herpes simplex virus infection. In: Viral Diseases of the Eyes. Lloyd-Luke, London 1985, pp. 135-178 Whitley, R.J. and Hutto, C. Neonatal herpes simplex virus infections. Paed. Rev. 1985, 7, 119-126 Gutman, L.T., Wilfort, C.M. and Eppes, R.B. Herpes simplex virus encephalitis in children: analysis of cerebrospinal fluid and progressive neurodevelopment deterioration. Stand. J. Inf. Dis. 1986, 154, 415-421 Quinnan, G.V., Masur, H., Rook, A.H. et al. Herpes virus infection in acquired immune deficiency. J. Am. Med. Assoc. 1984, 252,72-77 Whitley, R., Arvin, A., Prober, C. ef al. Predictors of morbidity and mortality in neonates with herpes simplex virus infection. N. Eng. J. Med. 1991, 154,415-421 Watson, R.J. and Enquist, L.W. Genetically engineered herpes simplex virus vaccines. frog. Med. Viral. 1985, 31, 84-108 Stanberry, L.R., Bernstein, D.I., Burke, R.L., Pachl, C. and Meyers, M.G. Recombinant herpes simplex virus glycoprotein vaccines protect against initial and recurrent genital herpes. J. Inf. Dis. 1987, 157, 914-920

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10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

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Kino, Y. Yeast-derived glycoprotein B-l of herpes simplex virus (HSV) as a candidate for an HSV vaccine. Adv. Exp. Med. Biol. 1990, 278, 183-190 Erturk, M., Jennings, R., Phillpotts, R.J. and Potter, C.W. Biochemical characterisation of herpes simplex virus type-l immunostimulating complexes (ISCOMs): a multi-glycoprotein structure. Vaccine 1991, 9, 668-674 Wachsman, M., Luo, J.H., Aurelian, L. and Paoletti, E. Protection from herpes simplex virus type-2 is associated with T cells involved in delayed type hypersensitivity that recognise glycosylation-related epitopes on glycoprotein D. Vaccine 1992, 10, 447-454 Nesburn, A.B., Burke, R.L., Ghiasi, H., Slanina, S., Bahri, S. and Wechsler, S.L. Vaccine therapy for ocular herpes simplex virus (HSV) infection: periocular vaccination reduces spontaneous ocular HSV type-l shedding in latently infected rabbits. J. Viral. 1994, 68, 5084-5092 Ghiasi, H., Kawar, R., Nesburn, A.B., Slanina, S. and Wechsler, S. Expression of seven herpes simplex virus type-l glycoproteins (gB, gC, gD, gE, gG, gH and gl): comparative protection against lethal challenge in mice. J. Viral. 1994, 68, 2118-2126 Stannard, L.M., Fuller, A.O. and Spear, P.G. Herpes simplex virus glycoproteins associated with different morphological entities projecting from the virion envelope. J. Gen. Viral. 1987, 68, 715-725 Meignier, B., Jourdier, T.M., Norrild, B., Pereira, L. and Roizman, B. lmmunisation of experimental animals with reconstituted glycoprotein mixtures of Herpes simplex virus 1 and 2: protection against challenge with virulent virus. J. Inf. Dis. 1987, 155,921-930 Erturk, M., Phillpotts, R.J., Welch, M.J. and Jennings, R. Efficacy of HSV-1 ISCOM vaccine in guinea-pig model of HSVP infection. Vaccine 1991, 9, 728-734 Burke, R.L. Development of a herpes simplex virus subunit vaccine for prophylactic and therapeutic use. Rev. Inf. Dis. 1991, 13Suppl. 11, 5906-5911 ErIurk, M., Jennings, R., Hockley, D. and Potter, C.W. Antibody responses and protection in mice immunised with herpes simplex virus type-l antigen immuno-stimulating complex preparations. J. Gen. Viral. 1989, 70, 2149-2155 Byars, N.E., Fraser-Smith, E.B., Pecyk, R.A. et al. Vaccinating guinea-pigs with recombinant glycoprotein D of herpes simplex virus in an efficacious adjuvant formulation elicits protection against vaginal infection. Vaccine 1994, 12, 200-209 Morein, B., Sundquist, B., Hoglund, S., Dalsgaard, K. and Osterhaus, A. ISCOM, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature 1984, 308,457-460 Claassen, I.J.T.M., Osterhaus, A.D.M.E. and Claassen, E. Antigen dectection in vivo after immunisation with different presentation forms of rabies virus antigen: involvement of marginal metallophilic macrophages in the uptake of immunestimulating complexes. Eur. J. Immun. 1995, 25, 1446-1452 De Vries, P., Uytdehaag, F.C.G.M. and Osterhaus, A.D.M.E. Canine distemper virus (CDV) immune-stimulating complexes (ISCOMs), but not measles virus ISCOMs, protect dogs against CDV infection. J. Gen. Viral. 1988, 69, 2071-2083 Lovgren, K., Kaberg, H. and Morein, B. An experimental subunit vaccine (ISCOM) induced protective immunity to influenza virus infection in mice after a single intranasal administration. C/in. Exp. Immun. 1990, 82, 435-439 Fekadu, M., Shaddock, J.H., Ekstrom, J. ef al. An immunestimulating complex subunit rabies vaccine protects mice and dogs against street rabies challenge. Vaccine 1992, 10, 192-l 97 Trudel, M., Naden, F., SBguin, C., Brault, S., Lusignan, Y. and Lemieux, S. Initiation of cytotoxic T-cell response and protection of Balb/c mice by vaccination with an experimental ISCOMs respiratory syncytial virus subunit vaccine. Vaccine 1992, 10, 107-127 Fossum, C., Bergstrom, M., Lovgren, K., Watson, D.L. and Morein, B. Effect of ISCOMs and their adjuvant moeity (matrix) on the initial proliferation and IL-2 responses: comparison of spleen cells from mice inoculated with ISCOMs and/or matrix. Cell. Immun. 1990, 129,414-425 Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R.N. and Berzofsky, J. Induction of CD8+ cytotoxic T cells by immunisation with purified HIV-1 envelope proteins in ISCOMs. Nature 1990, 344, 873-875

HSV-1 glycoprotein immune responses: Y Hassan et al. 29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

Villacress-Eriksson, M., Bergstrom-Mollaoglu, M., Kaberg, H. and Morein, B. Involvement of interleukin-2 and interferongamma in the immune response induced by influenza virus ISCOMs. Stand. J. Immun. 1992, 36, 421-426 Kensil, CR., Patel, U., Lennick, M. and Marciani, D. Separation and characterisation of saponins with adjuvant activity from Ouillaja saponaria molina cortex. J. Immun. 1991, 146, 43l437 Brewer, J.M. and Alexander, J. The adjuvant activity of nonionic surfactant vesicles (niosomes) on the Balb/c humoral response to bovine serum albumin. immunology 1992, 75, 570-575 Brewer, J.M., Roberts, C.W., Stimson, W.H. and Alexander, J. Accurate determination of adjuvant associated protein or peptide by ninhydrin assay. Vaccine 1995, 13, 1441-1444 Grun, J.L. and Maurer, P.H. Different T helper cell subsets elicited in mice utilising two different adjuvant vehicles: the role of endogenous IL-1 in proliferative responses. Cell. lmmun. 1989, 121, 134-145 Snapper, C.M. and Paul, W.E. Interferon-Y and B cell stimulatory factor-l reciprocally regulate immunoglobulin isotype control. Science 1987, 239, 944-947 Snapper, C.M. and Mond, J.J. Towards a comprehensive view of immunoglobulin class switching. Immun. Today 1993, 14, 15-17 Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A. and Coffman, R.L. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immun. 1986, 136, 2348-2357 Mosmann, T.R. and Coffman, R.L. Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immun. 1989, 7, 145-173 Suzuki, Y., Conely, F.K. and Remington, J.S. Importance of endogenous IFN-y for prevention of toxoplasmic encephalitis in mice. J. Immun. 1989,143, 2045-2050 Parker, S.J., Roberts, C.W. and Alexander, J. CD8+ T cells are the major lymphocyte population involved in protective immune response to Toxoplasma gondii in mice. C/in. Exp. Immun. 1991, 64, 207-212 Gazzinelli, R.T., Hakim, F.T., Hieny, S., Shearer, G.M. and Sher, A. Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-Y production and protective immunity induced by an attenuated T: aondii vaccine. J. Immun. 1991, 146. 286-292 Roberts, C.W., Brewer, J.M. and Alexander, J. Congenital toxoplasmosis in the Balb/c mouse: prevention of vertical disease transmission and fetal death by vaccination. Vaccine 1994,12,1389-l 394 Jennings, R. and Erturk, M. Comparative studies of HSV-1 antigens solubilised from infected cells by using non-ionic or zwitterionic detergents. J. Med. Viral. 1990, 31, 98-108 Erturk, M., Hill, T.J., Shimeld, C.A. and Jennings, R. Acute and latent infection of mice immunised with HSV-1 ISCOM vaccine. Arch. Virol. 1992, 125, 87-l 01 Pick, U. Liposomes with a large trapping capacity prepared by freezing and thawing of sonicated phospholipid mixtures. Arch. Biochem. Biophys. 1981, 212, 195-203 Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Analyf. Biochem. 1976, 72, 248-258 Walker, W., Brewer, J.M. and Alexander, J. Entrapment of antigen in lipid vesicles modulates the immune response of human-PBL reconstituted SCID mice to influenza A antigens. Eur. J. Immun. 1996, 26, 1664-1667 Allen, G. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 9. Sequencing of proteins and peptides (Eds Work, T.S. and Burdon R.H.). Elsevier Biomedical Press, North Holland, 1981, pp. 135-160 Boggs, J.M., Hashim, G.A., Day, E.G. and Moscarello, M.A. Lipid induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein. J. Immun. 1985, 133, 2617-2622 Lausch, R.N., Staats, H., Oakes, J.E., Cohen, G.H. and Eisenberg, R.J. Prevention of herpes keratitis by monoclonal antibodies specific for discontinous and continous epitopes on glycoprotein D. Invest. Opthamol. Vis. Sci. 1991, 32, 27352740

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

Spiegelberg, H.L. Biological activities of immunoglobulins of different classes and subclasses. Adv. Immun. 1974, 19, 259 294 Kipps, J., Parham, P., Punt, J. and Herzenberg, A. Importance of immunological isotype in human antibody-dependent cell-mediated cytotoxicity directed by murine monoclonal antibodies. J. Exp. Med. 1985, 161, 1-17 Coultier, J.-P., Van de Logt, J.T.M., Heesen, F.W.A., Warnier, G. and Van Snick, J. IgGPa restriction of murine antibodies elicited by viral infections. J. Exp. Med. 1987, 165, 64-69 Snapper, C.M., Waegell, W., Beernink, H. and Dasch, J.R. Transforming growth factor-61 is required for secretion of IgG of all subclasses by LPS-activated murine B cells in vifro. J. Immun. 1993, 151, 4625-4636 Nagelkerken, L., Gollob, K.J., Tielemans, M. and Coffman, R.L. Role of transforming growth factor-beta in the preferential induction of T helper cells of type-l by staphylococcal enterotoxin B. Eur. J. Immun. 1993, 23, 2306-2310 Cunningham, A.L. and Noble, J.R. Role of keratinocytes in human recurrent herpetic lesions. Ability to present herpes simplex virus antigens and act as a target for T lymphocyte cytotoxicity in vitro. J. C/in. Invest. 1989, 63, 490-496 Schmid, D.S. and Mawle, A.C. T cell responses to herpes simplex viruses in humans. Rev. Inf. Dis. 1991, 13Suppl. 11, 59465949 Simmons, A., Tscharke, DC. and Speck, P. The role of immune mechanisms in control of herpes simplex virus infections of the peripheral nervous system. Curr. Topics Microbial Immun. 1992,179,31-56 Martin, S. and Rouse, B.T. The mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: clearance of local infection. J. Immun. 1987, 138, 3431-3437 Al-Ghamdi, A., Jennings, R., Bentley, H. and Potter, C.W. Latent HSV-1 Infection in mice immunised with a zwitterionic detergent-extracted HSV-1 antigen preparation. Arch. Viro/. 1989, 108, 19-31 Kohl, S. Role of antibody-dependent cellular cytotoxicity in defense against herpes simplex virus infections. Rev. Inf. Dis. 1991,13,108-114 Ben-Ahmeida, E.T.S., Jennings, R., Erturk, M. and Potter, C.W. The IgG responses and protection in mice immunised with influenza antigens administered as ISCOMs with FCA ALH or as infectious virus. Arch. Viral. 1992, 125, 71-86 Heeg, K., Kuon, W. and Wagner, H. Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immune stimulating ovalbunim complexes enter the class I MHCrestricted antigen pathway and allow sensitization against the immunodominant peptide. Eur. J. Immun. 1991,21,1523-l 527 Valensi, J.M., Carlson, J.R. and Van Nest, G.A. Systemic cytokine profiles in Balb/c mice immunised with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J. Immun. 1994, 153, 4029-4039 de Waal Malefyt, R., Haanen, J., Spits, H. et a/. Interleukin 10 (ILlO) and viral IL10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via down regulation of class II major histocompatibility complex expression. J. Exp. Med. 1991, 174, 915-924 Clerici, M. and Shearer, G.M. A Thl-Th2 switch as a critical step in the aetiology of HIV infection. Immun. Today 1993, 14, 107-110 Osterhaus, A., Weijer, F., Uytdehaag, F.C.G.M., Jarrett, O., Sundquist, B. and Morein, B. Induction of immune response in cats by vaccination with feline leukemia virus ISCOM. J. Immun. 1985, 135,591-596 Mumford, J.A., Jessett, D.M., Rollinson, E.A., Hannant, D. and Draper, M.E. Duration of protective efficacy of equine influenza immunostimulating complex/tetanus vaccine. Vet. Record 1994, 134, 158-162

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