Immunostimulating complexes (ISCOMs) for nasal vaccination

Advanced Drug Delivery Reviews 51 (2001) 149–159 www.elsevier.com / locate / drugdeliv

Immunostimulating complexes (ISCOMs) for nasal vaccination ¨ Ke-Fei Hu*, Karin Lovgren-Bengtsson, Bror Morein Swedish University of Agricultural Sciences, College of Veterinary Medicine, Department of Veterinary Microbiology, Section of Virology, Box 585, BMC, S-751 23 Uppsala, Sweden

Abstract The immunostimulating complex (ISCOM) is documented as a strong adjuvant and delivery system for parenteral ¨ immunization. Its effectiveness for mucosal immunization has also been proven with various incorporated antigens. Lovgren et al. were the first to demonstrate the capacity of influenza virus ISCOMs to induce mucosal immune response and protection after one comparatively low nasal dose. Further studies show that similar to Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), ISCOMs break immunological tolerance and exert strong mucosal adjuvant activity, resulting in secretory IgA and systemic immune responses. Striking is the capacity of ISCOMs to induce CTL response also after nasal administration. In contrast to CT, ISCOMs initiate mucosal as well as systemic immune responses in an IL-12 dependent manner but independently of IL-4. The recombinant B subunit of cholera toxin (rCTB) was incorporated in the same ISCOM particle to explore symbiotic effects. The IgA response to rCTB in lungs was increased 100-fold when rCTB was administered nasally in ISCOMs and more than 10-fold in the remote mucosa of the genital tract. An enhanced IgA response to a passenger antigen OVA was recorded in the remote genital tract. After i.n. administration of the envelope proteins of respiratory syncytial virus in ISCOMs, high serum antibodies were induced, almost at the same levels as those following parenteral immunization and potent IgA responses were also evoked both at the local respiratory mucosa, and in the cases tested at the distant mucosae of the genital and intestinal tracts. Similar results have also been recorded with ISCOMs containing envelope proteins from Herpes simplex virus, Influenza virus and Mycoplasma mycoides. The mucosal targeting property of envelope proteins of RSV was utilized in an HIV-gp120 RSV ISCOM formulation. After nasal administration an enhanced mucosal IgA response to gp120 was observed in the female reproductive tract. In general, antigens derived from envelope viruses or cell membranes incorporated into ISCOMs retain their biological activity and conformation, encompassing the mucosal targeting and virus neutralizing properties.  2001 Elsevier Science B.V. All rights reserved. Keywords: Adjuvant; Delivery system; Quillaja saponin; Mucosal immunity; Intranasal; Subcutaneous; Oral immunization; Cholera toxin; IgA; IL-12

Contents 1. Introduction ............................................................................................................................................................................ 2. ISCOM concept ...................................................................................................................................................................... 2.1. Quillaja saponins ............................................................................................................................................................. 2.2. APC response to ISCOMs ................................................................................................................................................ 2.3. T-cell response to ISCOMs ...............................................................................................................................................

*Corresponding author. Tel.: 1 46-18-471-4573; fax: 1 46-18-504-603. E-mail address: [email protected] (K.-F. Hu). 0169-409X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00165-X

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2.4. B-cell response to ISCOMs............................................................................................................................................... 2.5. Protective immunity induced by ISCOMs .......................................................................................................................... 3. Nasal site is a preferred route for mucosal immunization ............................................................................................................ 3.1. Role of the induction site .................................................................................................................................................. 3.2. Parenteral administration for the induction of mucosal immune responses ............................................................................ 3.3. Mucosal administration .................................................................................................................................................... 3.3.1. Nasal route of administration .................................................................................................................................. 3.4. ISCOMs with targeting molecules and passenger (vaccine) antigens for nasal administration ................................................. 4. Conclusions and future considerations ...................................................................................................................................... References ..................................................................................................................................................................................

1. Introduction Most licensed vaccines are administered parenterally inducing mainly systemic immune response but hardly any mucosal response, essentially due to difficulties to obtain potent immune responses by mucosal routes particularly with non-replicating antigens. Consequently most vaccines for mucosal administration are still based on living microorganisms. However, live vaccines have many shortcomings, especially safety concerns. Although killed and subunit vaccines are generally safe, their efficacy relies on the involvement of proper mucosal adjuvants and delivery systems. Obviously, successful mucosal immunization with non-replicating systems offers added value in common with replicating systems, such as both mucosal and systemic immune responses are induced, and administration of vaccines by oral or nasal modes reduces the need for professionals to carry out the vaccination [1]. The most well-known non-replicating mucosal adjuvant systems are Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT). Their efficacy seems to be dependent on the existence of the toxic A subunit requiring modification of these toxins for safe use [2], which has resulted in development of subunit formulations and mutant products. There is evidence from different systems that the response to non-replicating antigens administered nasally or orally, is skewed toward a non-Th1 type of response. This is the case with CT inducing production of IL-10 and TGF-b [3]. Differently to CT, ISCOMs interact with the innate immune system to produce IL-12, which is a crucial modulator for a Th1 response and the ISCOM is not dependent on IL-4 or IFN-g for the induction of mucosal IgA and CTL responses [4].

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There are several requirements for a successful mucosal delivery and adjuvant system. First, the system should be resistant to the harsh mucosal environment containing proteolytic enzymes and able to guide the vaccine antigens through the mucus layer to the induction site containing antigen presenting cells (APCs), leading to immune responses rather than tolerance. Secondly and subsequently, immune responses have to be evoked at the local mucosa and in some cases at remote mucosal effector sites besides providing a potent systemic response. Thirdly, effector mechanisms, including the production of relevant cytokines, e.g. locally to support IgA response and systemically, often promote a Th1 profile including a CTL response, e.g. to protect against virus infections. ISCOMs to various degrees fulfil these criteria [5].

2. ISCOM concept In 1984, the ISCOM concept was first described [6] resulting from a search for an innocuous antigen delivery system with a satisfactory immunomodulatory capacity. The sum of experience indicated that an advantageous immunogen should be a multimeric formation of antigen(s) combined with an adjuvant. To minimize the side effects the dose of adjuvant should be kept optimally small and the adjuvant should be firmly associated or even bound to the antigen(s) [7]. Most of these ideas were met by the ISCOM technology. The typical ISCOM is a 40-nm cage-like structure exhibiting icosahedral symmetry [8] assembled from 12 morphological 10–12 nm subunits composed of Quillaja saponin and cholesterol. The assembly of the 40 nm ISCOM structure and the incorporation of

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the antigen is facilitated by the addition of a phospholipid and is mainly mediated by hydrophobic interactions. The ISCOM antigen can be an envelope protein of a virus, a cellular membrane protein, or peptides containing hydrophobic domains or any antigen that has a hydrophobic domain. Non-amphipathic proteins, such as gp120 of HIV, may be integrated into an ISCOM by structural modifications, such as partial denaturation of proteins with urea [9], exposure to low pH [9–11], or high temperature [12] to expose internal hydrophobic regions within proteins. Another strategy is to covalently attach fatty acid tails to soluble proteins allowing them to be incorporated into the ISCOMs [13] or the linkage of the antigen covalently to a preformed ISCOM-matrix (the assembled 40 nm particle without integrated antigens). A comprehensive technical review of the ISCOM technology has recently been published [14]. The immunogenic potency and utility of the ISCOM technology has been proven in over 250 publications covering most aspects of vaccine technology.

2.1. Quillaja saponins The size, stability and the particular form of ISCOMs together with the Quillaja saponins are the main mediators of the immunomodulating and immunoenhancing properties of ISCOMs. Quillaja saponins are plant glycosides consisting of an aglycone and one or two sugar chains. The basic structure of the aglycone part is a triterpenoid. The knowledge of adjuvant properties of Quillaja saponins dates back to the beginning of 1900. However, the last decade has revealed unique structures and fractions of saponins with diverse adjuvant and physical activities in the crude extracts [15–18].

2.2. APC response to ISCOMs Most of the immunological characterization of ISCOMs is done after parenteral administrations. ISCOMs increase the MHC class II expression on APCs [19,20]. The secretion of the proinflammatory cytokines IL-1 [21] and IL-6 is prominent when measured in murine spleen and peritoneal cells after in vitro stimulation and IL-12 can be measured in

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serum after the in vivo stimulation of mice with influenza ISCOMs. ISCOMs also induce in vitro TNF-a and GM-CSF, but less prominently [22–24]. ISCOM-borne antigens are presented by different APCs, as demonstrated in vitro by culturing T cells from mice primed with influenza ISCOMs with dendritic cells (DCs), B cells or peritoneal cells pulsed with the same ISCOMs. All these populations of APCs stimulated the T cells to proliferation and to cytokine production [23].

2.3. T-cell response to ISCOMs The purpose of using an adjuvant is to enhance and improve the magnitude and the characteristics of the immune response. In accord with a potent IL-12 production by the innate immune system, the ISCOMs have a strong immunomodulatory capacity activating murine Th cells to secrete the Th1 type cytokines IL-2 and IFN-g. This has been observed with a great variety of ISCOMs. e.g. these cytokines were induced by ISCOMs with influenza virus antigens [23,25–28], ovalbumin (OVA) [29,30], Herpes simplex virus type I (HSV-1) glycoproteins [31], and Epstein–Barr virus (EBV) gp340 [32]. As expected, ISCOMs up-regulate IgG2a antibody response. Most prominent is the capacity to induce CTL responses both after parenteral and mucosal administration (for review see Ref. [33]). The capacity to deliver antigen to the cytosol [34] paves the way for a MHC class I- restricted antigen presentation resulting in a strong CTL response [35,36]. The capacity of ISCOMs to induce a Th1 type of T cell response does not prevent ISCOMs from producing Th2 type cytokines. However, when measuring cytokine levels in the supernatants of cell cultures after antigen stimulation in vitro, the IL-4 production was low [25,26,29,37] despite the fact that high numbers of IL-4 secreting cells were detected by enzyme-linked immunospot assay (Elispot) [38] (this may reflect that it is unreliable to measure excreted IL-4 due to the consumption). In a direct comparison, OVA ISCOMs-activated T cells produced levels of IL-4 after restimulation comparable to those from cells primed with OVA in aluminium hydroxide; an adjuvant known to induce a Th2 like immune response [30]. Primates primed

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with gp120 ISCOMs and boosted with V2 and V3 peptides in ISCOMs were protected against a challenge infection with a chimer of HIV-1 and SIV in contrast to primates immunized with a DNA vaccine or primates immunized with gp120 cloned into a poxvirus. The protection was ascribed to coincide with IL-2, IFN-g and IL-4 production besides virus neutralizing antibodies [39]. In conclusion, ISCOMs induce a potent Th1 type response in a majority of the experiments carried out. Importantly, ISCOMs induce a concomitant Th2 response, resulting in a so-called balanced Th1 / Th2 response, which in part explains why ISCOMs also can induce the production of cytokines enhancing mucosal immunity.

2.4. B-cell response to ISCOMs It has been established in a great number of studies that the antibody responses induced by ISCOMs are usually ten times higher than those induced by non-adjuvanted particulate antigens like viruses or microparticles; such potentiation is also high compared to other adjuvants. Subcutaneous immunization of mice with influenza ISCOMs [40] could induce clear-cut antibody responses at antigen doses as low as 0.01 mg. ISCOMs containing OVA, gp340 of EBV or HSV-1 were highly immunogenic at doses ranging from 1 to 5 mg of protein [30,32,38]. The tiny amount of antigen required for induction of antibody- and cell-mediated immune responses might reflect the advantage of the incorporation of the antigen and adjuvant in the same ISCOM particle, thereby efficiently targeting APCs. The advantage of having several functions in one particle is also reflected by the low dose of adjuvant used i.e. 6- to 10-fold lower Quillaja saponins was required by influenza virus ISCOMs to induce the same magnitude of antibody responses as an influenza antigen co-administered with an ISCOMMatrix [25]. Studies have also shown that a balanced antibody response in mice, encompassing all immunoglobulin isotypes and IgG subclasses, is induced by ISCOMs containing various antigens. In general, up-regulation of IgG2a was recorded, which is in agreement with a prominent Th1 cytokine profile (for review, see references [33] and [29]).

2.5. Protective immunity induced by ISCOMs Protective immunity has so far been induced by ISCOMs against more than 20 pathogens encompassing viruses, bacteria including, mycoplasma and parasites. Particularly interesting is that protective immunity has been evoked against microorganisms for which vaccines are lacking. There are in general effective vaccines against acute infections, for instance rabies, measles, foot and mouth disease. For such type of pathogens ISCOMs induce protective immunity. ISCOMs also induce protection against acute infections causing pneumonia such as the influenza virus, bovine herpes virus 1 (BHV-1) and canine distemper viruses. More importantly, ISCOMs induce protection against persistent or chronic infections such as SIV, HIV-1, HIV-2 and Epstein– Barr virus in primates, feline leukaemia virus in cats and Trypanosoma cruzi lethal infection in mice as well as BHV-1 latent infection in cattle (for review see Ref. [41]).

3. Nasal site is a preferred route for mucosal immunization The first attempt to induce mucosal immune response with ISCOMs was carried out in mice by ¨ Lovgren et al. [42] with influenza virus antigens. High serum antibody titers, protective immunity, measured as the prevention of mortality, weight loss and virus isolation, were induced by one intranasal (i.n.) immunization with a comparatively low dose of 10 mg of antigen. Soon after, Jones et al. [36] showed that after i.n. administration, influenza virus ISCOMs induced IgA locally in the respiratory tract measured by ELISPOT, and CTL response.

3.1. Role of the induction site Induction of mucosal immunity is well studied in the gastrointestinal (GI) tract where the Peyer’s patch (PP) has such a specialized function. In the upper respiratory tract (URT), the pharyngeal rings are conceived to be an equivalent to PP. However, less information about induction sites is available for the respiratory tract where studies with ISCOMs are also lacking. Information with ISCOMs has, there-

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fore, to be taken from the GI tract, where Claassen et al. [43], using fluorochrome-labelled ISCOMs containing the G protein of rabies virus, showed that ISCOMs targeted PP more efficiently than rabies virus particles. Another route for targeting the lymphatic system in the gut is through the enterocytes, which may act as APCs [44]. Lazorova et al. [45] demonstrated, using Caco-2 cells — an human intestinal epithelial cell line, that influenza virus ISCOMs, in contrast to influenza virus micelles, applied on the apical side of the cells became processed to peptides with a molecular weight less than 5 kD and were transported to the basolateral side and excreted. Between 20 and 40% of these peptides were eluted by gel filtration at a position which indicated approximately ten amino acids. These peptides stimulated specifically spleen cells from mice primed with influenza virus antigens to proliferation. It is likely that epithelial cells in the respiratory tract likewise process antigen for immunological presentation.

3.2. Parenteral administration for the induction of mucosal immune responses Various routes of parenteral administration for ISCOMs to induce mucosal immune responses were tested in mouse by Thapar et al. [46], using a model ISCOM containing sheep erythrocyte membrane proteins. By administration of the ISCOMs to the pelvic presacral space (p.s.–p.s.) significantly higher anti-erythrocyte IgA ELISA titres were evoked in the vaginal fluid than after intraperitoneal (i.p.–i.p.), subcutaneous (s.c.–s.c.), intravaginal (i.vag.–i.vag.), or i.p.–i.vag. immunizations with the same antigen preparation. Specific IgG titers were less dependent on the route of immunization, and p.s.–p.s., i.p.–i.p. and s.c.–s.c. administrations all induced similar high titers, while i.p.–i.vag. immunizations gave lower titers. It is noteworthy that mucosal immune responses were elicited in the female reproductive tract against membrane proteins by non-mucosal administration at the pelvis presacral site. The p.s. immunization was an even more effective route for the induction of specific IgA than the local application of the ISCOMs to the vaginal mucosa. However, this is in agreement with the claim that the female re-

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productive organ is an inefficient route for the induction of a mucosal immune response [47]. A formulation for intradermal (i.d.) administration for the induction of mucosal immune responses in the respiratory tract was described by Chin et al. [48] as a hybrid liposome–ISCOM. This ISCOM was prepared from liposomes containing the vaccine antigen and the positively charged detergent DOTAB was used for solubilization and was incorporated in the ISCOM. By this route, the authors observed a series of co-ordinated reactions characterized initially by a rapid mobilization and recruitment of granulocytes to the lung. The activation of effector cells of the innate immune system has the potential to lend protective properties to the mucosal surfaces accompanied by a dramatic reduction in the numbers of T, B cell precursors and haematopoietic progenitor cells during the first 2–4 days postvaccination, similar to the events seen after infection with a pathogen. Initial decreases in cell numbers in the thymus and bone marrow (BM) are followed by a rapid increase in cellular proliferation in these organs. Vaccineinduced cell death (by apoptosis) in the thymus may provide one of many stimuli needed to up-regulate BM production of progenitor cells, and cells of the B, myeloid and monocytic lineages so that depleted peripheral compartments are replenished. Mice vaccinated i.d. with ISCOM-carried antigen also displayed increased rates of granulocyte and monocyte recruitment in the lung and spleen. These events occurred very rapidly (within 12–36 h of the challenge) and may be crucial in providing optimal protection by vaccines. Adaptive immunity elicited by intradermal vaccination may, therefore, be dependent on a more violent prior activation of the innate system than after mucosal and combined parenteral and mucosal modes of immunization.

3.3. Mucosal administration Due to the obvious advantages, there has been a focus on the administration of ISCOMs by the oral route. In 1991, Mowat et al. [49], in their review article titled ISCOMs — A Novel Strategy for Mucosal Immunization — suggested that immunostimulating complexes (ISCOMs) may provide an oral immunization vector for the induction of a wide range of immune responses to protein antigens. In

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their extensive studies, they evaluated ISCOMs for oral immunization with a model antigen-OVA. Like CT and LT, ISCOMs were shown to prevent the induction of immunological tolerance and to exert adjuvant activity in the digestive tract. Low but repeated oral doses with OVA ISCOMs induced secretory IgA, CTL and systemic immune responses [50]. This group also compared immune responses after oral administration of OVA entrapped in poly (lactide–co-glycolide, i.e. PLG) microparticles. The ISCOMs induced OVA-specific CTL responses more efficiently and fewer oral feeds were required than for the PLG formulation [29]. To unveil the interaction between ISCOMs and the innate immune system, Smith et al. [51] found in the mouse that ISCOMs after oral administration induced IL-2 production and an IL-12-dependent cascade of innate immune responses as occurred after parenteral immunization [29,33]. Only IL-12 knockout (KO) mice exhibited impaired response with regard to the recruitment of peritoneal exudate cells. On the contrary, IL-6 and inducible nitric oxide synthase (iNOS) knockout mice developed normal immune responses to OVA in ISCOMs. This IL-12dependent innate response to orally administered OVA ISCOMs is in agreement with the fact that ISCOMs enhance a Th1 response. A comparison of mucosal adjuvant properties between the classical mucosal adjuvant CT and ISCOMs was also made by the same group [52], demonstrating that CT is independent of, and ISCOM is dependent on IL-12 for the induction of an immune response. Also, in IL-4 and IFN-g receptor knockout mice, OVA ISCOMs induced a normal range of immune responses including delayed-type hypersensitivity (DTH), serum IgG and IgA antibodies, T-cell proliferation and cytokine production, MHC class I-restricted cytotoxic T lymphocyte (CTL) activity and specific intestinal IgA antibodies. These responses were of a similar magnitude to those found in the wild-type mice, indicating that the ISCOMs in contrast to CT are not dependent on the presence of IL-4 or IFN-g, although these cytokines were part of the immune response to ISCOMs in normal mice. These results suggest that separate and distinct antigen-processing pathways are involved for CT and ISCOMs. It also implies complementary properties between the two adjuvants, which may be used by the combination of two adjuvant systems or as alternatives.

ISCOMs containing somatostatin–avidin were tested by Estrada et al. for their capacity to induce a gastrointestinal response [53]. They measured the greatest IgA response in the mesenteric lymph node (MLN) and PPs after intraperitoneal (i.p.) / intragastric (i.g.) immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found after i.g. / i.g. immunization. They concluded that the ISCOM is an effective delivery system for protein–peptide antigens, especially by i.p. / i.g. administration, which offers another approach for the design and delivery of peptide vaccines to obtain a mucosal immune response.

3.3.1. Nasal route of administration A comparison between nasal and oral administrations of chimeric plant virus particles, alone or in the presence of an ISCOM matrix, was performed in the mouse by Brennan et al. [54]. The nasal mode of administration induced significantly higher and less variable serum IgG titers than those generated by oral immunisation, indicating that the effectiveness of the nasal route for the delivery of these nonreplicating particles. In various experiments, ISCOM-borne virus antigens induced specific local IgA by the nasal route. After i.n. administration of the envelope proteins of influenza virus in ISCOMs [41] a high serum antibody response was obtained, which was almost of the same order as that following parenteral immunization. A specific IgA response was also efficiently evoked both at the local respiratory mucosa and distant (genital and intestinal tract) mucosae. ISCOMs have also been used for the nasal delivery of membrane antigens from Mycoplasma mycoides subsp. mycoides (MmmSC), an organism causing a severe chronic disease in the lungs of the bovine species. In mice, ISCOMs induced high mucosal and systemic antibody responses. ISCOMs enhanced greatly the total Ig and IgG subclass (IgG1, IgG2a and IgG2b) responses in serum and in the lungs, and this enhancement was more prominent after i.n. than after s.c. immunisation. By the i.n. mode of immunisation ISCOMs containing mycoplasma antigens induced a 60-fold higher IgA response in the lungs than did the whole cell antigen. ISCOMs also induced substantially higher total Ig

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and IgG subclass responses in the lungs [55]. In this context, it should be noted that the whole cells naturally infect through the respiratory tract. The effectiveness of ISCOMs for nasal administration of parasitic antigens was verified by Carol et al. [56], using ISCOMs containing Echinococcus granulosus membrane antigens. The ISCOMs given intranasally to mouse evoked a significant antibody response. The i.n. route of immunization induced higher serum IgA titer in relation to IgG than the s.c. route and provided serum antibodies of slightly higher avidity. A ten-fold lower serum IgG2a:IgG1 as well as IgG3:IgG1 ratios were also recorded after the i.n. administration compared to s.c. immunization. Interestingly, the mucosal route of immunization induced more efficiently antibodies of both IgA and IgG isotypes directed to carbohydrate epitopes, in which protective antigens are thought to reside. There is a great need for the development of a vaccine against respiratory syncytial virus (RSV), a leading cause of infant respiratory disease. Early attempts with a formalin inactivated RSV vaccine applied parenterally resulted in exacerbation of disease upon a subsequent natural infection, which may partly be due to the absence of a mucosal immune response. Later studies also imply a role of the G protein generating a bias for a Th2 response, and a TNF-a enhanced inflammation. Thus, it is conceived that a RSV vaccine inducing a local mucosal immune response of the right kind in the respiratory tract with minimal side effects is required for immune protection. Trudel et al. were the first to demonstrate that the effectiveness of an experimental RSV ISCOMs vaccine in initiating humoral and cell-mediated immune responses and in providing overall protection upon live virus challenge in Balb / c mice. Their results also indicated that the i.n. route significantly reduced virus shedding [57]. Hu et al. [58] compared the immune responses to an experimental RSV ISCOM vaccine after i.n. and s.c. immunizations. The i.n. administration of ISCOMs for mice induced high levels of IgA antibodies both in the URT and lower respiratory tract. In the lungs, the IgA response was still significant at 22 weeks after the second immunization when the experiment was terminated. Also high systemic antibody levels were elicited comparable to those after s.c. immunization. Importantly, the functional virus neutralizing antibody was detected in various mucosal sites including the

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URT, lungs, gut and female genital tract as well as in serum, implicating that protective epitopes of RSV were well-preserved and recognized after i.n. administration. In contrast to our observations, Carol et al. [59] recorded only a mucosal IgA response, but not systemic antibody response after i.n. immunization of dogs with Echinococcus granulosus membrane antigens in ISCOMs. This may reflect the fact that the nasopharyngeal mucosal associated lymphoid tissue of dogs is more strictly compartmentalized than that of some other animal species. A common and economically important pathogen of cattle infecting the respiratory tract is bovine herpes virus 1 (BHV-1). In a Canadian vaccination experiment [60], ISCOMs containing the BHV-1 envelope proteins administered i.n. to cows induced protection to the disease and reduced virus excretion a 1000-fold after challenge via intubation of the respiratory tract. In a similar experiment carried out in Hungary [61], 2 weeks after the second vaccination, all the animals were challenge-infected i.n. with a virulent BHV-1 strain and 4 days later with a virulent Pasteurella multocida — in order to mimic harsh field conditions. When exposed to the challenge infection, all the animals vaccinated with the ISCOMs were fully protected, i.e. no virus could be recovered from their nasal secretions and no clinical symptoms were recorded. In contrast, the animals vaccinated with the commercial vaccine, responded to challenge with a moderate fever and a loss of appetite, and virus was isolated from the nasal secretions. The animals in the control group developed severe clinical symptoms. In the sera of ISCOM-vaccinated animals, the virus neutralization titers reached levels of 1 / 3500 or higher. Amongst the various mucosal sites so far tested for ISCOM administration, the nasal route seems to be the most effective and promising. The nasal passage is the invading route for large numbers of pathogens, and it is the choice for the induction of a common mucosal immune response and for the establishment of protective immunity at mucosal sites, e.g. for the induction of local IgA in the female reproductive tract, the nasal mode of administration has the potential to be the route of choice for ISCOMs as well as for other delivery systems designed for mucosal administration. In general, antigens derived from envelope viruses or cell

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membranes incorporated into ISCOMs retain their biological activity and conformation as measured by their reactivity to monoclonal antibodies and functional tests like hemagglutination, enzyme activity (neuraminidas) or the induction of virus neutralizing antibodies [6,12,62–67]. Merza et al. [68] showed, by use of monoclonal antibodies, that the neutralizing epitopes on gp51 of bovine leukaemia virus were preserved when mild detergents were used for the preparation of ISCOMs. Thus, functional epitopes and biological properties for targeting that are harbored in virus glycoproteins are retained, facilitating the penetration of mucus [69]; a prerequisite for the efficient induction of mucosal immune responses. These properties were used in ISCOMs following nasal administration to provide an immune response to passenger vaccine antigens in local and remote mucosal surfaces.

3.4. ISCOMs with targeting molecules and passenger (vaccine) antigens for nasal administration In mice, the envelope proteins from influenza and respiratory syncytial viruses and cholera toxin B subunit (CTB) have the capacity of targeting the corresponding pathogens in mucosal surfaces besides being vaccine antigens of interest. This property should also be useful for non-replicating antigen delivery systems. Consequently, these molecules were utilized for mucosal targeting after their incorporation into ISCOMs (together with vaccine antigens of interest lacking or having insufficient targeting properties) and to be delivered by nasal administration. In contrast to CTB [70], ISCOMs did not induce oral tolerance to a linked antigen by mucosal routes. To combine and compare the mucosal activities of CTB and influenza virus ISCOMs and ¨ et al. to explore possible symbiotic effects, Ekstrom [13] incorporated rCTB via the GM1 receptor into the ISCOMs. ISCOMs with and without envelope proteins enhanced the mucosal immune response to rCTB after i.n. administration by inducing an IgA response in the mucosa of the remote intestinal tract and a 100-fold increase in the specific IgA locally in the lung. Using two low i.n. doses of OVA as a passenger antigen in ISCOMs targeted with influenza virus envelope proteins, a significant mucosal anti-

OVA IgA response was induced locally in the lungs. However, ISCOM-matrix coadministered with OVA induced low or no mucosal IgA response to OVA in accordance with the results of others [71] who coadministered CTB as a separate entity with OVA. In serum, an enhanced IgG2a response was seen after i.n. administration of the ISCOMs, but not with the ISCOM matrix and antigen coadministered as separate entities. A synergism between ISCOMs and rCTB in the same particle could only be recorded as an enhanced IgA response to OVA in the remote genital tract. The genital tract is a main site for infection with sexually transmitted diseases, such as HIV, thus being an important effector site for a prospective vaccine. Using the envelope proteins of RSV for targeting gp120 of HIV-1, (a main candidate as a vaccine antigen), in ISCOMs, an enhanced IgA response was recorded after the i.n. immunisation of mice to gp120 in various mucosal organs and above all in the genital tract. The results underline that RSV envelope proteins as well as other surface proteins of respiratory viruses deserve further exploitation for enhancing and targeting of nasal vaccine antigens [72].

4. Conclusions and future considerations The ISCOM is a versatile delivery system, which can be tailored for mucosal administration particularly suited for the nasal route. The flexible structure allows interchange of the vaccine antigens, molecules for targeting local and distant mucosal sites as well as immunomodulating components. The ISCOM has different immunological properties to CT being dependent on IL-12 for the induction of mucosal immunity but not on IL-4 as is the case with CT. While CT promotes a strong Th2 response and its B subunit also induces tolerance to linked antigens, the ISCOM induces a balance between Th1 and Th2 and no tolerance. Thus, these two systems induce immune responses by two different pathways possibly with complementary properties, which may be used in combination or as alternatives. Vaccines are lacking for several infectious diseases, which infect through mucosal surfaces including those of the respiratory and genital tracts. Of

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particular interest are vaccines against sexually transmitted diseases including AIDS. For this purpose the nasal mode of administration to achieve protective immunity in the genital tract is an interesting approach, which requires further development of targeting and immunomodulating components. Age groups at the both ends of life-span, i.e. the newborn and elderly are the main targets of new vaccine strategies. In the newborn, there are two obstacles to overcome for successful immunization i.e. the immature immune system and the blocking effect of maternally derived antibodies. ISCOMs have been proven to induce an immune response in neonates and a nasal vaccination approach with ISCOMs may circumvent the blocking effect of maternal antibodies. In the elderly, the systemic immune system is becoming defect but the mucosal immune system remains active. ISCOM has been shown to efficiently induce immune response in elderly mice amongst others by restoring the capacity of APCs to express CD86 after parenteral administration of an influenza virus ISCOM in contrast to the poor effect of a commercial influenza virus vaccine [73]. The combination of an efficient adjuvant system and the nasal mode of immunization has the prospect to increase the efficacy of vaccines for the elderly. Too strong a Th1 response gives rise to severe inflammatory reactions and too strong a Th2 type of response causes allergic and atopic immune reactions, which are mainly localized in the respiratory tracts. Therefore, immune responses at mucosal sites have to be under a control probably requiring a balance between Th1 and Th2 including the production of TGF-b. Hence, delivery systems should be tailored to provide both Th1 and Th2 properties and future vaccines should be made to fit the antigen and disease to induce an immune protection but to evade severe side effects.

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