Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Abstract

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Figure 1

Generation of rLCMV vectors, replication in cultured cells and rapid elimination and lack of pathogenicity in vivo. (a) Wild-type LCMV with its two genome segments (S and L) and the four open reading frames (ORFs). Substitution of GP for a vaccine antigen or reporter gene creates the rLCMV vectors. (b) Expression cassettes of plasmids used for the recovery of rLCMV vectors. In pol-I-Sv and pol-I-L¹⁰, the mouse polymerase I promoter (pol-I-prom.) and terminator (pol-I-termin.) drive intracellular expression of the LCMV vector S segment and L segment, respectively; pol-II-L¹⁰ and pol-II-NP¹⁰ express the respective viral proteins under control of an actin promoter (Actin-prom.)-driven expression cassette with intron and polyadenylation (poly(A)) signal. ORF, open reading frame. (c) Process to recover rLCMV vectors in producer cells. LCMV-GP protein (GP) on the surface of producer cells is incorporated into vector particles. (d) Fluorescence microscopy of GFP expression in 293T cells and GP-expressing 293T cells (293T-GP) infected for 48 h with rLCMV-GFP at a multiplicity of infection of 0.05. Scale bars, 100 μm. (e) Infectious progeny particles in supernatants of the 293T cells and GP-expressing 293T cells in d at 0–72 h after infection. Data are representative of three individual culture wells (mean ± s.e.m.). (f) Real-time RT-PCR analysis of vector S segment copies, measured in total RNA from spleen of mice vaccinated with rLCMV-GFP. Each symbol represents an individual mouse; small horizontal lines indicate the mean. (f) Disease development in mice infected intracerebrally with wild-type LCMV or rLCMV-OVA (three mice per group). Animals displaying clinical signs of lymphocytic choriomeningitis were killed in accordance with the Swiss law for animal protection.

Figure 2

The rLCMV vectors elicit CD8⁺ T cell responses of high frequency and functionality that are efficiently amplified in homologous prime-boost vaccination. (a,b) SIINFEKL-specific CD8⁺ T cell frequencies in the blood of mice vaccinated (primed) with rAd-OVA or rLCMV-OVA (dose, horizontal axis) on day 0 and boosted with the same vector-dose combination on day 38, assessed on day 28 after vaccination (a) and 7 d after boost (b). (c) SIINFEKL-specific CD8⁺ T cell frequencies in peripheral blood: serum (500 μl) from mice vaccinated with rLCMV-OVA or rAd-OVA 4 weeks previously was transferred into naive mice; 1 d after transfer, both groups and controls without serum transfer were vaccinated with the same two vectors, and T cell frequencies were determined 14 d later. Data are representative of three mice per group (mean ± s.d.). (d) Intracellular cytokine assay of SIINFEKL-specific CD8⁺ T cells producing IFN-γ alone (IFN-γ⁺ only) or both IFN-γ and TNF-α (IFN-γ⁺TNF-α⁺) in spleen, assessed on day 14 after vaccination with rLCMV-OVA, rAd-OVA or VACC-OVA. Data are representative of three to six mice per group (mean ± s.e.m.). P values of less than 0.05 (*) were considered statistically significant, and P values of less than 0.01 (**) were considered highly significant.

Figure 3

The rLCMV vectors trigger rapid proliferation, T helper type 1 differentiation and memory formation of CD4⁺ T cells and elicit durable neutralizing antibody responses to vaccine antigen. (a) Proliferation (assessed as CFSE dilution) in blood of naive syngeneic recipients given CFSE-labeled SMARTA TCR-transgenic splenocytes carrying the Thy-1.1 marker, then vaccinated with rLCMV-INDG61 or left unvaccinated (No vaccine), assessed on days 3 and 6. Green indicates transferred epitope-specific (Thy-1.1⁺) cells. (b,c) Frequency of SMARTA1 CD4⁺ T cells in peripheral blood over time (b) and cytokine profile of SMARTA1 CD4⁺ T cells after restimulation with phorbol 12-myristate 13-acetate and ionomycin on day 9 (c), assessed for cells from the mice in a. (d) VSV-neutralizing titers of total immunoglobulin (Total Ig) and immunoglobulin G (IgG) in serum of C57BL/6 mice vaccinated with rLCMV-INDG61. nAb, neutralizing antibody. Data are representative of three to six mice per group (b,d; mean ± s.e.m.). (e) Disease development in type I interferon receptor–deficient mice vaccinated with rLCMV-INDG61 or left untreated, then challenged intravenously with 2 × 10⁶ PFU VSV 4 weeks later (four mice per group). Animals with terminal myeloencephalitis were killed.

Figure 4

Efficacy and protective capacity of rLCMV-induced CTL responses. (a) Bacterial titers in the livers of C57BL/6 mice vaccinated with rLCMV-OVA, rAd-OVA or VACC-OVA, then challenged with rLM-OVA 16 or 58 d after single immunization or 200 d after homologous prime-boost vaccination (days 0 and 38); titers were measured 3 d after challenge. Immunization with rLCMV-Cre (challenge on day 16 or 58) and no immunization (challenge on day 200) serve as controls. Each symbol represents an individual mouse; small vertical lines indicate the mean. (b) Blood glucose concentrations of RIP-OVA-transgenic mice transfused with 1 × 10³ OT-I TCR-transgenic splenocytes on day -1 and immunized with rLCMV-OVA, VACC-OVA or rAd-OVA on day 0. Data are from one representative of two experiments (mean ± s.e.m. of three to six mice). (c) Tumor volume and survival of C57BL/6 mice injected subcutaneously in both flanks with 5 × 10⁵ B16.F10 melanoma cells expressing the CTL epitope GP33 (ref. 19); 8 d later, tumor masses were palpable, and rLCMV-GP33, rAd-GP33 or VACC-GP33 was given for immunotherapy. Immunization with rLCMV-OVA serves as a control. Tumor volume was calculated by the formula V = π × abc/6, where a, b and c are the orthogonal diameters. For survival analysis, death and humane endpoints (tumor volume, tumor exulceration, cachexia) were counted as events. Broken lines (top) indicate loss of mice to follow-up (bottom), which caused a bias toward smaller tumors at subsequent time points. Data are representative of initially 18–22 tumors from 9–11 mice per group (mean ± s.e.m.). (d) Viral titers in the ovaries of C57BL/6 mice vaccinated with rLCMV-GP33 or rAd-GP33 (dose, horizontal axis) or left untreated, then given 2 × 10⁶ PFU of VACC-G2 intraperitoneally 18 d later and killed 6 d later. Statistical analysis identifies groups that differ significantly from unvaccinated controls. Data are representative of five mice (mean ± s.d.). P values of less than 0.01 (**) were considered highly significant.

Figure 5

The rLCMV vectors target and activate DCs. (a) Flow cytometry of GFP expression in DCs (CD11c⁺), macrophages (F4/80⁺), T cells (CD3⁺) and B cells (CD19⁺) in Z/EG transgenic reporter mice 3 d after vaccination with rLCMV-Cre or rLCMV-OVA (control vector). Numbers in outlined areas indicate total number of fluorescent cells per spleen (mean ± s.e.m.); gates were set such that no positive cells were recorded for mice vaccinated with rLCMV-OVA. Data are representative of four Z/EG mice per group. (b) CD86 surface expression on CD11c⁺ DC populations from the mice in a, identified as GFP⁺ (pos.) or GFP⁻ (neg.) and stained with antibody to CD86 (α-CD86) or isotype-matched control antibody (Isotype). (c) GP33-specific CD8⁺ T cell frequencies in blood of ST33 mice vaccinated with rLCMV-Cre or rLCMV-OVA control vector, assessed by major histocompatibility complex class I (H-2D^b) tetramer staining on days 10 and 27 after vaccination (left), and GP33-specific recall responses in spleen after mice were challenged with VACC-G2 on day 28 of the experiment, assessed 6 d later by intracellular cytokine assay. Numbers in plots indicate percent tetramer-positive CD8⁺ T cells (left; top right quadrant) or percent IFN-γ⁺ CD8⁺ T cells (right; top left quadrant) or IFN-γ⁺TNF-α⁺ CD8⁺ T cells (right; top right quadrant). Data are from one representative of two similar experiments (mean ± s.d. of three to four mice per group).