Attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type 1 vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice

Abstract

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

FIG. 1.

Design and protein expression of rVSV-gag vectors. (A) The genomes of the rVSV vectors created for this study are diagramed in the 3′-to-5′ orientation of the negative-stranded viral RNA next to the nomenclature used for each vector. (B) Generation of rVSV vector genomic cDNAs. Endonuclease sites used for the assembly of cDNA constructs are indicated. T7-Prom represents the T7 RNA polymerase promoter sequence. Le and Tr represent the virus untranslated leader and trailer sequences, respectively. Shaded bars represent viral transcription start signals, and each viral transcription unit is separated by the nontranscribed intergenic dinucleotides GT and CT, as indicated. A T7 RNA polymerase transcription-termination signal (T7 Term) and hepatitis delta virus (HDV) ribozyme lead to the generation of a precise viral 5′ end during the virus rescue process. (C) Western blots showing in vitro HIV-1 Gag expression of rVSV vectors 24 h postinfection of replicate BHK cell monolayers. Lanes 1 and 2 contain proteins from uninfected and “empty” rVSV-infected cells, respectively. Lanes 3 to 7 contain proteins from cells infected with the different Gag-expressing rVSV vectors. Protein size markers (24 kDa to 50 kDa) were run alongside test samples.

FIG. 2.

Neurovirulence properties of rVSV-gag vectors in mice following i.c. inoculation. Groups of 5-week-old Swiss Webster mice (n = 10) were inoculated i.c. with log₁₀-fold dilutions of rVSV_IN vectors. Mice were monitored for 3 weeks for mortality and morbidity (paralysis). (A) The LD₅₀ and PD₅₀ were determined by the method described previously by Reed and Muench (30). Arrowheads indicate where the LD₅₀ and PD₅₀ were not achieved at the highest dose tested of 10⁸ PFU. (B) Time to death was recorded for mice in the group receiving the dose immediately above the determined LD₅₀.

FIG. 3.

Immunogenicity of rVSV-gag vectors following i.n. inoculation. Mice (n = 5) were primed by i.n. inoculation of rVSV_IN vectors (1 × 10⁷ PFU) and boosted 8 weeks later by i.n. inoculation with the corresponding rVSV_NJ vectors. T-cell responses were assessed in splenocytes at 7 days postprime and both 5 days and 4 weeks postboost. Humoral responses were assessed in serum at 4 weeks postboost. (A) HIV-1 Gag tetramer responses. Isolated splenocytes were stained with Gag tetramer (H-2K^d) (AMQMLKETI) and assessed by flow cytometry for the percentage of CD3⁺ CD8⁺ T cells that were Gag tetramer positive. (B) HIV-1 Gag IFN-γ ELISPOT responses. Isolated splenocytes were cultured overnight with the H-2K^d Gag immunodominant peptide (AMQMLKETI), and IFN-γ secretion was determined by ELISPOT analysis. The data are normalized to 10⁶ spleen cells. (C) HIV-1 Gag ex vivo CTL responses at 5 days postboost. Isolated splenocytes were used in 3-h ex vivo CTL assays to lyse Eu-labeled P815 target cells loaded with the H-2K^d Gag immunodominant peptide. (D) HIV-1 Gag in vivo CTL responses at 4 weeks postboost. Gag-specific in vivo cytolytic activity was assessed by 18-h in vivo CTL assays in immunized mice. (E) HIV-1 Gag serum antibody responses at 4 weeks postboost. Serum was collected from immunized mice and assessed for Gag p24-specific total IgG by ELISA. Data are presented as the geometric mean titers. (F) VSV N IFN-γ ELISPOT responses. Vector-specific responses were assessed by measuring the T-cell response to VSV nucleoprotein (VSV N). Isolated splenocytes were cultured overnight with the H-2L^d VSV N immunodominant peptide (MPYLIDFGL), and IFN-γ secretion was determined by ELISPOT analysis. Immune responses induced by attenuated rVSVs were compared to immune responses induced by rVSV-gag5. *, P < 0.05; **, P < 0.001.

FIG. 4.

Immunogenicity of rVSV-gag vectors following i.m. inoculation. Mice (n = 5) were primed by i.m. inoculation of rVSV_IN vectors (1 × 10⁷ PFU) and boosted 8 weeks later by i.m. inoculation of the corresponding rVSV_NJ vectors. T-cell responses were assessed in splenocytes at 7 days postprime and both 5 days and 4 weeks postboost. Humoral responses were assessed in serum at 4 weeks postboost. Assays were performed as described in the legend of Fig. 3. (A) HIV-1 Gag tetramer responses. (B) HIV-1 Gag IFN-γ ELISPOT responses. (C) HIV-1 Gag ex vivo CTL responses 5 days postboost. (D) HIV-1 Gag in vivo CTL responses 4 weeks postboost. (E) HIV-1 Gag serum antibody responses 4 weeks postboost. (F) VSV N IFN-γ ELISPOT responses. Immune responses induced by attenuated rVSVs were compared to immune responses induced by rVSV-gag5. *, P < 0.05; **, P < 0.001.

FIG. 5.

Dose response of N4CT9-gag1 following i.m. inoculation. Mice (n = 5) were primed by i.m. inoculation with ascending doses of N4CT9_IN-gag1 and boosted 8 weeks later by i.m. inoculation with the corresponding dose of N4CT9_NJ-gag1. T-cell responses in splenocytes and humoral responses in serum were assessed 5 days postboost. Assays were performed as described in the legend of Fig. 3. (A) HIV-1 Gag tetramer responses. (B) HIV-1 Gag IFN-γ ELISPOT responses. (C) HIV-1 Gag in vivo CTL responses. (D) HIV-1 Gag serum antibody responses.

FIG. 6.

Detection of in vivo gag mRNA transcription following i.m. priming and boosting with rVSV-gag vectors. Mice (n = 5 per time point) were primed by i.m. inoculation of rVSV_IN vectors (1 × 10⁷ PFU) and boosted 8 weeks later by i.m. inoculation of the corresponding G_NJ protein exchange vectors. At 4 h and 1, 2, 4, and 8 days postpriming and postboosting, muscle (A) and draining lymph nodes (B) were excised from mice and homogenized in sucrose-phosphate-glutamic acid buffer. Tissue homogenates were frozen and then assessed later for Gag mRNA by quantitative real-time PCR. The limit of detection for the quantitative real-time PCR assay is 5 × 10³ Gag mRNA copies per 10 mg tissue.

FIG. 7.

Effect of gag gene position on immunogenicity. Mice (n = 5) were primed by i.m. inoculation of rVSV_IN vectors (1 × 10⁷ PFU) and boosted 8 weeks later by i.m. inoculation of rVSV_NJ vectors. T-cell responses were assessed in splenocytes at 7 days postprime and at 5 days and 4 weeks postboost. Humoral responses were assessed in serum at 4 weeks postboost. Assays were performed as described in the legend of Fig. 3. (A) HIV-1 Gag IFN-γ ELISPOT responses. (B) HIV-1 Gag serum antibody responses 4 weeks postboost. Immune responses induced by rVSV-gag5 and the attenuated rVSVs were compared to immune responses induced by rVSV-gag1. *, P < 0.05; **, P < 0.001.