Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Abstract

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

FIG. 1.

Immunization with chimeric HBsAg DNA encoding an RSV CTL epitope at ΔIPQ and/or ΔGLS elicits RSV-directed CTL responses. (a to e) Epitope-specific IFN-γ-secreting T-cell response following immunization of BALB/c mice (three per group) immunized twice i.d. (a) with pM2₉ΔIPQ, (b) with pM2₁₅ΔIPQ, or (c) with pM2₁₅ΔGLS. Control mice were immunized (d) once with M2₁₅ peptide plus adjuvant s.c. or (e) twice with wild-type HBsAg DNA (pHBsAg) i.d. IFN-γ-secreting cells were quantified by ELISPOT assay in splenocytes harvested at 14 days (DNA immunizations) or 10 days (peptide immunizations) after immunization and incubated for 15 to 18 h with specific peptide (M2₁₅ or IPQ) or without peptide, as shown. Bars represent means ± standard deviations of three replicates. (f to j) Epitope-specific CTL memory responses. Groups of mice (three per group) were immunized as described above. Percent cytotoxicity of splenocytes restimulated with RSV-specific M2₁₅ or HBsAg-specific IPQ peptide was measured in a ⁵¹Cr release assay using target cells pulsed with M2₁₅ or IPQ peptide or without peptide as shown. Data points represent means of three replicates ± standard deviations. (Note that in some cases standard deviation bars, though plotted, are too small to appear.) (k and l) BALB/c mice (three per group) were immunized three times i.d. with chimeric HBsAg DNAs encoding the M2₁₅ CTL epitope inserted either at one location (pM2₁₅ΔIPQ.HWI or pM2₁₅ΔGLS.HWI) or at two locations (pM2₁₅ΔIPQ.ΔGLS.HWI). Control mice were immunized with pHBsAg wild-type DNA. (k) IFN-γ-secreting cells were quantified by ELISPOT assay using ex vivo splenocytes incubated with or without M2₁₅ peptide. (l) Percent cytotoxicity of splenocytes restimulated for 6 days with M2₁₅ peptide was quantified in a ⁵¹Cr release assay using target cells pulsed with M2₁₅ peptide or without peptide as shown.

FIG. 2.

Chimeric HBsAg DNA encoding RSV M2 CTL epitope elicits protection against RSV infection and disease. BALB/c mice (five per group) were (ii) immunized twice i.d. with pM2₁₅ΔGLS DNA or (i and iii) unimmunized and (i and ii) challenged 2 weeks later with 8 × 10⁵ PFU RSV or (iii) left unchallenged. Four days later, mice lungs were processed for (a) hematoxylin-eosin staining and (b) immunostaining with goat anti-RSV antibody.

FIG. 3.

Chimeric HBsAg DNA encoding a CTL epitope of HPV16 E7 elicits E7-directed CTL responses and protects against challenge with E7-expressing tumor. (a to c) A2.1K^b mice (three per group) were immunized twice i.d. with pRAHΔIPQ DNA or with pHBsAgWT DNA or once s.c. with RAH peptide plus adjuvant. IFN-γ-secreting splenocytes harvested at 14 days (DNA immunizations) or 10 days (peptide immunizations) were quantified by ELISPOT assay with or without specific peptide (RAH) as shown. Bars are means ± standard errors of three replicates. (d to f) CTL responses following immunization with pRAHΔIPQ DNA. Mice (three per group) were immunized as described above. Percent cytotoxicity of splenocytes restimulated with RAH peptide was measured in a ⁵¹Cr release assay using EL4.A2 target cells pulsed with or without RAH peptide. Data points are means ± standard deviations of three replicates (although plotted, standard deviation bars are too small to appear). (g and h) Growth of E7-expressing tumor in immunized mice and controls. Mice immunized with pRAHΔIPQ DNA, with pHBsAg wild-type DNA, or with RAH peptide plus adjuvant and unimmunized mice were challenged with 2 × 10⁵ E7-expressing TC-1 tumor cells on day 14 postimmunization. Results are expressed as tumor-free mice (%) at the indicated time points. Groups of tumor-challenged mice were compared by use of the log rank statistic.

FIG. 4.

Immunization with chimeric HBsAg DNA encoding two protective CTL epitopes from distinct pathogens elicits CTL responses to both epitopes. (a and b) Epitope-specific IFN-γ-secreting response of ex vivo splenocytes following two i.d. immunizations of mice (BALB/c × A2.1K^b; three per group) with chimeric HBsAg DNA encoding both M2₁₅ and RAH epitopes (pM2₁₅ΔIPQ.RAHΔGLS.HWI), the M2₁₅ epitope alone (pM2₁₅ΔIPQ), or the RAH epitope alone (pRAHΔGLS). IFN-γ-secreting cells were quantified by ELISPOT assay using splenocytes incubated for 15 to 18 h with or without specific peptide M2₁₅ or RAH as shown. Histogram bars represent means ± standard deviations of three replicates. (c and d) Percent cytotoxicity of splenocytes restimulated with RAH peptide or with M2₁₅ peptide measured in a ⁵¹Cr release assay using P815 or EL4.A2 target cells pulsed with M2₁₅ peptide or RAH peptide, respectively, or without peptide, as shown. Data points represent means ± standard deviations of three replicates (standard deviation bars, although plotted, are too small to appear in some cases). (e) Growth of E7-expressing tumor in mice immunized with pM2₁₅ΔIPQ.RAHΔGLS.HWI DNA, with pRAHΔ IPQ DNA, or with control DNA as indicated. Mice (five per group) were challenged with 2 × 10⁵ E7-expressing TC-1 tumor cells on day 14 postimmunization. Results are expressed as tumor-free mice (%) at the indicated time points. Differences between means (*, **, ***) were calculated using Student's t test. Differences between survival curves (****) were calculated using the log rank statistic.

FIG. 5.

Chimeric HBsAgs containing foreign CTL epitopes package HDAg-L for secretion. Culture fluid from HuH-7 cells cotransfected with plasmids encoding chimeric or wild-type HBsAg proteins, and a plasmid encoding HDAg-L, was pelleted through sucrose cushions and resuspended in sample buffer, and supernatants (a) and cell pellets (b) were analyzed by immunoblotting using a human serum antibody specific for HDAg-L. Lanes: 1, pM2₉ΔIPQ; 2, pM2₁₅ΔIPQ; 3, pM2₁₅ΔGLS; 4, wild-type pHBsAg; 5, wild-type pHBsAg without pHDAg-L; 6, pHDAg-L without pHBsAg; 7, neither pHDAg-L nor pHBsAg.