Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines

Fish & Shellfish Immunology (2001) 11, 437–458 doi:10.1006/fsim.2001.0351 Available online at http://www.idealibrary.com on

REVIEW ARTICLE Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines JOHANNES M. DIJKSTRA1, UWE FISCHER2, YOSHIHIRO SAWAMOTO3, MITSURU OTOTAKE1* AND TERUYUKI NAKANISHI1† 1

Immunology Section, National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan, 2Institute for Diagnostic Virology, Friedrich-Loeffler-Institutes, D-17498 Insel Riems, Germany and 3Nagano Prefectural Experimental Station of Fisheries, Akashina, Nagano 399-7102, Japan (Received 17 August 2000, accepted after revision 23 March 2001, published electronically 15 June 2001) An MHC class I restricted cytotoxic T lymphocyte (CTL) activity assay has recently been established for rainbow trout. MHC class I restricted cytotoxicity probably plays a critical role in immunity to most viral diseases in mammals and may play a similar role in fish. Therefore, it is very important to investigate what types of vaccines can stimulate this immune response. Although logical candidates for vaccine components that can stimulate an MHC class I restricted response are live attenuated viruses and DNA vaccines, these materials are generally not allowed in fish for commercial vaccine use due to potential safety issues. In mammals, however, a number of interesting vaccination strategies based on exogenous antigens that stimulate MHC class I restricted cytotoxicity have been described. Several of these strategies are discussed in this review in the context of fish vaccination.  2001 Academic Press

Key words:

MHC class I, cell-mediated cytotoxicity, exogenous antigen, adjuvant, teleost.

I. MHC class I restricted cytotoxicity in fish Knowledge of the fish immune system is limited compared with that of mammals. Current data, however, indicate that it is very comparable to that of other vertebrates. Specific and innate responses have been found involving macrophages, natural killer cells, neutrophils and B-lymphocytes, as well as antibodies and cytokines (Iwama & Nakanish, 1996). Molecular data *Corresponding author. E-mail: ototake@a#rc.go.jp † Present address: Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8570, Japan. 1050–4648/01/060437+22 $35.00/0

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confirm the similarity of the fish-specific immune system to that of mammals: polymorphic MHC (major histocompatibility complex) class I and class II genes, as well as T cell receptor (TCR), CD8 and immunoglobulin genes have been described for a number of fish species (Dixon et al., 1995; Stet et al., 1996; Manning & Nakanishi, 1996; Rast & Litman, 1998; Hansen & Strassburger, 2000). On the functional level, research on fish-specific immune responses has been mainly focused on antibody production (Kaattari & Piganelli, 1996) and the relatively few investigations of specific cell-mediated cytotoxicity have mainly concentrated on allogeneity (Nakanishi et al., 1999). In higher vertebrates the primary specific response to many viral disease agents is cell-mediated cytotoxicity involving classical MHC class I molecules (Oldstone, 1997; Rodriguez et al., 1997). Classical MHC class I molecules present endogenous peptides at the cell surface for recognition by the TCR/CD8 receptor complex of cytotoxic T-lymphocytes (CTLs) (Townsend & Bodmer, 1989; Germain, 1994). Recognition can lead to stimulation and clonal expansion (proliferation) of the T-cell, and to specific lysis of the peptidepresenting cells. Classical MHC class I molecules are polymorphic and TCRs recognise the combination of antigenic peptide plus MHC class I molecule. Therefore, activated cytotoxic T-lymphocytes can only recognise and lyse cells with identical combinations of MHC class I and peptide as that with which they were initially stimulated (Simpson, 1988; Kourilsky & Claverie, 1989). This has been a great impediment in establishing MHC class I restricted cytotoxicity assays in fish, as it requires target cells and donors of e#ector cells with an identical MHC class I allele (this is called ‘MHC class I restriction’). This requires the knowledge of the classical MHC class I sequences, preferably genetically identical fish, and a target cell line with matching MHC class I and suitable properties for assay systems. The study of Somamoto et al. (2000), was probably the first that investigated MHC class I restricted cytotoxicity in fish. They isolated peripheral blood leukocytes (PBL) of ginbuna carp (Carassius auratus langsdorfii) that were immunised with syngeneic virus-infected cells, and showed in vitro cytotoxicity against the infected cells. The possibility of antibody involvement in this cytotoxicity was excluded. However, as there is no knowledge on MHC class I sequences in ginbuna crucian carp and their study failed to include allogeneic controls, the study could not be conclusive regarding MHC class I restriction. We fortuitously obtained all the elements essential to establish an MHC class I restricted cytotoxicity assay in rainbow trout (Oncorhynchus mykiss). Hansen et al. (1996, 1999), Shum et al. (1999) and Aoyagi et al. (submitted) described di#erent classical MHC class I sequences, which were probably derived from the same locus Onmy-UBA. Aoyagi et al. (submitted) showed that Onmy-UBA is the single major expressed classical MHC class I locus in rainbow trout by Northern and Southern blotting and by sequence analysis of the 3 untranslated regions of the di#erent alleles. The clonal rainbow trout strain C25 and the commonly used RTG-2 (rainbow trout gonad; fibroblastlike cells) were found to share the Onmy-UBA*501 allele (Dijkstra et al., 2001).

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Fig. 1. Summary of the MHC class I restricted cytotoxicity assay in rainbow trout. Clonal C25 rainbow trout and the permanent cell line RTG-2 express the MHC class I sequence Onmy-UBA*501. MHC class I characterised outbred fish and the permanent cell line RTE that do not express Onmy-UBA*501 were used as negative controls. The dots within the upper drawings of RTE and RTG-2 cells represent infection with IHN virus. Peripheral blood leukocytes were isolated from naïve C25 fish, and from C25 fish and outbred fish that were immunised twice with an IHNV-G plasmid. This plasmid encoded the glycoprotein of IHN virus under control of a human cytomegalovirus promotor. Cytotoxicity was determined by lactate dehydrogenase release from lysed target cells. PBL from DNA immunised C25 fish only killed RTG-2 cells that were infected with IHN virus (indicated by solid arrow); the other analysed target cells were not killed (indicated by dashed arrows). The PBL from naïve C25 fish or from DNA immunised outbred fish did not kill IHNV infected RTG-2 cells, nor any of the other investigated target cells.

When the C25 fish were immunised with DNA encoding for the G protein of infectious hematopoietic necrosis virus (IHNV), isolated lymphocytes killed IHNV infected RTG-2 cells, and not cells with di#erent classical MHC class I alleles or uninfected RTG-2 cells (Fischer et al., submitted). Lymphocytes from naïve C25 fish or fish with di#erent MHC class I alleles did not show specific killing of IHNV infected RTG-2 cells. An outline of the experimental method is schematically shown in Fig. 1. As clonal fish and derived cell lines are being more commonly applied in fish research, other research groups may establish MHC class I restricted cytotoxicity assays soon. Therefore we believe this is a good moment to discuss how these assays might be used for the establishment of safe and commercially acceptable vaccines. This review focuses on rainbow trout, but the data can probably be extrapolated to other fish.

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II. MHC class I presentation of exogenous antigens The traditional viewpoint was that only endogenous proteins are o#ered by MHC class I at the cell surface. After being degraded to small peptides by the proteasome in the cytoplasm, these peptides are transported by transporters associated with antigen processing (TAP) to the endoplasmic reticulum where they form a complex with MHC class I and
2m, and this heterotrimer is transported to the cell surface through the intracellular membrane system (Pamer & Cresswell, 1998; van Endert, 1999; Reits et al., 2000). Exogenous antigens, on the other hand, were believed to be only presented by MHC class II molecules at the cell surface, stimulating an antibody response (Weenink & Gautam, 1997). This viewpoint has changed in recent years. Exogenous antigens generally do not stimulate CTL activity (Takahashi et al., 1990). However, if they are packed into or connected to a structure that delivers them into the cytoplasm, they are treated as endogenous proteins and can be o#ered by MHC class I molecules (for examples see below). Recently it was also discovered that primarily macrophages, but also dendritic cells, mast cells and B-cells can present peptides from exogenous proteins by MHC class I after phagocytosis or endocytosis (Bachmann et al., 1996; Ke & Kapp, 1996; Reimann & Kaufmann, 1997). In some cases the exogenous antigen must have gained access to the cytosol from a phagosome or endosome (Oh et al., 1997). In other cases the process must be di#erent from the normal MHC class I processing pathway, because no sensitivity to brefeldin A (which inhibits MHC class I transport from the endoplasmic reticulum) or proteasome inhibitors was found (Pfeifer et al., 1993; Schirmbeck et al., 1995a,b; Song & Harding, 1996). The way this alternative MHC class I processing pathways (or pathways) works has not been clarified, but it has been hypothesised that peptides of exogenous antigens can be loaded on MHC class I molecules in vacuolar compartments, similar to the situation for MHC class II (Pfeifer et al., 1993; Raychaudhuri & Morrow, 1993; Song & Harding, 1996; Reimann & Kaufmann, 1997). The advantage for an organism to present antigens by MHC class I after phagocytosis is probably that professional antigen presenting cells (APCs) are actively phagocytic cells. This ensures a stronger induction of a CTL response against viruses that do not infect APCs because presentation by MHC class I on APCs stimulates the CTL response more strongly than presentation by other cells (Jondal et al., 1996; Mellman et al., 1998). Favouring this hypothesis are observations that antigens associated with cellular debris can stimulate a CTL response (Bachmann et al., 1994b, 1996; Wijburg et al., 1998). It has been shown that the alternative MHC class I processing pathway can present identical antigen epitopes as the conservative pathway, but also di#erent ones (Schirmbeck et al., 1998). To further indicate the complexity of the MHC class I loading pathways, we quote a sentence of Reimann and Schirmbeck in a 1999 review: ‘‘What makes the field di$cult to understand at this stage is that there is not one but many alternative pathways, that the data supporting each of them are fragmentary, and that their biological importance is uncertain’’ (Reimann & Schirmbeck, 1999).

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Despite the uncertainties about the mechanisms of CTL induction by exogenous antigens, a number of promising vaccine strategies have been described for mammals that are interesting to analyse in fish. III. Potential vaccines which induce CTL responses Both viral infection and DNA immunisation cause the production of intracellular antigens, and therefore these antigens can be presented by the classical MHC class I pathway (Fujii et al., 1994a; Rodriguez et al., 1997). However, live viruses are undesirable as vaccines as they may escape into natural waters. That DNA vaccines induce a MHC class I restricted cytotoxic response in fish was shown in our study with the IHNV-G gene (mentioned above), and has also been suggested by a study by Lorenzen et al. (1998) who showed protection against viral haemorrhagic septicemia (VHS) after DNA immunisation with the VHS-N gene in the absence of neutralising antibodies. However, there is concern over potential risks of DNA vaccines inducing the expression of oncogenes or creating transgenic animals, although neither has been shown. Recently we showed that DNA injection in fish can result in antigen expression for at least 2 years (Dijkstra et al., 2001), which bodes poorly for the acceptance of DNA vaccines in aquaculture because an ideal vaccine should only be present for a limited time period. For mammals a number of di#erent vaccines that can stimulate MHC class I restricted cytotoxicity have been described, and they might be better accepted than DNA vaccines or live attenuated viruses for commercial use. These vaccines direct exogenous, non-replicating antigen into either the conservative MHC class I loading pathway or into a lesser-defined MHC class I pathway for which antigen delivery into the cytosol is not necessary (see above). Generally, the vaccines that can stimulate these responses are particulate and are taken up by macrophages, and/or deliver antigen into the cytoplasm by association with hydrophobic agents or membrane translocating proteins (reviewed by Raychaudhuri & Morrow, 1993; Morein et al., 1996; Schirmbeck et al., 1996; Liu, 1997; Jondal et al., 1996; Reimann & Kaufmann, 1997). Table 1 summarises some representative studies on the following vaccines. One group of CTL-inducing vaccines consists of antigens associated with hydrophobic agents that probably help them to pass through the cellular membrane. Antigens can be entrapped in liposomes (Kawano et al., 1990; Collins et al., 1992; Reddy et al., 1991, 1992; Zhou et al., 1992; Wijburg et al., 1998), virosomes (Wijburg et al., 1998; Arkema et al., 2000), or immunestimulating complexes (ISCOMs) (Takahashi et al., 1990; Heeg et al., 1991; Mowat et al., 1991; van Binnendijk et al., 1992; Villacres et al., 1998), conjugated or complexed with a lipid component (Deres et al., 1989; Walker et al., 1992; Allsopp et al., 1996; Nixon et al., 1996), emulsified in Freund’s adjuvant (Aichele et al., 1990; Ke et al., 1995) or associated with squalene (Allsopp et al., 1996; Wijburg et al., 1998) or saponins (Newman et al., 1992; Wu et al., 1992, 1994). Other vaccines seem to depend on their particulate nature for CTL stimulation. Antigens entrapped in or bound to microparticles (Nixon et al., 1996; Song & Harding, 1996; Oh et al., 1997; Partidos et al., 1997), antigens in

Incomplete Freund’s adjuvant

Complete Freund’s adjuvant

Squalene/tween 80/pluronic L121 Saponin

Adjuvants Monophosphoryl lipid A/ squalene

Strategy

N.D.


+

+





9 aa peptide

9 aa peptide Ovalbumin absorbed to particulate Al(OH)3 Ovalbumin

HIV-1 gp160 9 aa peptide

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

+

Ovalbumin

Antigen

CTLactivity

Macrophages (mouse in vivo): macrophage paralysis inhibits CTL stimulation. Macrophages (mouse in vivo): macrophage depletion inhibits CTL stimulation.

Macrophages (mouse in vitro): macrophage depletion inhibits CTL stimulation. N.D. (mouse in vivo)

Cells presenting antigen by MHC class I

Takahashi et al., 1990 Allsopp et al., 1996

Ke et al., 1995

Allsopp et al., 1996 Wu et al., 1994

Allsopp et al., 1996

Wijburg et al., 1998

Reference

Table 1. Representative studies of strategies that induce CTL-activity in mammals by use of exogenous antigens. Strategy: the strategies are grouped into di#erent sections (underlined). Antigen: for small peptides the antigen source is not mentioned. CTL-activity: CTL-activity was measured by the lysis of target cells or by the stimulation of cytotoxic T lymphocytes. Location in the cytosol: describes if the antigen was detected in the cytosol. Cytosol location important for CTL stimulation: describes if it was found that cytosol location of the antigen is important for CTL stimulation. Method of detection: describes how the location in the cytosol was detected and how its importance for CTL stimulation was shown. Cells presenting antigen by MHC class I: describes those cells that present peptides of the exogenous antigen by MHC class I and in some cases the method of detection

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N.D./
/CTL

+

activity not blocked by brefeldin A

activity not blocked by brefeldin A

N.D.

N.D./
/CTL

+

Ovalbumin

N.D.

N.D.

N.D.

N.D.

(mouse in vivo) (mouse in vivo)

Cells presenting antigen by MHC class I

Mouse macrophages (in vitro)

N.A. mouse neuroblastoma cells (in vitro) L929 mouse cells (in vitro) Macrophages (mouse in vivo): macrophage depletion inhibits CTL stimulation. N.D. (mouse in vivo): macrophage depletion does not inhibit CTL stimulation. N.D. (mouse in vivo) Mouse macrophages (in vitro) D2SC/1 mouse dendritic cells (in vitro) N.D. (mouse in vivo): macrophage depletion does not inhibit CTL stimulation. Mouse macrophages (in vitro)

N.D.

N.D. N.D.

N.D.

N.D.

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

+

+ +

VSV N protein LCMV nucleoprotein

+

Ovalbumin

+

+

Rabies virus G protein

Ovalbumin

+ +

CTLactivity

9–12 aa peptides 9 aa peptide

Antigen

21 aa peptide Expression in bacteria as fusion protein (Salmonella typhimurium and Escherichia coli) 21 aa peptide

Virosomes derived from influenza A virus Cellular debris

Particles Liposomes

Lipopeptides Lipopeptides

Strategy

Table 1. (Continued)

Song & Harding, 1996

Pfeifer et al., 1993

Wijburg et al., 1998

Bachmann et al., 1994b Bachmann et al., 1996

Wijburg et al., 1998

Wijburg et al., 1998

Kawano et al., 1990

Nixon et al., 1996 Allsopp et al., 1996

Reference

EXOGENOUS ANTIGENS AND MHC CLASS I

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Antigen

Yeast derived virus-like particles formed by self assembling modified

HIV-1 gp160 Ovalbumin HIV-1 V3 loop (40 aa)

Heat aggregation Hepatitis B surface antigen Antigen coupled Ovalbumin to latex beads Antigen coupled Ovalbumin to polystyrene particles Antigen coupled Ovalbumin to poly-e-caprolactone particles 9–12 aa peptides, Entrapment in microparticles lipid modified prepared from peptides (9–12 aa) (lactide-coglycolide) polymers ISCOM Influenza envelope proteins

Strategy

N.D.

N.D.

N.D.

+/N.D./histology

+

+ + +

N.D.

activity not blocked by brefeldin A N.D./
/CTL activity not blocked by brefeldin A +/+/histology and CTL activity is blocked by brefeldin A +/+/histology and CTL activity is blocked by brefeldin A

N.D./
/CTL

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

+

+

+

+

+

CTLactivity

Table 1. (Continued)

(mouse in vivo)

In vitro: mouse macrophages, B-lymphocytes and dendritic cells take up Iscoms. MHC class I presentation shown for mouse P815 mastocytoma cells. N.D. (mouse in vivo) N.D. (mouse in vivo) Mouse macrophages (in vitro) D2SC/1 mouse dendritic cells (in vitro)

N.D.

Mouse macrophages (in vitro)

Mouse macrophages (in vitro) Mouse macrophages (in vitro) Mouse macrophages (in vitro)

Cells presenting antigen by MHC class I

Takahashi et al., 1990 Mowat et al., 1991 Bachmann et al., 1996

Villacres et al., 1998

Nixon et al., 1996

Oh et al., 1997

Oh et al., 1997

Song & Harding, 1996

Schirmbeck et al., 1995a

Reference

444 J. M. DIJKSTRA ET AL.

9 aa peptide

Antigen

15 aa peptide Virus like particles formed by self-assembling modified porcine parvovirus VP2 protein Fusions with bacterial toxins HIV gp120 Anthrax toxin: 8–10 aa peptide fusion to N-terminal half of lethal factor, second factor necessary for uptake. Non-toxic. Translocating 12 aa peptide domain of Pseudomonas exotoxin. Non-toxic. 12–15 aa peptide

Ty protein p1

Strategy

+

+

++

+

+

CTLactivity Reference

P815 mouse mastocytoma Allsopp et al., 1996 cells (in vitro) N.D. (mouse in vivo) Lo-man et al., 1998

Cells presenting antigen by MHC class I

N.D.

activity is not blocked by brefeldin A

N.D./
/CTL

Ulmer et al., 1994

U2-OS human epithelial Donnelly et al., 1993 cells (in vitro) P815 mouse mastocytoma cells (in vitro)

U2-OS human epithelial cells (in vitro)

+/+/CTL activity is blocked P815 mouse mastocytoma Goletz et al., 1997 by a proteasome inhibitor cells (in vitro) Ballard et al., 1998 N.D. N.D. (mouse in vivo)

N.D.

N.D.

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

Table 1. (Continued)

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Antigen

Fusions with di#erent proteins Tat(37–72) Ovalbumin peptide
-galactosidase horseradish peroxidase RNase A domain III of Pseudomonas exotoxin A

N-terminal end of 12–30 aa peptide diphtheria toxin A fragment. B fragment necessary for translocation. Reduced cytotoxicity by manipulation B-fragment of 14 aa peptide Shiga toxin. Non-toxic.

Strategy

N.D.

+

+

N.D.

CTLactivity

In vitro: human PBMC, BM21 and LB705 B lymphoma cells, and primary dendritic cells

Vero green monkey fibroblasts (in vitro)

Cells presenting antigen by MHC class I

Lee et al., 1998

Stenmark et al., 1991

Reference

EL4 mouse T lymphoma Moy et al., 1996 cells (in vitro) Fawell et al., 1994 +/N.D./histology and enzyme Mouse endothelial and activity phagocytic cells (in vivo) in vitro: human HeLa epithelial cells, H9 T lymphoblasts, primary keratinocytes and umbilical vein endothelial cells, monkey COS-1 fibroblasts, hamster CHO fibroblasts and mouse NIH 3T3 fibroblasts N.D.

+/+/histology and CTL activity is blocked by brefeldin A

+/N.D./fusion protein behaves as cytosol protein upon saponin fractionation

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

Table 1. (Continued)

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+

Large part of Heat shock protein 70 ovalbumin (hsp70) Pore forming toxins Ovalbumin Mixing with lysteriolysin (builds pores in the target cells, no toxicity detected at concentrations used) Influenza nucleoprotein N.D.

+/+/CTL activity blocked by brefeldin A (preliminary result)

N.D.

+/+/histology and CTL activity is blocked by brefeldin A

Location in the cytosol/ cytosol location important for CTL stimulation/ method of detection

In vitro: mouse P815 mastocytoma cells, EL4 T lymphoma cells and J774 macrophages

EL4 mouse T lymphoma cells (in vitro)

In vitro: P815 mouse mastocytoma cells, D2.1 mouse fibroblasts, E36 chinese hamster lung cells and LKd mouse fibroblasts N.D. (mouse in vivo)

Cells presenting antigen by MHC class I

Bruder et al., 1998

Darji et al., 1995

Suzue et al., 1997

Schutze-Redelmeier et al., 1996

Reference

HIV-1=human immunodeficiency virus 1; VSV=vesicular stomatitis virus; LCMV=lymphocytic choriomeningitis virus; + =detected;
=not detected; N.D. =not determined.

+

+

+

Antigen

Third helix of the 10 aa peptide Antennapedia homeodomain protein

Strategy

CTLactivity

Table 1. (Continued)

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recombinant bacteria (Pfeifer et al., 1993; Song & Harding, 1996), and heataggregated antigen without additional adjuvants (Schirmbeck et al., 1995a; Speidel et al., 1997) were found to stimulate this response. Virus-like particles (VLPs) are also structures that might depend at least partly on their particulate character for CTL stimulation. VLPs are stable, multimeric, lipid-containing protein particles. They usually contain self-assembling viral proteins. Some have been shown to induce a CTL response to a loaded heterologous antigen (Allsopp et al., 1996; Bachmann et al., 1996; Lo-man et al., 1998). How the VLPs are taken up by cells is largely undefined. VLPs based on proteins from parvovirus, pappilomavirus, HIV, hepatitis B virus or yeast and their induction of a CTL response have been reviewed by Schirmbeck et al. (1996) and Reimann & Schirmbeck (1999). For some of the above-mentioned particles, the involvement of an alternative pathway in MHC class I presentation was demonstrated (Schirmbeck et al., 1995a) but other particulate structures apparently need the conservative pathway for MHC class I presentation (Oh et al., 1997; see Table 1). Other vaccines depend on fusion of antigen to a protein that helps it to pass through the cellular membrane, and thus allows them to enter the conservative proteasome/TAP/MHC class I pathway. Most of these proteins are bacterial toxins that are e$ciently delivered into the cytoplasm. By chemical or genetic fusion of (portions of) these toxins with antigens, the antigen can also be delivered into the cytoplasm. Examples include the toxins of Corynebacterium diphtheriae (Stenmark et al., 1991), Pseudomonas aeruginosa (Prior et al., 1992; Donnelly et al., 1993; Ulmer et al., 1994), Bacillus anthracis (Milne et al., 1995; Goletz et al., 1997; Ballard et al., 1998), Ricinus communis (Beaumelle et al., 1997) or Shigella dysenteriae (Lee et al., 1998). For some of these toxins induction of an MHC I restricted CTL response against the associated antigen has indeed been shown (Table 1). Surprisingly, the CTL response induced by fusion proteins with the translocating domain of pseudomonas exotoxin does not need the conservative MHC class I presentation pathways (Ulmer et al., 1994). Via site-directed mutations or choice of domains the toxin-based delivery systems generally lack the cytotoxicity of the original toxin. Other fusion proteins that deliver antigens into the cytoplasm are a 36 amino acid (AA) peptide derived from the Tat protein of HIV (Fawell et al., 1994; Moy et al., 1996), the 16 aa peptide derived from the antennapedia protein of Drosophila (Theodore et al., 1995; Derossi et al., 1996; Schutze-Redelmeier et al., 1996; Fenton et al., 1998) and the HSP70 heat shock protein (Suzue et al., 1997). HSP70 can also induce CTL responses for non-covalently bound peptides (Román & Moreno, 1996). A unique method of bringing antigens into the cytoplasm is by coinjection with listeriolysin, a pore forming toxin of Listeria monocytogenes that allows transmembrane transport of whole proteins (Darji et al., 1995; Bruder et al., 1998). For many of the vaccine strategies mentioned above macrophages may be essential for CTL stimulation, as in several studies macrophage depletion abrogated the CTL response (Wu et al., 1994; Ke et al., 1995; Wijburg et al., 1998; see Table 1).

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IV. Vaccine-induced CTL activity against rhabdovirus infections IHNV and VHSV are rhabdoviruses causing severe economic losses in salmonid farming (Lorenzen & Olesen, 1997; Winton, 1997). In this section we discuss data on MHC class I restricted CTL activity induced by other rhabdoviruses and their vaccines, and compare them with data from fish when appropriate. Vesicular stomatitis virus (VSV) and rabies virus are extensively investigated rhabdoviruses of mammals (Frenia et al., 1992; Letchworth et al., 1999). CTL induction by the structural proteins of rabies virus and VSV was measured in mice vaccinated with recombinant vaccinia virus that expressed individual genes. The expression of genes for rabies proteins G, N, NS and M (Fujii et al., 1994a,b) and genes for the proteins G and N of VSV (Yewdell et al., 1986) were able to elicit MHC class I restricted CTL activity. Transfer of a cytotoxic T cell clone directed against rabies protein G induced significant protection against rabies in syngeneic mice (Kawano et al., 1990). However, studies in knockout mice indicated that the humoral response against VSV was most important in protection, and CD8-positive cytotoxic T-cells only played a minor role (Thomsen et al., 1997). Apparently the importance of CTL activity varies with di#erent rhabdoviruses. Studies on rhabdoviruses showed that MHC class I restricted cytotoxic responses could be induced by several vaccine strategies distinct from live attenuated viruses or DNA vaccines. For example,
-propiolactoneinactivated rabies virus (Fujii et al., 1994a; Wiktor et al., 1977) or UV-inactivated VSV (Bachmann et al., 1994a) induced a CTL response. These treatments mainly damage the viral RNA, which probably abrogates viral replication but still allows fusion with the cellular membrane. Interesting in this respect is that heat- or formalin-inactivated VHS virus did not induce protection in rainbow trout, but
-propiolactone-inactivated VHS induced significant protection (Lorenzen & Olesen, 1997). Heat and formalin treatment influence the protein structures of the virus and probably inhibit fusion with the cellular membrane. MHC class I restricted cytotoxicity was also obtained when mice were immunised with liposomes containing protein G of rabies virus, whereas protein G alone did not (Macfarlan et al., 1986). Although as far as we know liposomes have not been considered as ingredients of vaccines for fish, administration of liposomes into fish and their uptake by macrophages has been described (Espenes et al., 1997). The N protein of VSV contained in cellular debris elicits a CTL response in mice (Bachmann et al., 1994b). Interesting in this respect is that immunisation of trout fry with recombinant IHNV G protein in freeze/thawed insect cells induced some protection, although no neutralising antibodies could be detected (Cain et al., 1999). Several studies indicate that ISCOMs containing rhabdovirus antigens stimulate a CTL response in mammals and that these are promising vaccine components. Rabies virus antigen in ISCOMs was taken up by macrophages and to a much lesser extent by follicular-dendritic or B-cells (Claassen et al., 1995). Rabies virus antigen-containing ISCOMs induced protection (Fekadu et al., 1992; Claassen et al., 1995) and a proliferative T-cell response suggesting

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CTL activation (Claassen et al., 1998). Macrophage depletion largely inhibited the proliferative T-cell response, although it did not interfere with the humoral response (Claassen et al., 1998). Injection of rabies virus glycoprotein integrated in ISCOMs caused minimal lesions in mice, and no signs of prolonged distress were observed (Leenaars et al., 1995). In fish, local and systemic humoral responses were observed after oral immunisation with antigens entrapped in ISCOMs (Jenkins et al., 1994). In summary, a CTL response can be important in the control of rhabdoviruses, and several vaccine strategies based on exogenous antigen have been shown to induce this response. V. Which approaches toward CTL-stimulating vaccines should be taken? Considering the studies discussed above it is di$cult to decide what types of vaccine should be developed and analysed for their ability to stimulate a CTL response in fish. Easily preparable vaccines such as antigens associated with cellular debris, or currently existing vaccines such as
-propiolactoneinactivated viruses should be analysed. In addition, adjuvants that can be delivered orally such as microparticles (Partidos et al., 1999) or ISCOMs (Mowat et al., 1991, 1999) should be examined. Microparticles have already been described as oral vaccines in rainbow trout (Lavelle et al., 1997), and ISCOMs have been shown to induce a humoral immune response after oral application in Oreochromis mossambicus (Jenkins et al., 1994). In contrast to many of the vaccine strategies mentioned in this study that are only barely investigated, ISCOMs must have been promising enough to tempt a large number of researchers to investigate their protective value. In mammals ISCOMs have been shown to yield protection against a number of viruses, namely orthomyxo-, paramyxo-, herpes-, rhabdo-, retro-, flavi- and enteroviridae (reviewed in Osterhaus & Rimmelzwaan, 1998). The spectrum of antigens that can be integrated into these vaccines is large. ISCOMs are cage-like micelles formed by the spontaneous coalescence of phosphatidyl choline and cholesterol in the presence of Quil A (saponin) (Mowat et al., 1991; Osterhaus & Rimmelzwaan, 1998). Although ISCOMs do not fuse with cell membranes as liposomes can, they probably penetrate them and direct the antigen into the cytoplasm (Takahashi, 1990; Raychaudhuri & Morrow, 1993; Morein & Bengtsson, 1998). ISCOMs have been shown to stimulate both humoral and CTL responses (Jones et al., 1988; Trudel et al., 1992; Hulskotte et al., 1995). Whole bacteria expressing foreign antigens might also be a valuable vaccine strategy to induce CTL responses in fish. Interesting in this respect is that a bacterial vaccine consisting of Aeromonas salmonicida expressing IHNV or VHSV epitopes induced a protective immune response against these rhabdoviruses (Noonan et al., 1995). The ability of emulsifying agents such as Freund’s adjuvant to stimulate CTL activity seems restricted to certain antigens (Table 1). However, oil-based vaccines are now commonly and successfully used in fish (Markestad & Grave, 1997), and their potential to induce CTL activity should be examined.

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Bacterial toxins as fusion proteins are promising, although information on protection levels induced by these vaccines is very limited. This is also true for fusion systems based on the antennapeptedia peptide, the Tat peptide or the HSP70 protein. Whether the small Tat peptide, as derived from HIV, would be accepted by the public in fish vaccines is questionable, but this fusion system has the advantage of being able to deliver a broad spectrum of antigens (Table 1). Liposomes or lipoprotein particles are interesting delivery systems, because they allow the administration of a wide variety of intact protein antigens (Table 1). Liposomes may also be used in immersion-vaccination in fish, as has been shown by successful delivery of DNA by a marker DNA-liposome formulation (Fernandez-Alonso et al., 1999). Another advantage of liposomes is that findings from mammals are likely to be successfully extrapolated to fish. For vaccine strategies that are based on protein function such as fusion protein systems, virosomes or virus-like particles, this might be less likely because of di#erences between species and e#ects of di#erent temperatures. VI. Conclusion and discussion Viral diseases cause major losses in fish farming and for most viral diseases, there are no satisfactory vaccines. As trial and error experiments over several decades has not resulted in acceptable vaccines, it is important to study how to stimulate the immune response against viruses at a more fundamental level. For this, not only humoral, but also specific cellular immune mechanisms must be investigated. The establishment of an MHC class I restricted cell-mediated cytotoxicity assay in rainbow trout o#ers the possibility to study that portion of the immune response which may be of primary importance for many viral diseases. This new assay system provides a mechanism by which various vaccine formulations and vaccination techniques/schedules can be tested for their potency to stimulate CTL activity. Combined with previously existing knowledge on stimulation of the humoral response in fish, this will lead to further improvement of fish vaccines. Studies performed in mammals provide a basis to identify vaccine strategies which should be examined in fish. This review concerns vaccine strategies based on exogenous antigens that are likely to be accepted for commercial use in fish, in contrast to live attenuated viruses and DNA vaccines. We have described a number of promising vaccine strategies based on targeting exogenous antigens into the MHC class I pathway. Because the field of research is young, data on many of the vaccine strategies mentioned in this review are limited. Typically there are only a few studies and the mechanisms involved in the cellular response often remain unclear. In addition, for several vaccine strategies no data on the protective value upon challenge are available and limitations in the spectrum of antigens capable of CTL stimulation exist. Despite these limitations, the combination of the positive data shown in Table 1 and the need for commercially acceptable vaccines that can stimulate CTL responses in fish advocate the investigation of CTL stimulation by exogenous antigen vaccines in fish. Particularly interesting candidates in our opinion are antigens complexed in ISCOMs, expressed in bacteria or enclosed in microparticles, as

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these can be administered orally. Some of the vaccine strategies mentioned in this review include the antigen being o#ered by alternative MHC class I loading pathways. Whether these pathways also exist in fish and whether their relative importance compared to the conservative MHC class I loading pathway is higher or lower than in mammals remains unclear. This will naturally influence the success of these vaccine strategies in fish. An MHC class I restricted CTL response has also been shown to be important in the control of several intracellular bacteria infecting mammals (reviewed by Harty & Bevan, 1999). This may also be true for intracellular bacteria in fish. The CTL assay can analyse this matter and also which types of bacterial vaccines are able to induce this response. It is to be hoped that the analysis of CTL stimulation by exogenous antigens will be of great benefit in the development of e#ective and safe vaccines against diseases in fish. This study was supported by ‘the promotion of basic research activities for innovative biosciences’ funded by Bio-oriented Technology Research Advancement Institution (BRAIN) and supported by a postdoctoral fellowship for J. M. Dijkstra from the Science and Technology Agency of Japan. We thank Jim Moore for helping to edit this article.

References Aichele, P., Hengartner, H., Zinkernagel, R. M. & Schulz, M. (1990). Antiviral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide. Journal of Experimental Medicine 171, 1815–1820. Allsopp, C. E., Plebanski, M., Gilbert, S., Sinden, R. E., Harris, S., Frankel, G., Dougan, G., Hioe, C., Nixon, D., Paoletti, E., Layton, G. & Hill, A. V. (1996). Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization. European Journal of Immunology 26, 1951–1959. Aoyagi, K., Dijkstra, J. M., Xia, C., Denda, I., Ototake, M., Hashimoto, K. & Nakanishi, T. Classical MHC class I genes composed of highly divergent sequence lineages share a single locus in rainbow trout (Oncorhynchus mykiss). Submitted to the Journal of Immunology. Arkema, A., Huckriede, A., Schoen, P., Wilschut, J. & Daemen, T. (2000). Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes. Vaccine 18, 1327–1333. Bachmann, M. F., Bast, C., Hengartner, H. & Zinkernagel, R. M. (1994a). Immunogenicity of a viral model vaccine after di#erent inactivation procedures. Medical Microbiology and Immunology 183, 95–104. Bachmann, M. F., Hengartner, H. & Zinkernagel, R. M. (1994b). Immunization with recombinant protein: conditions for cytotoxic T cell and/or antibody induction. Medical Microbiology and Immunology 183, 315–324. Bachmann, M. F., Lutz, M. B., Layton, G. T., Harris, S. J., Fehr, T., Rescigno, M. & Ricciardi-Castagnoli, P. (1996). Dendritic cells process exogenous viral proteins and virus-like particles for class I presentation to CD8+ cytotoxic T lymphocytes. European Journal of Immunology 26, 2595–2600. Ballard, J. D., Collier, R. J. & Starnbach, M. N. (1998). Anthrax toxin as a molecular tool for stimulation of cytotoxic T lymphocytes: disulfide-linked epitopes, multiple injections, and role of CD4(+) cells. Infection and Immunity 66, 4696–4699. Beaumelle, B., Taupiac, M. P., Lord, J. M. & Roberts, L. M. (1997). Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol. Journal of Biological Chemistry 272, 22097–22102.

EXOGENOUS ANTIGENS AND MHC CLASS I

453

Bruder, D., Darji, A., Gakamsky, D. M., Chakraborty, T., Pecht, I., Wehland, J. & Weiss, S. (1998). E$cient induction of cytotoxic CD8+ T cells against exogenous proteins: establishment and characterization of a T cell line specific for the membrane protein ActA of Listeria monocytogenes. European Journal of Immunology 28, 2630–2639. Cain, K. D., LaPatra, S. E., Shewmaker, B., Jones, J., Byrne, K. M. & Ristow, S. S. (1999). Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect calls. Diseases of Aquatic Organisms 36, 67–72. Claassen, I. J., Osterhaus, A. D. & Claassen, E. (1995). Antigen detection in vivo after immunization with di#erent presentation forms of rabies virus antigen: involvement or marginal metallophilic macrophages in the uptake of immunestimulating complexes. European Journal of Immunology 25, 1446–1452. Claassen, I. J., Osterhaus, A. D., Poelen, M., Van Rooijen, N. & Claassen, E. (1998). Antigen detection in vivo after immunization with di#erent presentation forms of rabies virus antigen, II. Cellular, but not humoral, systemic immune responses against rabies virus immune-stimulating complexes are macrophage dependent. Immunology 94, 455–460. Collins, D. S., Findlay, K. & Harding, C. V. (1992). Processing of exogenous liposomeencapsulated antigens in vivo generates class I MHC-restricted T cell responses. Journal of Immunology 148, 3336–3341. Darji, A., Chakraborty, T., Wehland, J. & Weiss, S. (1995). Listeriolysin generates a route for the presentation of exogenous antigens by major histocompatibility complex class I. European Journal of Immunology 25, 2967–2971. Deres, K., Schild, H., Wiesmuller, K. H., Jung, G. & Rammensee, H. G. (1989). In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342, 561–564. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. & Prochiantz, A. (1996). Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. Journal of Biological Chemistry 271, 18188–18193. Dijkstra, J. M., Okamoto, H., Ototake, M. & Nakanishi, T. (2001). Luciferase expression 2 years after DNA injection in glass catfish (Kryptopterus bicirrhus). Fish & Shellfish Immunology 11, 199–202. Dixon, B., van Erp, S. H., Rodrigues, P. N., Egberts, E. & Stet, R. J. (1995). Fish major histocompatibility complex genes: an expansion. Developmental & Comparative Immunology 19, 109–133. Donnelly, J. J., Ulmer, J. B., Hawe, L. A., Friedman, A., Shi, X. P., Leander, K. R., Shiver, J. W., Oli#, A. I., Martinez, D., Montgomery, D. & Liu, M. (1993). Targeted delivery of peptide epitopes to class I major histocompatibility molecules by a modified Pseudomonas exotoxin. Proceedings of the National Academy of Sciences of the United States of America 90, 3530–3534. Espenes, A., Press, C. M., Rooijen, N. V. & Landsverk, T. (1997). Apoptosis in phagocytotic cells of lymphoid tissues in rainbow trout (Oncorhynchus mykiss) following administration of clodronate liposomes. Cell & Tissue Research 289, 323–331. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B. & Barsoum, J. (1994). Tat-mediated delivery of heterologous proteins into cells. Proceedings of the National Academy of Science of the United States of America 91, 664–668. Fekadu, M., Shaddock, J. H., Ekstrom, J., Osterhaus, A., Sanderlin, D. W., Sundquist, B. & Morein, B. (1992). An immune stimulating complex (ISCOM) subunit rabies vaccine protects dogs and mice against street rabies challenge. Vaccine 10, 192–197. Fenton, M., Bone, N. & Sinclair, A. J. (1998). The e$cient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line. Journal of Immunological Methods 212, 41–48. Fernandez-Alonso, M., Alvarez, F., Estepa, A., Blasco, R. & Coll, J. M. (1999). A model to study fish DNA immersion vaccination by using the green fluorescent protein. Journal of Fish Diseases 22, 237–241.

454

J. M. DIJKSTRA ET AL.

Fischer, U., Dijkstra, J. M., Aoyagi, K., Xia, C., Ototake, M. & Nakanishi, T. Specific cell-mediated cytotoxicity against virus-infected MHC class I matching cells in rainbow trout (Oncorhynchus mykiss). Submitted to the Journal of Virology. Frenia, M. L., Lafin, S. M. & Barone, J. A. (1992). Features and treatment of rabies. Clinical Pharmacology 11, 37–47. Fujii, H., Mannen, K., Takita-Sonoda, Y., Hirai, K., Cruz-Abrenica, M. S., Kawano, Y., Nichizono, A. & Mifune, K. (1994a). Target cells of cytotoxic T lymphocytes directed to the individual structural proteins of rabies virus. Microbiology and Immunology 38, 721–726. Fujii, H., Takita-Sonoda, Y., Mifune, K., Hirai, K., Nishizono, A. & Mannen, K. (1994b). Protective e$cacy in mice of post-exposure vaccination with vaccinia virus recombinant expressing either rabies virus glycoprotein or nucleoprotein. Journal of General Virology 75, 1339–1344. Germain, R. N. (1994). MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76, 287–299. Goletz, T. J., Klimpel, K. R., Arora, N., Leppla, S. H., Keith, J. M. & Berzofsky, J. A. (1997). Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein. Proceedings of the National Academy of Sciences of the United States of America 94, 12059–12064. Hansen, J. D., Strassburger, P. & Du Pasquier, L. (1996). Conservation of an alpha 2 domain within the teleostean world, MHC class I from the rainbow trout Oncorhynchus mykiss. Developmental and Comparative Immunology 20, 417–425. Hansen, J. D., Strassburger, P., Thorgaard, G. H., Young, W. P. & Du Pasquier, L. (1999). Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss. Journal of Immunology 163, 774–786. Hansen, J. D. & Strassburger, P. (2000). Description of an ectothermic TCR coreceptor, CD8 alpha, in rainbow trout. Journal of Immunology 164, 3132–3139. Harty, J. T. & Bevan, M. J. (1999). Responses of CD8 T cells to intracellular bacteria. Current Opinion in Immunology 11, 89–93. Heeg, K., Kuon, W. & Wagner, H. (1991). Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immunostimulating-ovalbumin complexes enter the class I MHC-restricted antigen pathway and allow sensitization against the immunodominant peptide. European Journal of Immunology 21, 1523–1527. Hulskotte, E. G., Geretti, A. M., Siebelink, K. H., van Amerongen, G., Cranage, M. P., Rud, E. W., Norley, S. G., de Vries, P. & Osterhaus, A. D. (1995). Vaccine-induced virus-neutralizing antibodies and cytotoxic T cells do not protect macaques from experimental infection with simian immunodeficiency virus SIVmac32H (J5). Journal of Virology 69, 6289–6296. Iwama, G. & Nakanishi, T. (1996). The Fish Immune System. San Diego, CA: Academic Press Inc. Jenkins, P. G., Wrathmell, A. B., Harris, J. E. & Pulsford, A. L. (1994). Systemic and mucosal immune responses to enterically delivered antigen in Oreochromis mossambicus. Fish & Shellfish Immunology 4, 255–271. Jondal, M., Schirmbeck, R. & Reimann, J. (1996). MHC class I-restricted CTL responses to exogenous antigens. Immunity 5, 295–302. Jones, P. D., Tha Hla, R., Morein, B., Lovgren, K. & Ada, G. L. (1988). Cellular immune responses in the murine lung to local immunization with influenza A virus glycoproteins in micelles and immunostimulatory complexes (iscoms). Scandinavian Journal of Immunology 27, 645–652. Kaattari, S. L. & Piganelli, J. D. (1996). The specific immune system: humoral defense. In The Fish Immune System (G. Iwama & T. Nakanishi, eds) pp. 159–205. San Diego, CA: Academic Press Inc. Kawano, H., Mifune, K., Ohuchi, M., Mannen, K., Cho, S., Hiramatsu, K. & Shichijo, A. (1990). Protection against rabies in mice by a cytotoxic T cell clone recognizing the glycoprotein of rabies virus. Journal of General Virology 71, 281–287.

EXOGENOUS ANTIGENS AND MHC CLASS I

455

Ke, Y., Li, Y. & Kapp, J. A. (1995). Ovalbumin injected with complete Freund’s adjuvant stimulates cytolytic responses. European Journal of Immunology 25, 549–553. Ke, Y. & Kapp, J. A. (1996). Exogenous antigens gain access to the major histocompatibility complex class I processing pathway in B cells by receptor-mediated uptake. Journal of Experimental Medicine 184, 1179–1184. Klein, J. & Sato, A. (1998). Birth of the major histocompatibility complex. Scandinavian Journal of Immunology 47, 199–209. Kourilsky, P. & Claverie, J. M. (1989). MHC restriction, alloreactivity, and thymic education: a common link? Cell 56, 327–329. Lavelle, E. C., Jenkins, P. G. & Harris, J. E. (1997). Oral immunization of rainbow trout with antigen microencapsulated in poly(DL-lactide-co-glycolide) microparticles. Vaccine 15, 1070–1078. Lee, R. S., Tartour, E., van der Bruggen, P., Vantomme, V., Joyeux, I., Goud, B., Fridman, W. H. & Johannes, L. (1998). Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin. European Journal of Immunology 28, 2726–2737. Leenaars, P. P., Hendriksen, C. F., Koedam, M. A., Claassen, I. & Claasen, E. (1995). Comparison of adjuvants for immune potentiating properties and side e#ects in mice. Veterinary Immunology and Immunopathology 48, 123–138. Letchworth, G. J., Rodriguez, L. L. & Barrera, J. C. (1999). Vesicular stomatitis. Veterinary Journal 157, 239–260. Liu, M. A. (1997). The immunologist’s grail: vaccines that generate cellular immunity. Proceedings of the National Academy of Sciences of the United States of America 94, 10496–10498. Lo-Man, R., Rueda, P., Sedlik, C., Deriaud, E., Casal, I. & Leclerc, C. (1998). A recombinant virus-like particle system derived from parvovirus as an e$cient antigen carrier to elicit a polarized Th1 immune response without adjuvant. European Journal of Immunology 28, 1401–1407. Lorenzen, N., Lorenzen, E., Einer-Jensen, K., Heppell, J., Wu, T. & Davis, H. L. (1998). Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish & Shellfish Immunology 8, 261–270. Lorenzen, N. & Olesen, N. J. (1997). Immunization with viral antigens: viral haemorrhagic septicaemia. Developments in Biological Standardization 90, 201–209. Macfarlan, R. I., Dietzschold, B. & Koprowski, H. (1986). Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain. Molecular Immunology 23, 733–741. Manning, M. J. & Nakanishi, T. (1996). The specific immune system: cellular defenses. In The Fish Immune System (G. Iwama & T. Nakanishi, eds) pp. 159–205. San Diego, CA: Academic Press Inc. Markestad, A. & Grave, K. (1997). Reduction of antibacterial drug use in Norwegian fish farming due to vaccination. Developments in Biological Standardization 90, 365–369. Mellman, I., Turley, S. J. & Steinman, R. M. (1998). Antigen processing for amateurs and professionals. Trends in Cell Biology 8, 231–237. Milne, J. C., Blanke, S. R., Hanna, P. C. & Collier, R. J. (1995). Protective antigenbinding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus. Molecular Microbiology 15, 661–666. Morein, B., Villacres-Eriksson, M., Sjolander, A. & Bengtsson, K. L. (1996). Novel adjuvants and vaccine delivery systems. Veterinary Immunology and Immunopathology 54, 373–384. Morein, F. & Bengtsson, K. L. (1998). Functional aspects of iscoms. Immunology and Cell Biology 76, 295–299. Mowat, A. M., Donachie, A. M., Reid, G. & Jarrett, O. (1991). Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted

456

J. M. DIJKSTRA ET AL.

T lymphocytes in vivo and are immunogenic by the oral route. Immunology 72, 317–322. Mowat, A. M., Smith, R. E., Donachie, A. M., Furrie, E., Grdic, D. & Lycke, N. (1999). Oral vaccination with immune stimulating complexes. Immunology Letters 65, 133–140. Moy, P., Daikh, Y., Pepinsky, B., Thomas, D., Fawell, S. & Barsoum, J. (1996). Tat-mediated protein delivery can facilitate MHC class I presentation of antigens. Molecular Biotechnology 6, 105–113. Nakanishi, T., Aoyagi, K., Xia, C., Dijkstra, J. M. & Ototake, M. (1999). Specific cell-mediated immunity in fish. Veterinary Immunology and Immunopathology 72, 101–109. Newman, M. J., Wu, J. Y., Gardner, B. H., Munroe, K. J., Leombruno, D., Recchia, J., Kensil, C. R. & Coughlin, R. T. (1992). Saponin adjuvant induction of ovalbuminspecific CD8+ cytotoxic T Lymphocyte responses. Journal of Immunology 148, 2357–2362. Nixon, D. F., Hioe, C., Chen, P. D., Bian, Z., Kuebler, P., Li, M. L., Qiu, H., Li, X. M., Singh, M., Richardson, J., McGee, P., Zamb, T., Ko#, W., Wang, C. Y. & O’Hagan, D. (1996). Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity. Vaccine 14, 1523–1530. Noonan, B., Enzmann, P. J. & Trust, T. J. (1995). Recombinant infectious hematopoietic necrosis virus and viral haemorrhage septicaemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss). Applied and Environmental Microbiology 61, 3586–3591. Oh, Y. K., Harding, C. V. & Swanson, J. A. (1997). The e$ciency of antigen delivery from macrophage phagosomes into cytoplasm for MHC class I-restricted antigen presentation. Vaccine 15, 511–518. Oldstone, M. B. (1997). How viruses escape from cytotoxic T lymphocytes: molecular parameters and players. Virology 234, 179–185. Osterhaus, A. D. & Rimmelzwaan, G. F. (1998). Induction of virus-specific immunity by iscoms. Developments in Biological Standards 92, 49–58. Pamer, E. & Cresswell, P. (1998). Mechanisms of MHC class I-restricted antigen processing. Annual Review of Immunology 16, 323–358. Partidos, C. D., Vohra, P., Jones, D., Farrar, G. & Steward, M. W. (1997). CTL responses induced by a single immunization with peptide encapsulated in biodegradable microparticles. Journal of Immunological Methods 206, 143–151. Partidos, C. D., Vohra, P., Jones, D. H., Farrar, G. & Steward, M. W. (1999). Induction of cytotoxic T-cell responses following oral immunization with synthetic peptides encapsulated in PLG microparticles. Journal of Controlled Release 62, 325–332. Pfeifer, J. D., Wick, M. J., Roberts, R. L., Findlay, K., Normark, S. J. & Harding, C. V. (1993). Phagocytic processing of bacterial antigens for class I MHC presentation to T cells. Nature 361, 359–362. Prior, T. I., FitzGerald, D. J. & Pastan, I. (1992). Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol. Biochemistry 31, 3555–3559. Rast, J. P. & Litman, G. W. (1998). Towards understanding the evolutionary origins and early diversification of rearranging antigen receptors. Immunological Reviews 166, 79–86. Raychaudhuri, S. & Morrow, W. J. (1993). Can soluble antigens induce CD8+ cytotoxic T-cell responses? A paradox revisited. Immunology Today 14, 344–348. Reddy, R., Zhou, F., Huang, L., Carbone, F., Bevan, M. & Rouse, B. T. (1991). pH sensitive liposomes provide an e$cient means of sensitizing target cells to class I restricted CTL recognition of a soluble protein. Journal of Immunological Methods 141, 157–163. Reddy, R., Zhou, F., Nair, S., Huang, L. & Rouse, B. T. (1992). In vivo cytotoxic T lymphocyte induction with soluble proteins administered in liposomes. Journal of Immunology 148, 1585–1589.

EXOGENOUS ANTIGENS AND MHC CLASS I

457

Reimann, J. & Kaufmann, S. H. (1997). Alternative antigen processing pathway in anti-infective immunity. Current Opinion in Immunology 9, 462–469. Reimann, J. & Schirmbeck, R. (1999). Alternative pathways for processing exogenous and endogenous antigens that can generate peptides for MHC class I-restricted presentation. Immunological Reviews 172, 131–152. Reits, E. A., Vos, J. C., Gromme, M. & Neefjes, J. (2000). The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404, 774–778. Rodriguez, F., Zhang, J. & Whitton, J. L. (1997). DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. Journal of Virology 71, 8497–8503. Román, E. & Moreno, C. (1996). Synthetic peptides non-covalently bound to bacterial hsp 70 elicit peptide-specific T-cell responses in vivo. Immunology 88, 487–492. Schirmbeck, R., Bohm, W., Melber, K. & Reimann, J. (1995a). Processing of exogenous heat-aggregated (denatured) and particulate (native) hepatitis B surface antigen for class I-restricted epitope presentation. Journal of Immunology 155, 4676– 4684. Schirmbeck, R., Melber, K. & Reimann, J. (1995b). Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. European Journal of Immunology 25, 1063–1070. Schirmbeck, R., Bohm, W. & Reimann, J. (1996). Virus-like particles induce MHC class I-restricted T-cell responses. Intervirology 39, 111–119. Schirmbeck, R., Wild, J. & Reimann, J. (1998). Similar as well as distinct MHC class I-binding peptides are generated by exogenous and endogenous processing of hepatitis B virus surface antigen. European Journal of Immunology 28, 4149– 4161. Schutze-Redelmeier, M. P., Gournier, H., Garcia-Pons, F., Moussa, M., Joliot, A. H., Volovitch, M., Prochiantz, A. & Lemonnier, F. A. (1996). Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo. Journal of Immunology 157, 650–655. Simpson, E. (1988). Function of the MHC. Immunology Supplement 1, 27–30. Somamoto, T., Nakanishi, T. & Okamoto, N. (2000). Specific cell-mediated cytotoxicity against a virus-infected syngeneic cell line in isogeneic ginbuna crucian carp. Developmental and Comparative Immunology 24, 633–640. Song, R. & Harding, C. V. (1996). Roles of proteasomes, transporter for antigen presentation (TAP), and beta 2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway. Journal of Immunology 156, 4182–4190. Speidel, K., Osen, W., Faath, S., Hilgert, I., Obst, R., Braspenning, J., Momburg, F., Hammerling, G. J. & Rammensee, H. G. (1997). Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: conditions, e$ciency, and relation to antibody responses. European Journal of Immunology 27, 2391–2399. Stenmark, H., Moskaug, J. O., Madshus, I. H., Sandvig, K. & Olsnes, S. (1991). Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol. Journal of Cell Biology 113, 1025–1032. Stet, R. J. M., Dixon, B., van Erp, S. H. M., van Lierop, M. C., Rodrigues, P. N. S. & Egberts, E. (1996). Inference of structure and function of fish major histocompatibility complex (MHC) molecules from expressed genes. Fish & Shellfish Immunology 6, 305–318. Suzue, K., Zhou, X., Eisen, H. N. & Young, R. A. (1997). Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway. Proceedings of the National Academy of Sciences of the United States of America 94, 13146–13151. Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R. N. & Berzofsky, J. A. (1990). Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature 344, 873–875.

458

J. M. DIJKSTRA ET AL.

Theodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P. & Prochiantz, A. (1995). Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. Journal of Neuroscience 15, 7158–7167. Thomsen, A. R., Nansen, A., Andersen, C., Johansen, J., Marker, O. & Christensen, J. P. (1997). Cooperation of B cells and T cells is required for survival of mice infected with vesicular stomatitis virus. International Immunology 9, 1757–1766. Townsend, A. & Bodmer, H. (1989). Antigen recognition by class I-restricted T lymphocytes. Annual Review of Immunology 7, 601–624. Trudel, M., Nadon, F., Seguin, C., Brault, S., Lusignan, Y. & Lemieux, S. (1992). Initiation of cytotoxic T-cell response and protection of Balb/c mice by vaccination with an experimental ISCOMs respiratory syncytial virus subunit vaccine. Vaccine 10, 107–112. Ulmer, J. B., Donnelly, J. J. & Liu, M. A. (1994). Presentation of an exogenous antigen by major histocompatibility complex class I molecules. European Journal of Immunology 24, 2590–2596. van Binnendijk, R. S., van Baalen, C. A., Poelen, M. C., de Vries, P., Boes, J., Cerundolo, V., Osterhaus, A. D. & UytdeHaag, F. G. (1992). Measles virus transmembrane fusion protein synthesized de novo or presented in immunostimulating complexes is endogenously processed for HLA class I- and class II-restricted cytotoxic T cell recognition. Journal of Experimental Medicine 176, 119–128. van Endert, P. M. (1999). Genes regulating MHC class I processing of antigen. Current Opinion in Immunology 11, 82–88. Villacres, M. C., Behboudi, S., Nikkila, T., Lovgren-Bengtsson, K. & Morein, B. (1998). Internalization of iscom-borne antigens and presentation under MHC class I or class II restriction. Cellular Immunology 185, 30–38. Walker, C., Selby, M., Erickson, A., Cataldo, D., Valensi, J. P. & Van Nest, G. V. (1992). Cationic lipids direct a viral glycoprotein into the class I major histocompatibility complex antigen-presentation pathway. Proceedings of the National Academy of Sciences of the United States of America 89, 7915–7918. Weenink, S. M. & Gautam, A. M. (1997). Antigen presentation by MHC class II molecules. Immunology and Cell Biology 75, 69–81. Wijburg, O. L., van den Dobbelsteen, G. P., Vadolas, J., Sanders, A., Strugnell, R. A. & van Rooijen, N. (1998). The role of macrophages in the induction and regulation of immunity elicited by exogenous antigens. European Journal of Immunology 28, 479–487. Wiktor, T. J., Doherty, P. C. & Koprowski, H. (1977). In vitro evidence of cell-mediated immunity after exposure of mice to both live and inactivated rabies virus. Proceedings of the National Academy of Sciences of the United States of America 74, 334–338. Winton, J. R. (1997). Immunization with viral antigens: infectious haematopoietic necrosis. Developments in Biological Standardization 90, 211–220. Wu, J. Y., Gardner, B. H., Kushner, N. N., Pozzi, L. A., Kensil, C. R., Cloutier, P. A., Coughlin, R. T. & Newman, M. J. (1994). Accessory cell requirements for saponin adjuvant-induced class I MHC antigen-restricted cytotoxic T-lymphocytes. Cellular Immunology 154, 393–406. Wu, J. Y., Gardner, B. H., Murphy, C. I., Seals, J. R., Kensil, C. R., Recchia, J., Beltz, G. A., Newman, G. W., Newman, M. J. (1992). Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine. Journal of Immunology 148, 1519–1525. Yewdell, J. W., Bennink, J. R., Mackett, M., Lefrancois, L., Lyles, D. S. & Moss, B. (1986). Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes. Journal of Experimental Medicine 163, 1529–1538. Zhou, F., Rouse, B. T. & Huang, L. (1992). Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles. Journal of Immunology 149, 1599–1604.