Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity

Abstract

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.

FIG. 1

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified CyaA constructs carrying multiple epitopes. The individual constructs were expressed in E. coli XL-1, extracted with 8 M urea, and purified close to homogeneity by a combination of DEAE-Sepharose and phenyl-Sepharose chromatography, as described in Materials and Methods. Two micrograms of each purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 7.5% acrylamide gel and visualized by Coomassie blue staining.

FIG. 2

Presentation of detoxified CyaAs carrying multiple epitopes to anti-LCMV LC3A10 and anti-OVA B3Z class I-restricted T-cell hybridomas. APCs (splenocytes from BALB/c [A] or C57BL/6 mice [B]) were incubated in the presence of various concentrations of the CyaA toxoids, harboring either OVA (●) or LCMV (□) epitope at site 224 or the three epitopes (MEP) at different sites (108 [■], 233 [▴], or 336 [▾]) and were cocultured, respectively, with the anti-LCMV CD8⁺ hybridoma LC3A10 (A) or the anti-OVA CD8⁺ hybridoma B3Z (B). IL-2 secretion by the stimulated hybridoma was determined by the CTL proliferation assay. Results are expressed in cpm of incorporated [³H]thymidine (cpm in the presence of CyaA − cpm in the absence of CyaA). Data represent the mean values of duplicate samples (standard error of the mean, <10%) and are representative of two (A) and three (B) experiments.

FIG. 3

CTL induction by the detoxified CyaAs bearing multiple epitopes. BALB/c (A and B) and C57BL/6 (C) mice (n = 3 for each group) were immunized i.p. on days 0 and 14 with 50 μg of CyaA toxoids carrying the polyepitope at different sites (108 [●], 233 [▴], or 336 [▾]) or with either the LCMV (▿) or OVA (▵) epitope at site 224 or with control detoxified CyaA (CyaA w.t.-E5) (■) mixed with alum. Seven days later, the animals were sacrificed, the splenocytes were restimulated in vitro for 5 days with 1 μg of the LCMV (A), V3 (B), or OVA (C) peptide per ml in the presence of irradiated syngeneic splenocytes, and used as effectors against unsensitized target cells (P815 for panels A and B and EL4 for panel C) or against target cells sensitized with the same peptide as used for in vitro stimulation. Target lysis was evaluated by ⁵¹Cr release. Lysis of unsensitized target cells was less than 10% and is not shown. (A and B) LCMV (p118–132)-specific CTL (A) and V3 (p316–327)-specific CTL (B) responses in the same BALB/c mice injected with the detoxified recombinant CyaAs. (C) OVA (p257–264)-specific CTL response in C57BL/6 mice injected with the detoxified recombinant CyaAs. CTL responses of control mice injected with the wild-type CyaA-E5 are shown to illustrate the specificity of the responses. The data represent the mean percentage of the specific lysis values from duplicate samples and standard error of the mean and are representative of four experiments. E:T ratio, effector-to-target-cell ratio.

FIG. 4

Immunization of mice with detoxified CyaA toxoids bearing multiple epitopes in the absence of adjuvant induces high specific CTL responses. BALB/c (A and B) and C57BL/6 (C) mice (n = 3 for each group) were immunized i.p. on days 0 and 14 with 50 μg of CyaA toxoids carrying the polyepitope at different sites (108 [●], 233, [▴], or 336) [▾] or with either the LCMV (▿) or OVA (▵) epitope at site 224 or with control detoxified CyaA (CyaA wt-E5) (■) in PBS. Seven days later, animals were sacrificed, the splenocytes were restimulated in vitro for 5 days with 1 μg of the LCMV (A), V3 (B), or OVA (C) peptide per ml in the presence of irradiated syngeneic splenocytes and used as effectors against unsensitized target cells (P815 for panels A and B and EL4 for panel C) or against target cells sensitized with the same peptide as used for in vitro stimulation. Target lysis was evaluated by ⁵¹Cr release. Lysis of unsensitized target cells was less than 10% and is not shown. (A and B) LCMV (p118–132)-specific CTL (A) and V3 (p316–327)-specific CTL (B) responses in the same BALB/c mice injected with the detoxified recombinant CyaA toxoids. (C) OVA (p257–264)-specific CTL response in C57BL/6 mice injected with the detoxified recombinant CyaAs. CTL responses of control mice injected with the wild-type CyaA-E5 are shown to illustrate the specificity of the responses. The data represent the mean percentage of the specific lysis values from duplicate samples and standard error of the mean and are representative of two experiments. E:T ratio, effector-to-target-cell ratio.

FIG. 5

Detection of LCMV-specific IFNγ-producing cells after immunization with the CyaA toxoids carrying multiple epitopes. BALB/c mice (n = 6 for each group) were immunized i.p. on days 0 and 14 with 50 μg of detoxified CyaAs carrying either the polyepitope at different positions (108, 233, or 336), the LCMV epitope alone at position 224, or control CyaA toxoid, mixed with alum. On day 21, spleen cells isolated from immunized mice were cultured in vitro for 36 h without stimulation (i.e., no peptide) or with 1 μg of the LCMV peptide per ml in the presence of syngeneic irradiated splenocytes and 10 U of recombinant IL-2 per ml. The data are expressed as the number of SFC per spleen and represent the mean and standard error of the mean obtained with six mice in three independent experiments.

FIG. 6

Immunization with the detoxified CyaA toxoids carrying multiple epitopes induces full protection against a lethal LCMV challenge. On days 0 and 14, BALB/c mice were immunized with 50 μg of wild-type CyaA E5 (mock control, CyaA wt-E5) or the recombinant CyaA carrying the LCMV epitope alone (CyaA224LCMV-E5) or the polyepitope at different positions (CyaA108MEP-E5, CyaA233MEP-E5, or CyaA336MEP-E5), mixed with 1 mg of alum. Control groups received either PBS or 10⁵ foci of LCMV i.p. On day 21, the mice were challenged intracerebrally with 10^1.7 foci of LCMV. Mortality was monitored for 21 days. The percentage of mice that survived and the number of surviving mice out of the total number of challenged mice are shown for each group. The data represent the cumulative results of two experiments.

FIG. 7

Effect of prior priming with the mock CyaA vector on the induction of CTL responses by a recombinant CyaA carrying a CD8⁺ T-cell epitope. BALB/c mice were primed with PBS (□, ■) or 50 μg of wild-type CyaA (○, ●, ▵, ▴) mixed with 1 mg of alum by i.p. injection on days 0 and 14. After 15 days, 1 month, or 3 months, all mice were immunized twice i.p. with 50 μg of CyaA224LCMV (□, ■, ○, ●) or wild-type CyaA (▵, ▴), mixed with 1 mg of alum, at a 3-week interval. At 7 days after the last injection, spleen cells were stimulated in vitro with the p118–132 peptide in the presence of syngeneic spleen cells. The cytotoxic activity of these effector cells was measured on ⁵¹Cr-labeled P815 target cells pulsed with the same peptide (solid symbols) or incubated with medium alone (open symbols). The data represent the mean percentage of the specific lysis values from duplicate samples and standard error of the mean and are representative of three experiments. E:T ratio, effector-to-target-cell ratio.

FIG. 8

Effect of prior priming with a detoxified recombinant CyaA on the induction of CTL responses by a recombinant CyaA toxoid carrying another CTL epitope. BALB/c mice were primed with either PBS (○, ●), 50 μg of detoxified wild-type CyaA (CyaA wt-E5) (□, ■, ▿, ▾), or 50 μg of detoxified CyaA224OVA-E5 mixed with 1 mg of alum (▵, ▴) by i.p. injection on days 0 and 14. After 15 days, all mice were immunized twice i.p. with 50 μg of CyaA224LCMV-E5 (□, ■, ▵, ▴, ○, ●) or CyaA wt-E5 (▿, ▾), mixed with 1 mg of alum, at a 3-week interval. Seven days after the last injection, spleen cells were stimulated in vitro with the p118–132 peptide in the presence of syngeneic spleen cells. The cytotoxic activity of these effector cells was measured on ⁵¹Cr-labeled P815 target cells pulsed with the same peptide (solid symbols) or incubated with medium alone (open symbols). The data represent the mean percentage of the specific lysis values from duplicate samples and standard error of the mean and are representative of three experiments. E:T ratio, effector-to-target-cell ratio.