Recombinant parvovirus-like particles as an antigen carrier: a novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

Abstract

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8(+) T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8(+) class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.

Figure 1

Analysis of the expression in insect cells of the recombinant PPV:VLP carrying the 118–132 LCMV CD8⁺ epitope. (a) Coomassie blue-stained SDS/polyacrylamide gels showing the different steps of PPV:VLP-(LCMV) purification. Lanes: M, molecular mass markers; 1, mock-infected Sf9 cells; 2, crude extract from infected cells; 3, supernatant from lysed cells; 4, pellet from 20% ammonium sulfate precipitation. (b) Immunoblotting analysis of PPV:VLP-(LCMV) with anti-PPV rabbit antiserum (lane 1) and anti-LCMV 118–132 peptide mouse antiserum (lane 2). (c) Electron microscopy analysis of PPV:VLP-(LCMV) particles negatively stained with 2% uranyl acetate.

Figure 2

In vivo induction of CTL responses by recombinant PPV:VLP carrying the 118–132 LCMV CD8⁺ epitope. BALB/c mice were immunized i.p. on day 0 (A) or on days 0 and 21 (B) with 10 μg of control PPV:VLP or PPV:VLP-(LCMV). Fourteen (A) or 7 (B) days later, spleen cells were in vitro-stimulated for 5 days with the p118–132 peptide and irradiated syngeneic spleen cells. Cytotoxic activity of the effector cells was measured on ⁵¹Cr-labeled P815 target cells pulse-labeled with the p118–132 peptide (•) or incubated with medium alone (▴). Data represent the mean of percent specific lysis from duplicate samples.

Figure 3

Immunization of mice with PPV:VLP-(LCMV) induces a long-lasting memory CD8⁺ CTL activity. (A) Mice were immunized i.p. on days 0 and 21 with 10 μg of PPV:VLP-(LCMV) in PBS (□) or with alum (▪). Spleens were removed 7 days later. (B) Mice were treated on days −1, 0, +1, and 9 with anti-CD4 (•) or anti-CD8 (▵) mAbs or with PBS (□) and were injected on day 0 with 10 μg PPV:VLP-(LCMV). Control mice received 10 μg of control PPV:VLP (⧫). Spleens were removed on day 14. (C) Mice were immunized i.p. on days 0 and 21 with 10 μg of either PPV:VLP (hatched bars) or PPV:VLP-(LCMV) (solid bars). Spleens were removed at various times after the second PPV:VLP injection as indicated. CTL activity was determined after in vitro stimulation of splenocytes as described in Fig. 2. Lysis of uncoated target cells was less than 10% and is not shown. Data represent the mean of percent specific lysis from duplicates (SD < 5%). Data in C were obtained at a 100:1 effector/target (E/T) ratio.

Figure 4

In vivo immunization with PPV:VLP-(LCMV) stimulates a strong virus-specific CTL response. BALB/c mice were injected i.p. on days 0 and 21, with 10 μg of PPV:VLP, 10 μg of PPV:VLP-(LCMV), PBS, or 10⁵ pfu of LCMV. Seven days later, splenocytes were in vitro-stimulated and cytotoxic activity of the effector cells was measured on ⁵¹Cr-labeled J774 target cells infected with LCMV (▪) or incubated with medium (□). Data represent the mean of percent specific lysis from triplicate samples.

Figure 5

CD8⁺ T cells induced by recombinant PPV:VLP-(LCMV) fully protect mice against a lethal LCMV challenge. BALB/c mice were injected i.p. on days 0 and 21 with 10 μg of PPV:VLP, 10 μg of PPV:VLP-(LCMV), PBS, or 10⁵ pfu of LCMV. Two groups were also treated on days −1, 0, +1, 7, 14, and 20 with anti-CD4 or anti-CD8 mAbs. After 7 (A) or 49 (B) days, mice were challenged i.c. with 10^1.7 pfu of LCMV. Survival was recorded for 21-days period. The percent survival and the number of surviving mice/the number of total mice (∗) are the cumulative results of four experiments. ND, not determined.