Characterization of a single-cycle rabies virus-based vaccine vector

Abstract

Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-DeltaG-Gag). Passage of SPBN-DeltaG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-DeltaG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8(+) T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 10(3) focus-forming units for both RV vaccine vectors. CD8(+) T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.

FIG. 1.

Construction of the single-cycle RV, SPBN-ΔG-Gag. Shown are the negative-sense RNA genomes of the recombinant RV utilized in subsequent experiments. The cDNA copy of full-length SPBN (A) has a deletion of RV-G, and HIV-1-Gag is inserted between RV-M and RV-L to make single-cycle SPBN-ΔG-Gag (B). A replication-competent control was generated by inserting HIV-1 Gag between RV-N and RV-P of the full-length BNSP cDNA (C) to make BNSP-Gag (D).

FIG. 2.

SPBN-ΔG-Gag is limited to single-cell infection in vitro. (A) Anti-RV-G surface staining of BSR-RVG trans-complementing cells induced to express RV-G in the absence of doxycycline (top) and not induced in the presence of doxycycline (bottom). (B) Virus spread assay on BSR or induced BSR-RVG cells infected with SPBN-ΔG-Gag or BNSP-Gag virus at an MOI of 0.001 for 72 h and stained for intracellular RV-N. Images in panels A and B are shown at ×10 fluorescent microscope magnification. (C) Viral growth kinetics were demonstrated by multistep growth curves in which BSR or BSR-RVG cells are infected at an MOI of 0.01 with either BNSP-Gag or SPBN-ΔG-Gag virus, and viral titers were determined from samples taken at 0, 24, 48, and 72 h postinfection.

FIG. 3.

Single-cycle RV induces antibody profiles comparable to replication-competent RV. (A and C) Relative serum concentrations of anti-RV-G and anti-RNP total IgG, IgG2a, and IgG1 antibodies from serum diluted 1:60 and their respective IgG2a/IgG1 isotype ratios (B and D) from mice 15 days postimmunization with 1 × 10⁶ FFU of SPBN-ΔG-Gag^RVG or BNSP-Gag. (E) Mice immunized i.m. with different titers of SPBN-ΔG-Gag^RVG or BNSP-Gag were assayed at 1 month postimmunization for anti-RV-G total IgG antibody. (F) A time course showing total anti-RV-G IgG antibody levels from mice immunized i.m. with 1 × 10⁵ FFU of SPBN-ΔG-Gag^RVG or BNSP-Gag. (E and F) Results shown are for sera diluted 1:50. Significance between virus groups was measured by a t test. Values are the means ± standard errors. OD, optical density. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

FIG. 4.

The magnitude and kinetics of primary T-cell responses are comparable for single-cycle and replication-competent RVs. (A) Schematic of mouse experiments for analysis of primary immune responses. BALB/c mice immunized i.m. with 1 × 10⁶ FFU of SPBN-ΔG-Gag^RVG or BNSP-Gag were sacrificed at 7, 10, 15, or 20 days postimmunization. Splenocytes were analyzed for activated Gag tetramer-positive T cells (Gag-tetramer⁺ CD62L^lo) (B) and IFN-γ-producing cells by ELISPOT assay (C). Sample groups were composed of 8 to 13 mice at each time point for each virus, with the exception of the naïve group and day 20 time point, which had 4 to 5 mice for each virus. Significance between time points within viral immunization groups was measured by one-way ANOVA with a Bonferroni correction. (D) Mice immunized i.m. with different titers of SPBN-ΔG-Gag^RVG or BNSP-Gag were assayed at 10 days postimmunization, and splenocytes were analyzed for IFN-γ-producing cells by ELISPOT assay. *, P < 0.05; **, P < 0.01.

FIG. 5.

CTL recall responses are equal in magnitude and functionality in mice primed with single-cycle and replication-competent RVs. (A) Schematic of prime-challenge schedule. BALB/c mice were immunized i.m. with 2 × 10⁶ FFU of SPBN-ΔG-Gag or BNSP-Gag and challenged i.p. with 1 × 10⁶ PFU of VV-Gag 10 weeks postimmunization. Splenocytes were analyzed at 4.5 days postchallenge for activated Gag-tetramer-positive T cells (Gag-tetramer⁺ CD62L^lo), as shown in a representative experiment on individual mice (B) and in summary for five mice per group (C). IFN-γ-producing cells were assayed by ELISPOT assay (D). (E and F) BALB/c mice were immunized i.m. with 1 × 10⁶ FFU of SPBN-ΔG-Gag^RVG or BNSP-Gag and challenged i.p. with 1 × 10⁶ PFU of VV-Gag 4 weeks postimmunization. Naïve mice were challenged as controls. After 4.5 days splenocytes were harvested and cultured with Gag-stimulated targets or irrelevant antigen Env-stimulated targets and assayed for lytic activity by a nonradioactive CTL assay. (E) The percent specific lysis was evaluated on individual samples and is shown as a mean ± standard error. (F) The same splenocytes used in the nonradioactive CTL assay were analyzed for activated Gag-tetramer-positive T cells, and a positive correlation between cytolytic activity and tetramer-positive staining was observed. Cytolytic data are representative of three independent experiments.

FIG. 6.

Robust proliferation of antigen-specific T-cell responses following low-titer immunization with SPBN-ΔG-Gag^RVG. (A and B) Mice were immunized i.m. with different titers of SPBN-ΔG-Gag^RVG or BNSP-Gag and challenged i.p. with 1 × 10⁶ PFU of VV-Gag at 5 weeks postimmunization. Splenocytes were analyzed at 4.5 days postchallenge for activated Gag tetramer-positive T cells (Gag-tetramer⁺ CD62L^lo) (A) and IFN-γ-producing cells by ELISPOT assay (B). *, P < 0.05.

FIG. 7.

Production of SPBN-ΔG-Gag^VSV-RVG complemented with chimeric VSV-RVG for heterologous prime-boost immunization. (A) Anti-VSV-G surface staining of BSR-VSV-RVG trans-complementing cells induced to express VSV-RVG in the absence of doxycycline (top) and not induced in the presence of doxycycline (bottom). (B) Virus spread assay on induced (top) or uninduced (bottom) BSR-VSV-RVG cells infected with SPBN-ΔG-Gag at an MOI of 0.001 for 72 h and stained for intracellular RV-N. Images in panels A and B are shown at ×20 fluorescent microscope magnification. (C) Virus growth kinetics were demonstrated by multistep growth curves in which BSR or BSR-VSV-RVG cells were infected at an MOI of 0.01 with either BNSP-Gag or SPBN-ΔG-Gag virus, and virus titers were determined from samples taken at 0, 24, 48, and 72 h postinfection.

FIG. 8.

Heterologous boost with single-cycle RV generates enhanced Gag-specific immune responses. (A) Schematic of prime-boost schedule. Five BALB/c mice per group were immunized i.m. with 1 × 10⁶ FFU of SPBN-ΔG-Gag^RVG and boosted i.m. at 6 weeks postimmunization with 1 × 10⁶ FFU of SPBN-ΔG-Gag^VSV-RVG (heterologous boost), 1 × 10⁶ FFU of SPBN-ΔG-Gag^RVG (homologous boost), or PBS (mock boost). Splenocytes were analyzed by ICS at 0, 5, 10, and 15 days postboost for IFN-γ-producing cells by ELISPOT assay (B), for activated Gag-tetramer-positive T cells (Gag-tetramer⁺ CD62L^lo) (C), for generally activated CD8⁺ T cells (D), and for cytokine production (E to H). Significant differences between heterologous and homologous boosting groups at a single time point were measured by a t test (indicated by an asterisk above a time point). *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Significant differences between time points for heterologously boosted mice only were measured by one-way ANOVA with a Bonferroni correction (indicated by pound signs spanning two time points). #, P < 0.05; ##, P < 0.01; ###, P < 0.001.