Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems

Vaccine 18 (2000) 2083±2094

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Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems S.A. Mohamedi a, J.M. Brewer b, J. Alexander b, A.W. Heath a, R. Jennings a,* a

Sheeld Institute for Vaccine Studies, Division of Molecular and Genetic Medicine, Floor `F', University of Sheeld Medical School, Beech Hill Road, Sheeld S10 2RX, UK b Department of Immunology, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G4 0NR, UK Received 7 July 1999; received in revised form 10 November 1999; accepted 7 December 1999

Abstract The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen speci®c total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-g)) production by in vitro restimulated spleen cells. The degree of protection a€orded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The ®ndings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, in¯uenced the immunogenicity of the ®nal preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-g levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration±rehydration NISV, or entrapped in NISV by freeze±thawing at 308C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-g levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze±thawing with NISV at 608C (HSV-2 NISV 60) did not. There were no di€erences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was a€orded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was a€orded by the HSV-2 NISV 30 vaccine formulation. The ®ndings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: HSV-2 vaccine; Immunostimulating complexes (ISCOMs); Non-ionic surfactant vesicles (NISV)

1. Introduction Infections with herpes simplex virus type 2 (HSV-2) are a major public health problem and continue to be so partly because of the high proportion of sub-clinical * Corresponding author. Tel.: +44-114-272-4072; fax: +44-114273-9926. E-mail address: [email protected] (R. Jennings).

or unrecognised cases and the frequency of viral shedding in the absence of symptoms [1,2]. Although neonatal herpes infections are relatively rare, they can give rise to serious systemic disease [3]. HSV-2 is also a common cause of encephalitis in the United States a€ecting both children and adults [4]. The mortality rate of neonatal HSV infection has declined with current antiviral therapy, although the mortality rate in CNS disease (15%) and disseminated

0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 5 6 7 - 8

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disease (57%) remains high [3]. Furthermore, genital herpes causes considerable psychological and psychosexual morbidity [5], while it is now recognised that this disease is a co-factor in the transmission of the human immunode®ciency virus [6]. It is clear, therefore, that genital herpes is an important disease to control. Although antiviral agents such as acyclovir, can reduce disease severity, prevent recurrences and shorten periods of viral shedding, and while various treatment options continue to be explored, resistance of HSV to antiviral drugs is an emerging problem in immuno-compromised patients [7,8], and suggests that ultimately the only way to impact incidence of this disease is by vaccination. Although traditional approaches to virus vaccination such as live attenuated viruses and inactivated whole virus vaccines theoretically o€er promise, concerns regarding safety issues surround the development of a live viral vaccine for HSV, since attenuated HSV may theoretically retain its potential for latency and reactivation, and the ability to revert to a virulent form [9]. Therefore, the knowledge that major antigenic sites of HSV are located on the envelope glycoproteins, together with opportunities for separating surface viral proteins from detergent-solubilised infected cells, have led many interested in HSV vaccination towards development of protein subunit vaccines which in terms of safety, ecacy and antigenic speci®city, show considerable promise for control of genital herpes infection [10]. In addition, although in general, subunit virus vaccines are relatively expensive to produce and are poorly immunogenic, with the use of suitable adjuvants less antigen may be required to stimulate high immune responses, thus saving on vaccine cost [11]. The manner in which viral antigens are presented to the immune system is an important factor governing the nature and level of the subsequent immune response [12±17]. In the development and evaluation of vaccines against infections caused by the herpes simplex viruses, it is of considerable importance to establish the nature of the immune responses elicited by di€erent vaccine formulations, as some responses may be more relevant for protection against primary infection, the establishment of latent infection or the prevention of recurrent disease from the latent state. T lymphocytes have been implicated as major contributors to the immunological control of HSV as severe herpetic infections often occur in patients with T cell de®ciencies [18±20]. Animal studies show that both CD4+ and CD8+ T lymphocytes play crucial roles in the protection of cutaneous [21±23], mucosal [24,25] and peripheral nervous tissues [26,27] against both primary and recurrent HSV infection, whilst optimal protection against a lethal intraperito-

neal challenge occurs in the presence of both subpopulations [28]. HSV-speci®c CD4+ T cell clones are bifunctional, exhibiting both accessory function, as helper cells to mount e€ective antibody responses [28], and cytolytic activity [29]. CD4+ T cells play a dominant role in the resolution of cutaneous HSV infection and interferon-gamma (IFN-g) has a profound e€ect on the level of protection a€orded by these cell types [30]. The primary function of CD8+ cytotoxic T lymphocytes (CTLs) is to limit the spread of HSV to the peripheral nervous system [21,31]. These cells also produce IFN-g, which serves to recruit macrophages to the site of infection, induce an antiviral state in bystander cells [32] and may clear an established HSV infection [30]. CD8+ T lymphocytes have also been implicated in the protection of mucosal surfaces against primary HSV infection. Severe genital infection is observed in mice following the in vivo depletion of vaginal CD8+ T cells [25]. Recent studies have shown high numbers of CD8+ CTL precursors in patients with recurrent genital herpes [33,34]. Koelle et al. [35] have demonstrated that NK (natural killer) cells and CD4+ T cells predominate early in lesion formation, followed by an in¯ux of CD8+ T cells as the lesion develops. The consensus opinion is that an essentially cell-mediated immune response e€ected through cytotoxic T lymphocytes (CTLs) may be pivotal in eliciting protection against primary HSV infections in both experimental animals and humans [19,21,36±38], although antibody may also be involved [39,40]. Such protection, in mice at least, may be brought about through Th1- rather than Th2-type responses [19]. The former response in these animals is associated with high levels of IgG2a antibodies and the cytokines interferon-gamma (IFN-g) and interleukin-2 (IL-2), as opposed to interleukin-4 (IL-4), and the production of IgG1 antibodies [41,42]. Previous studies from this laboratory have reported on the nature of the immune response and the protection induced by HSV-1 antigen preparations incorporated into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) [43±46]. Both ISCOMs and NISV are e€ective stimulators of IFN-gdependent, antigen-speci®c IgG2a production [12,47± 49], and both HSV-1 ISCOMs and HSV-1 NISV can induce protection against homologous and heterologous HSV challenge infection in mice [46]. The present paper is concerned with additional assessment of ISCOMs and NISV as delivery systems for HSV-2 antigens in the murine model of primary lethal HSV infection. The e€ect of these delivery systems on the nature and level of the cytokine and subclass antibody responses of these animals to HSV-2 has also been evaluated.

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2. Materials and methods 2.1. Virus strains and virus glycoprotein preparation The virus strains used, their growth and titration by plaque assay in Vero cells have all been described in detail elsewhere [46]. HSV glycoprotein antigens for use as a vaccine preparation were obtained by extraction with zwitterionic detergent using procedures described earlier for HSV-1 [45]. These procedures were found to be e€ective for preparation of HSV-2 glycoproteins from virus cultured in Vero cells. Partial characterisation of the HSV-2 glycoprotein preparations was carried out following centrifugation of the preparations on sucrose density gradients and probing of individual fractions for the presence of the common HSV glycoproteins B, D and E and the HSV2 speci®c glycoprotein G-2, using monoclonal antibodies speci®c for the various glycoproteins. These were kindly supplied from the University of Goteborg, Sweden, through Dr Stig Jeansson. 2.2. HSV-2 vaccine preparations The HSV-2 ISCOM vaccine was prepared using methodologies similar to those used for HSV-1 and described in detail elsewhere [50]. ISCOM formation from the HSV-2 glycoprotein preparation using Quil A (kindly donated by Professor Bror Morein, National Veterinary Institute, Division of Vaccine Research, Uppsala, Sweden), was con®rmed by electron microscopic visualisation of the characteristic cage-like structures [50,51]. Non-ionic surfactant vesicles incorporating HSV-2 antigens (NISV HSV-2) were also prepared under aseptic conditions, essentially by methods previously described in detail [46,48]. However, in this study, the HSV-2 antigen preparation was also entrapped into NISV using the usual freeze±thaw cycle [52], but with thawing at temperatures of 308C followed by 2 h agitation again at 308C. Alternatively, HSV-2 antigens were entrapped into preformed NISV using the dehydration±rehydration (DRV) method described elsewhere [53,54]. In this experiment therefore, three NISV HSV-2 preparations were investigated, HSV-2 NISV 608 and HSV-2 NISV 308, termed NISV 60 and NISV 30 respectively, and dehydration±rehydration NISV termed DR-NISV. 2.3. Anti-HSV antibody determinations in mouse sera These were carried out by two procedures, enzymelinked immunosorbent assay (ELISA) for both total and subclass IgG1 and IgG2a antibodies using an antibody sandwich technique, and the neutralisation test (NT). Both procedures have been detailed elsewhere

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[55,56]. To determine endpoint titres of total or subclass IgG antibodies in sera from mice immunised with various HSV-2 antigen preparations, an ELISA procedure was performed according to the methods used by the workers in our laboratories [57]. The endpoint titre of each serum was considered as that dilution at which the OD (optical density) of the tested serum was at least equal to, or less than, the OD reading of negative control sera. 2.4. Lymphoproliferative responses of mouse splenocytes in vitro Spleens obtained from three representative animals in each immunised or control group of mice were homogenised in RPMI 1640 medium (Life Technologies Ltd, Paisley, Scotland) +10% foetal calf serum (FCS) and plated in 96-well culture plates at a concentration of 4  105 viable cells in 100 ml/well. The cells were then restimulated in vitro with live or heat-inactivated HSV-2, or HSV-2 subunit antigen preparation. After 72 h, supernatant ¯uids were removed to ÿ708C for subsequent assay for the cytokines IL-2, IL-4 and IFN-g, and the remaining cells pulsed with 50 ml (0.5 mCi/well) of [3H]thymidine at 1 mCi mmolÿ1 (Amersham International, Amersham, UK). The amount of incorporated label was determined by liquid scintillation counting using a scintillation counter. Cell proliferation was expressed as mean counts per minute (c.p.m) in the presence of antigen minus c.p.m. in the absence of antigen2standard error. 2.5. Assays for cytokines These were carried out on supernatants collected from mouse spleen cells cultured as set out above, by an ELISA procedure described in detail elsewhere [46]. Standard curves were produced using recombinant mouse IFN-g (Cambridge Bioscience Ltd, Cambridge), recombinant mouse IL-2 (Pharmingen Diagnostics Ltd, Cambridge) or recombinant mouse IL-4 (kindly donated by Dr. S. Menon, DNAX Research Institute, Palo Alto, USA). Other reagents, including puri®ed rat anti-mouse IFN-g, rat anti-mouse IL-2 for coating the plates, and their biotin-labelled equivalents for use as the second antibody were all purchased from Cambridge Bioscience Ltd (Pharmingen). Coating and secondary antibody for use in the IL-4 assay were kindly donated by DNAX Research Institute. Results are expressed as units/ml for IL-2 and IL-4, and as pg/ml for IFN-g. 2.6. Immunisation and infection procedures Female Balb/c mice aged 8±10 weeks were used throughout, and were supplied from the closed, ran-

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3. Results

domly-bred colony at the University of Sheeld. A small, representative number of mice were pre-bled, and groups of 25±30 animals immunised subcutaneously using 0.2 ml volumes of the various HSV-2 vaccine preparations, or mock-immunised with PBS (phosphate-bu€ered saline, pH 7.2). Each vaccine preparation was administered in two doses three-weeks apart, and each dose contained 5 mg of HSV-2 antigen preparation. Seven days subsequent to the second immunisation, i.e. 48 h prior to challenge infection, three mice in each group were killed, their spleens removed and cell cultures set up as described earlier to assess lymphoproliferative responses and cytokine levels. On the following day a blood sample was collected from each of the remaining mice, the sera separated and stored individually for ELISA and NT antibody determinations and the mice in each vaccine-immunised or PBS control group, subdivided into two further groups. Twenty-four hours following the blood sampling one of these groups was challenge-infected with homologous HSV-2 (strain 333) at 6 mouse lethal doses (MLD50), while the other was challenge-infected with HSV-1 (strain WAL) at the same dosage level. Both challenge infections were given intraperitoneally (i.p.) in volumes of 0.2 ml. The animals were then observed daily over a period of 21 days for evidence of severe illness. The intraperitoneal route of challenge infection as used in the present studies in mice, although an unnatural route in man, is valuable for evaluating the potential of experimental vaccine preparations and for screening new drugs [58], whilst mice are extensively used in studies on the immune response to HSV vaccines, often providing useful immunological data.

3.1. Antiviral antibodies in mouse sera Table 1 shows the levels of total IgG, subclass IgG1 and IgG2a HSV-speci®c antibodies, as measured by ELISA endpoint titration, in sera obtained from immunised or mock-immunised mice following two doses of the various HSV-2 vaccine preparations. The results indicate that there were no signi®cant di€erences with respect to the levels of total IgG antibody induced three weeks following two immunisations with the di€erent vaccine preparations. Although there were no statistically signi®cant di€erences in levels of the IgG1 subclass antibody between the vaccine groups, the HSV-2 ISCOM vaccine preparation induced signi®cantly greater (P < 0.01) IgG2a responses in mouse sera compared to each of the other preparations. It is of interest to note that the ratio of IgG1 to IgG2a antibodies for the HSV-2 ISCOM preparation, as calculated from the actual endpoint titres, was near to equivalence (1.27) compared to all other preparations; IgG1:IgG2a ratios for HSV-2 NISV 30, HSV-2 NISV 60 and HSV-2 DR-NISV were 2.14, 2.77 and 2.07 respectively. Table 1 also shows the mean NT antibody titres in sera from immunised mice and these results also indicate that signi®cantly greater (P < 0.05) NT antibody titres were present in sera from mice immunised with the HSV-2 ISCOM vaccine than for all other preparations, including the NISV 30 preparation. Signi®cantly higher NT antibody titres were also induced in comparison to either the HSV-2 NISV 60 or the HSV2 DRV-NISV vaccine formulation (P < 0.05), by the HSV-2 NISV 30 vaccine.

2.7. Statistical analyses

3.2. Lymphoproliferative responses

The Mann±Whitney `U' test was employed to analyse the experimental data relating to the cytokine levels in spleen culture supernatant ¯uids, lymphoproliferative results and antibody levels in mouse sera. Values of P < 0.05 were taken as signi®cant.

These were assessed in cell cultures prepared from spleens collected seven days following the second immunisation of mice in the presence of three di€erent in vitro stimulating agents, live HSV-2, heat-inactivated

Table 1 ELSIA and neutralising antibody titres in sera from mice immunised with HSV-2 vaccine preparations Mean antibody titres (range) 3 weeks post-second immunisation Immunising agent

No. of sera tested

Total IgG

IgG1

HSV-2 HSV-2 HSV-2 HSV-2 PBS

19 21 19 21 21

4.0720.21b (3.7±4.31) 4.0220.18 (3.71±4.31) 3.9320.31 (3.41±4.31) 3.9120.32 (3.41±4.31) 2.1720.21 (1.90±2.50)

3.9620.23 3.9220.33 3.8520.38 3.8020.27 2.0020.26

a b

ISCOMs NISV 30 NISV 60 DR-NISV

NTa

IgG2a (3.71±4.31) (3.41±4.31) (3.11±4.31) (3.11±4.01) (1.60±2.20)

3.8520.21 3.5920.26 3.4120.35 3.4720.37 2.0020.26

NT=Neutralising antibody. Reciprocal of the log of the endpoint dilution for ELISA or NT antibody (2standard deviation).

(3.71±4.31) (3.41±4.01) (3.11±4.01) (3.11±4.01) (1.60±2.20)

2.5720.41 2.0620.31 1.7520.23 1.8320.30 0.7020.14

(1.80±3.01) (1.80±2.71) (1.50±2.11) (1.50±2.41) (0.60±0.90)

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Fig. 1. Lymphoproliferation responses and levels of IL-2, IL-4 and IFN-g detected in spleen cell culture supernatants from mice immunised with various HSV-2 vaccine formulations. (a) Lymphoproliferation responses, (b) IL-2, (c) IL-4, (d) IFN-g levels in mouse spleen culture supernatants obtained from mice 7 days subsequent to the second immunisation. The in vitro stimulants used were HSV-2 subunits ; heat inactivated HSV-2 ; or live HSV-2 Q. PBS=negative control group, inoculated with diluent through the experiment.

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HSV-2 and HSV-2 subunit antigen preparation. When live virus was used as the in vitro stimulant, the levels of lymphoproliferation obtained were signi®cantly greater (P < 0.05) for the group immunised with HSV2 ISCOMs than for all other groups, except that receiving the HSV-2 NISV 30 vaccine, where P = 0.05 (Fig. 1a). No signi®cant lymphoproliferative responses were detected in spleen cell cultures established from mock-immunised mice. The greatest responses to the HSV-2 ISCOM vaccine preparation were observed in spleen cell cultures stimulated in vitro with live HSV-2 (Fig. 1a), with counts 1.2- to 1.4-fold greater than those seen in cultures stimulated with heat-inactivated HSV-2, but this was not uniformly observed in cultures of spleen cells from mice immunised with the HSV-2 NISV 30 or HSV-2 NISV 60 vaccine preparations. 3.3. Cytokine levels in in vitro stimulated spleen cell cultures The levels of IL-2, IL-4 and IFN-g detected in supernatant ¯uids from cultures of spleen cells taken from mice immunised with the various vaccine preparations are shown in Fig. 1b, c and d. 3.3.1. Interleukin-2 (IL-2) Fig. 1b shows that the greatest levels of IL-2 were detected in cultured spleen cells from mice immunised with the HSV-2 ISCOM vaccine, and these levels were 2.5-fold greater (following in vitro stimulation with live HSV-2), than those present in cultures from mice immunised with all other preparations and these di€erences were signi®cant (P < 0.05). In addition, as noted for the lymphoproliferation results, in vitro stimulation with live virus elicited greater levels of IL-2 than those detected when heat-inactivated virus or HSV-2 subunits were used as in vitro stimulants. 3.3.2. Interleukin-4 (IL-4) Fig. 1c shows the IL-4 levels detected in spleen cell cultures from HSV-2 immunised mice, and it can be seen that these levels were essentially similar for each of the immunised groups, and no particular vaccine elicited IL-4 levels, following in vitro stimulation with live HSV-2, heat-inactivated HSV-2 or HSV-2 subunit antigen preparations, that were markedly higher or lower than any other, and no signi®cant di€erences were recorded. However, the greatest levels of IL-4 were recorded in spleen cell culture supernatants stimulated in vitro with live HSV-2, and the lowest following stimulation with the subunit antigen preparation. 3.3.3. Interferon-gamma (IFN-g) These results are presented in Fig. 1d and show that

signi®cantly greater levels of IFN-g were present in spleen cell culture supernatants from mice immunised with the HSV-2 ISCOM vaccine in comparison to those from mice immunised with all other preparations (P < 0.05). Thus, in spleen cell culture supernatants stimulated in vitro with live HSV-2, IFN-g levels were at least 3.1-fold greater from HSV-2 ISCOM immunised mice than from mice given any other vaccine preparation. 3.4. Protection against HSV-1 or HSV-2 challenge infection In this study, mice were challenge-infected with 6 MLD50 of heterologous HSV-1 (strain WAL), or homologous HSV-2 (strain 333) three weeks following booster immunisation. The results, shown in Fig. 2, indicate that following homologous HSV-2 challenge infection (Fig. 2a), eight of ten mice immunised with the HSV-2 ISCOM vaccine preparations survived; in contrast, only three of nine (33%) animals vaccinated with the HSV-2 NISV 60 preparation survived the homologous challenge. The HSV-2 NISV 30 preparation was intermediate in the extent of protection provided against homologous HSV-2 challenge, with ®ve of eleven (46%) of mice surviving infection. Challenge infection with the heterologous HSV-1 (strain WAL) resulted in lower protection rates than those observed following HSV-2 challenge, and only ®ve of nine (56%) mice (Fig. 2b), immunised with two doses of HSV-2 ISCOM vaccine survived the challenge. Protection rates in all other immunised groups were lower than this, and indeed, in the group of mice vaccinated with the HSV-2 NISV 60 preparation there were no survivors of heterologous challenge. 4. Discussion Strategic approaches for immunisation against infections caused by the herpes simplex viruses must take into account a number of factors. These include the complexity of the virus, in particular the large number of surface glycoproteins [59], the HSV type (1 or 2) that is to be the target of immunisation, the type of disease, whether primary or recurrent, that is to be controlled [60], and the nature of the immune response to be induced. Pre-clinical evaluations of candidate HSV vaccines are often carried out in murine models where many of these factors can be tested. Because of the high number of HSV surface glycoproteins, strategies for vaccines against this virus may be divided into those that opt for immunisation with no more than one or two highly-puri®ed or recombinant glycoproteins, usually the quantitatively most abundant ones, gB and gD [61±63], which are also type-common

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[64,65], or more recently, the DNA coding for such proteins [66±68]. Alternatively, some vaccines are based on a `blunderbuss' approach where all or as many as possible of the surface glycoproteins are incorporated into the vaccine. These latter types of HSV vaccine include live, attenuated and genetically-disabled vaccine preparations [69±71], in addition to those consisting of inactivated whole virus [72] or mixtures of several partly-characterised and puri®ed HSV antigens [44,73,74]. Support for this latter approach is gained from the work of Ghiasi et al. [75], showing that greater protection against homologous HSV-1 challenge can be achieved using a mixture of seven HSV-1 glycoproteins, rather than any one alone. It is

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believed that genetically-disabled HSV vaccines or potential live attenuated vaccines, may promote broader and more relevant immune responses due in part to the manner of their presentation and processing through the immune system [17,60]. In contrast, single or multiple HSV glycoprotein vaccine preparations require some form of adjuvant, carrier or delivery system to optimise or appropriately channel their presentation to, and handling by, the immune system [12,60]. In the present study, Empigen extracted, gradient puri®ed HSV-2 infected Vero cell cultures were used to prepare HSV-2 antigen preparations with a high degree of purity [76,77]. This preparation was used as the antigen in vaccine formulations; as the capture

Fig. 2. Protection of mice against 6 MLD50 of homologous HSV-2, strain 333 (a), or 6 MLD50 of heterologous HSV-1, strain WAL (b), following challenge at 3 weeks subsequent to the second immunisation. Mice were immunised with HSV-2 ISCOMs (*), HSV-2 NISV 30 (R), HSV-2 NISV 60 (r), HSV-2 DR-NISV (w) or given PBS (q).

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antigen for the ELISA, and also as a source of antigen in the T cell proliferation assays. To assess any potential impact of anti-Vero cell responses, mock ISCOMs prepared in Vero cells were used as antigen in representative ELISAs; as a stimulant in representative proliferation assays, and in mice as mock vaccine preparations in certain experiments. The results indicated that both in vitro and in vivo responses to mock ISCOMs were similar to those of negative controls, and con®rmed previous reports that the proteins present in such antigen preparations are primarily of viral rather than cellular origin [77]. The HSV-2 ISCOM vaccine used here, and containing several HSV glycoproteins, produced greater protection against both homologous HSV-2 and heterologous HSV-1 challenge infection in comparison to the protection observed following two immunisations with either of the HSV-2 NISV preparations, or with the HSV-2 DR-NISV preparation. These ®ndings are in general agreement with earlier studies comparing HSV-1 ISCOM and NISV preparations [46]. The studies described here con®rmed previous work on HSV-2 vaccine formulations, and present the data from one of two experiments giving identical results. Although not statistically signi®cant, the levels of protection between the groups given di€erent types of NISV formulations or the HSV-2 ISCOM preparation were reproducible (Mohamedi, PhD thesis, 1999). During formulation of the HSV-1 NISV vaccine used previously, a cycle of liquid nitrogen freezing accompanied by thawing at 608C was employed [46,52], and it is possible that such harsh treatment may have caused some denaturation of the antigenic determinants of the HSV-1 antigens. This e€ect may have resulted in a decreased capacity to elicit the appropriate immune responses, in particular the generation of virus neutralising antibodies. In attempts to o€set this, the HSV-2 NISV vaccines in the current study were prepared using a freeze±thaw cycle that involved temperatures of only 308C, and in addition, HSV-2 antigen entrapment into NISV by the dehydration± rehydration method [53,54] was also performed. In theory, preparation of antigen into NISV using the DR method should avoid denaturation as this method does not require subjecting the antigen/NISV mixture to great variations in temperature associated with the freeze±thaw method. Furthermore, earlier work has indicated that incorporation of viral glycoproteins into DRV liposomes can elicit protective antibodies in both mice and hamsters [54]. However, the current ®ndings indicate that although the NISV-30 preparation induced greater protection against homologous and heterologous challenge with lethal HSV-2 or HSV-1 than the HSV-2 NISV 60 vaccine preparation, this level of protection was also markedly greater than that a€orded to mice by the HSV-2 DR-NISV vaccine.

This would suggest that the stability of the antigen during the procedures used to formulate the antigen into adjuvants is an important issue in adjuvant selection. Antigen stability is also a problem for certain other carrier or delivery systems, as in the encapsulation of antigen into biodegradable microspheres [11]. The di€erences in protective levels achieved by these di€erent vaccine formulations, correlated with the neutralising antibody levels elicited by the various vaccine preparations, the levels of the cytokines IL-2 and IFN-g induced and the subclass IgG2a antibody levels. In each case signi®cantly higher levels of each parameter were induced by the HSV2 ISCOM vaccine preparation. However, there are di€erences compared to the earlier study [46], with regard to the IgG2a and IFN-g levels elicited by the HSV ISCOMs and HSV NISV preparations. Thus, the HSV-NISV preparation used previously induced markedly higher IFN-g levels than the HSV-1 ISCOMs, while the levels of IgG2a elicited by both preparations were similar [46]. The reasons for the di€erences are unknown and studies on the immune responses and protective capacity associated with HSV-1, as compared to HSV-2 ISCOM and NISV vaccine formulations are required. It is known that there are di€erences between the two HSV types in terms of their interaction with the immune system [78,79]. Thus, in vitro experiments have shown that HSV-2 infected cells are much less susceptible to lysis by cytotoxic T lymphocytes (CTLs) than are HSV-1 infected cells, due, at least in part, to a much greater reduction in expression of class 1 MHC antigens in cells infected with HSV-2 [78]. However, whether such di€erences would be re¯ected through immunisation procedures is uncertain. In the current work the relatively high levels of neutralising antibody, subclass IgG2a antibody and IFN-g present in mice immunised with the HSV-2 ISCOM vaccine, may all be factors contributing to the higher level of protection observed. A number of workers have reported that antibody can prevent severe illness and death, as well as the establishment of latent infection in mice challengeinfected with lethal HSV [39,80±82]. In the murine model, IgG2a predominates in the antiviral immune response against a number of viruses, including HSV, whereas soluble protein antigens produce primarily IgG1 [83,84]. Murine IgG2a exhibits di€erent e€ector functions to the IgG1 subclass, is a potent mediator of complement-®xation [85,86], and has relatively high viral neutralising capacity [83,87]. IgG2a is also the most e€ective murine subclass for the induction of macrophages and ADCC (antibody-dependent cell cytotoxicity), making these antibodies most e€ective for opsonisation and phagocytosis whereas IgG1 has more limited ADCC

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activity [88,89]. The current results showing that the ratios of IgG1 to IgG2a antibodies for the HSV-2 ISCOM vaccine are nearer to equivalence (1.27) compared to all other vaccines tested, indicate a bias towards a Th1 type response. In addition, high levels of the cytokines IL-2 and IFN-g, indicative of a bias towards Th1 immune responses [41], have been correlated with protection against HSV [19,36]. Di€erences in levels of both IL-4 and subclass IgG1 antibodies elicited by the di€erent vaccine preparations in the present study were not marked, suggesting these factors may correlate less well with resistance to HSV infection in these animals. Other workers have reported similar ®ndings [90]. Incorporation of viral antigens into ISCOMs can stimulate mechanisms re¯ecting a broad response of the immune system, including antibody and cytokine pro®les representing both Th1 and Th2 cell activity [46,91±95]. On the other hand, there is previous evidence that antigen-bearing NISV may preferentially stimulate a Th1 response pro®le in mice [46,48,49], although this was not the case in the present study, in which HSV-2 antigens were used to stimulate the immune response. Although no attempts were made here to use the mucosal route of immunisation or to assess speci®c IgA responses, studies in our laboratory have shown that HSV-ISCOMs represent an appropriate formulation for oral delivery of HSV antigens, promoting long-lasting immune responses in mice, including secretory IgA (Mohamedi, submitted for publication). However, recent murine studies have demonstrated that IgG in vaginal secretions confers protection against virulent intravaginal challenge with HSV-2 whereas virus-speci®c sIgA contributed little to protection [96,97]. The results of the present study thus indicate that the incorporation of a mixture of HSV-2 antigens into ISCOMs, or into NISV by di€erent procedures, evokes di€erent levels of immune responses and protection in mice. The ®ndings using the HSV-2 antigen preparation are somewhat at variance with those obtained earlier using an HSV-1 antigen preparation formulated into ISCOMs or NISV by similar procedures [46], suggesting that antigen preparations, even from closely related viruses, may exhibit di€erences in their immunogenicity on incorporation into particular adjuvant vehicles. This observation is supported by known di€erences between HSV-1 and HSV-2 in their surface glycoproteins, particularly glycoproteins C [65,98] and G [99], probably in¯uencing the physical nature of the antigen preparation used for incorporation, and the interaction with the murine immune system [48,49]. Further, comparative studies to investigate the detailed interaction of HSV-1 and HSV-2 viral glycoproteins with the carriers or delivery systems used to enhance

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the immune response to these glycoproteins, are required.

Acknowledgements This work was supported by a grant from the Iranian Ministry of Health and Medical Education to one of the authors (SAM).

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