Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 2, 2000, pp. 115-116

New Non-replicative Vectors to Induce Protective
Anti-viral and Anti-tumor Cytotoxic T Cell Responses

Claude Leclerc

Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 28 rue du Docteur Roux,
75724 Paris Cedex 15, France.

Papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: ba00028

Introduction

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the elimination of cells infected by pathogens and in the regression of tumors. CTLs recognize antigen-derived peptides presented by major histocompatibility (MHC) class I molecules on the cell surface and are usually activated by peptides resulting from the processing of endogenous intracellular proteins. Because antigens have to gain access to the cytosol to enter the class I-restricted presentation pathway, exogenous soluble proteins are usually unable to stimulate CTL responses. Therefore, several strategies have been developed to deliver exogenous antigens into the cytosol. Protein or peptide antigens delivered in association with appropriate adjuvants efficiently stimulate CTL responses. However, alum is still the only adjuvant currently licensed for use in human vaccines. Several recombinant bacterial or viral live vectors have also been shown to sensitize CTLs in vivo but are risk-prone. DNA vaccination may also represent a powerful strategy to activate CTL responses, but the safety of this method remains to be determined. Therefore, the development of safe strategies to induce CTL responses with non-replicating antigens is still an important prerequisite for the design of new efficient vaccines.

Recently, we have developed new and efficient non-replicative recombinant vectors able to deliver antigens into the MHC class I pathway based either on the invasive property of a bacterial toxin or on the capacity of parvovirus-like particles to deliver foreign CTL epitopes into the cytosol of cells.

Recombinant adenylate cyclase
toxins of Bordetella pertussis

The adenylate cyclase toxin (CyaA) of B. pertussis is able to invade a large number of eukaryotic cells and to deliver its N-terminal catalytic domain (400 amino acid residues) directly into the cytosol through the cytoplasmic membrane [1, 2]. Foreign peptides can be inserted into various permissive sites of the catalytic domain of CyaA without altering its main properties such as stability, and catalytic and invasive activities [3, 4]. Purified CyaA toxins carrying CTL epitopes from the nucleoprotein of LCMV or from the V3 region of HIV-1 gpl20 were shown to stimulate strong specific CTL responses, mediated by class I-restricted CD8+ T cells and able to kill target cells coated with the relevant peptide [5].

Mice immunized with the recombinant toxin carrying the LCMV epitope developed strong CTL responses against LCMV-infected target cells. Moreover, these mice were protected against an intracerebral challenge with a virulent strain of LCMV that killed all non-immunized mice within 7 days [6]. This protection was abolished after in vivo elimination of CD8+ T cells. A mutant toxin devoid of adenylate cyclase activity (i.e., cAMP synthesizing activity) was constructed by insertion of a dipeptide into the catalytic site of the molecule. This genetically detoxified invasive toxin carrying the LCMV epitope also stimulated a strong CTL response against both peptide-coated and virus-infected target cells. Mice immunized with this detoxified molecule were fully protected against a lethal intracerebral LCMV challenge [6].

The capacity of CyaA to also induce anti-tumor therapeutic T cell responses was recently demonstrated using melanoma cells transfected with ovalbumin. Mice immunized with a recombinant CyaA toxin carrying a CTL epitope derived from ovalbumin were able to reject tumors that express ovalbumin [7].

These results, which represent the first demonstration of the in vivo induction of protective CTL responses by a detoxified invasive toxin illustrate the strong potential of this strategy to develop new preventive or therapeutic vaccines against viral infections or cancers. Recent results will be also presented which further document the efficiency of recombinant adenylate cyclases to stimulate responses against various CTL epitopes. Mechanisms by which the epitopes carried by the recombinant toxin are delivered to MHC class I molecules have also been recently deciphered [8].

In particular, we have established that the recombinant CyaA is currently processed and presented like an endogenous cytosolic antigen by a mechanism requiring: 1) processing by the proteasome, 2) the TAP molecules, 3) the neosynthesis of MHC class I molecules [8].

Recombinant parvovirus-like particles

Hybrid recombinant parvovirus-like particles (PPV:VLP) are formed by the self-assembly of the VP2 capsid protein of PPV carrying a foreign epitope at its N-terminus. Chimeric PPV:VLPs expressing a CD4+ T cell epitope from the VP1 protein of poliovirus type 1 or from the PreS2 region of hepatitis B virus stimulated CD4+ T cell proliferative responses and cytokine production [9, 10].

Hybrid PPV:VLP carrying a CD8+ T cell epitope from the LCMV nucleoprotein induced in mice strong CTL responses against both peptide-coated or virus-infected target cells [11]. These CTL responses were obtained in the absence of any adjuvant but immunization of mice with hybrid PPV:VLP in the presence of alum did not prevent CTL activation. lnterestingly, the CD8+ class I-restricted cytotoxic activity induced by these recombinant particles persisted in vivo for at least 9 months.

Furthermore, the hybrid parvovirus-like particles carrying the LCMV epitope were able to induce a complete protection of mice against a lethal LCMV infection. These results represent the first demonstration that hybrid non-replicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge in the absence of any adjuvant [11].

The capacity of these pseudo-particles to stimulate cellular or humoral mucosal responses has also been recently investigated. Intranasal immunization of mice by PPV:VLP expressing the LCMV epitope induces CTL responses. Moreover, anti-PPV IgA antibody responses can also be evidenced in mucosal fluids of intranasally immunized animals [12].

Altogether, these results illustrate the strong potential of these alternative new strategies to develop vaccines able to stimulate cellular immunity. Moreover, these new vectors represent important new tools to dissect the various MHC class I presentation pathways.

References

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