VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications

Abstract

Antivector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication-competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA and VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was possible only for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA-specific cytotoxic T-lymphocyte (CTL) responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication-competent vaccine vector that does not lose efficacy upon repeated application.

Importance: Although there has been great progress in treatment and prevention of infectious diseases in the past several years, effective vaccines against some of the most serious infections, e.g., AIDS, malaria, hepatitis C, or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing, repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e., neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and therefore can serve as a platform technology for the development of novel vaccines against a broad spectrum of diseases.

FIG 1

VSV vectors containing OVA antigen. (A) Genomes of VSV-OVA and GP-pseudotyped VSV-GP-OVA vectors containing ovalbumin (OVA). The sequence coding for full-length OVA, C terminally fused to eGFP, was introduced at position 5 into both vectors. (B) BHK-21 cells were infected with VSV-OVA or VSV-GP-OVA at an MOI of 0.1, and 24 h later, cell lysates were prepared. The eGFP_OVA fusion protein (69 kDa) was detected by Western blotting using a GFP-specific antibody. (C) BHK-21 cells were infected with VSV-OVA or VSV-GP-OVA at an MOI of 0.1 in duplicate. Virus production at the indicated time points was determined by TCID₅₀ assay.

FIG 2

Cellular and humoral immune responses induced by VSV-OVA and VSV-GP-OVA. C57BL/6 mice were immunized intramuscularly on days 0 and 26 with 1 × 10⁶ PFU of VSV-OVA or VSV-GP-OVA. Mice were sacrificed at the indicated time points, and splenocytes and plasma samples were collected. n was ≥5 mice per group and time point. (A) OVA-specific CD8⁺ T cell responses were determined in an IFN-γ–ELISpot assay after restimulation of splenocytes with OVA_257–264 peptide (SIINFEKL). Values for unstimulated cells were subtracted as background. Statistical significances were determined in a one-way analysis of variance followed by Bonferroni's multiple comparison test (*, P ≤ 0.05; ***, P ≤ 0.001; n.s., not significant). (B) OVA-specific IgG titers in mouse plasma were determined by ELISA. Data were analyzed by nonparametric statistics (two-tailed, Mann-Whitney test), and statistically significant differences are marked (*, P ≤ 0.05; **, P ≤ 0.01).

FIG 3

VSV-GP-OVA containing the less cytopathic M mutant (ΔM51) induces reduced anti-OVA immune responses. C57BL/6 mice (n = 5) were immunized in a prime/boost schedule intramuscularly with either VSV-GP-OVA or VSV-ΔM51-GP-OVA. Seven days after the second boost, mice were sacrificed and splenocytes were harvested. Cells were stimulated with the OVA-specific CTL peptide SIINFEKL and analyzed via intracellular cytokine staining for production of IFN-γ and TNF-α. (A and B) Means ± standard deviations for IFN-γ⁺ (A) and IFN-γ⁺/TNF-α⁺ cells within CD8⁺ T cells (B) are depicted. Statistical significances were determined using a two-tailed t test, and asterisks indicate statistical significance (**, P = 0.01). (C) Plasma was collected at indicated time points, and the titer of anti-OVA antibodies was determined by ELISA. Statistical significances were determined by nonparametric statistics (two-tailed, Mann-Whitney test).

FIG 4

Vaccination with VSV-GP does not induce neutralizing antibodies against the vector. Mice were immunized intramuscularly on days 0 and 26 with 1 × 10⁶ PFU of VSV-OVA or VSV-GP-OVA in a prime/boost regimen, and plasma samples collected at the indicated time points were analyzed for the titer of neutralizing antibodies against VSV (A) or VSV-GP (B). Titers of neutralizing antibodies are given as the highest dilution which completely inhibited the cytopathic effect induced by 100 PFU of VSV-GFP or VSV-GP-GFP. At least 5 mice were analyzed per time point and virus. Plasma samples from VSV- or VSV-GP-immunized mice were collected at the indicated time points and were analyzed for the titer of LCMV-GP1 binding antibodies by ELISA using recombinant LCMV-GP1 (C). Plasma samples from 4 mice per group were pooled, and data show means ± standard deviations from 4 independent experiments. Statistical significances were determined using nonparametric statistics (two-tailed, Mann-Whitney test; *, ≤0.05). For serum transfer, naive mice received either immune serum from VSV-OVA- or VSV-GP-OVA-immunized mice or nonimmune serum from naive mice as a control. Subsequently, mice were immunized intramuscularly with 1 × 10⁶ PFU of VSV-OVA or VSV-GP-OVA, and OVA-specific CTL responses were determined via tetramer (D) and intracellular cytokine (E) staining on day 7 postimmunization. Four mice per group were analyzed. Means ± standard deviations are shown. Statistical significances were determined using an unpaired, two-tailed t test (*, P ≤ 0.05; ***, P ≤ 0.01; ns, not significant).

FIG 5

VSV-GP-OVA is at least as potent as a state-of-the-art adenoviral vaccine vector. Mice (n = 5) were immunized with VSV-GP-OVA, AdiOVA (expressing intracellular OVA), or AdsOva (expressing secreted OVA) in a prime/boost schedule with two boost immunizations. (A) Seven days after the second boost immunization, mice were sacrificed and splenocytes were harvested. Cells were stimulated with the OVA-specific CTL epitope SIINFEKL and analyzed by intracellular cytokine staining for production of IFN-γ, TNF-α, and IL-2. Means ± standard deviations for IFN-γ⁺, IFN-γ⁺/TNF-α⁺, and IFN-γ⁺/TNF-α⁺/IL-2⁺ cells within CD8⁺ T cell population are shown. Statistical significances were determined using an unpaired, two-tailed t test (**, P ≤ 0.01; *, P ≤ 0.05; n.s., not significant). (B) Plasma was collected at the indicated time points, and the titer of OVA-specific antibodies was determined by ELISA. Statistical significances were determined using nonparametric statistics (two-tailed, Mann-Whitney test; *, P ≤ 0.05). (C) Mice (n ≥ 4) were immunized with AdiOVA in a prime/boost schedule with two boost immunizations. Plasma was collected at indicated time points, and the titer of antiadenovirus antibodies was determined via ELISA.

FIG 6

VSV-GP-OVA-immunized mice were protected from challenge with Listeria monocytogenes expressing OVA. Mice were immunized in a prime/boost regimen with the indicated vectors or PBS as a control and challenged 7 days later with Lm_OVA. Three days after challenge, mice were sacrificed. (A) Bacterial load in the spleen was determined (n ≥ 5). Data from two independent experiments were combined in the graph. (B) Frequency of OVA-specific CTLs was determined by tetramer staining in the second experiment (n ≥ 2).