Heterologous boosting of recombinant adenoviral prime immunization with a novel vesicular stomatitis virus-vectored tuberculosis vaccine

Abstract

Pulmonary tuberculosis (TB) remains a serious health problem worldwide. Effective vaccination strategies are needed. We report the development of a novel TB vaccine using vesicular stomatitis virus (VSV) as a viral vector system to express Ag85A. VSVAg85A was shown to be immunogenic when given to mice by either an intranasal or an intramuscular (i.m.) route. Although distinct T-cell profiles resulted from both routes of immunization, only intranasal delivery generated a mucosal T-cell response that was protective upon pulmonary Mycobacterium tuberculosis (M.tb) challenge. While this protection manifested at an early time-point after immunization, it was not sustained. The potential of VSVAg85A to be used as a mucosal booster for parenteral priming by an adenoviral TB vaccine expressing Ag85A (AdAg85A) was investigated. VSVAg85A immunization markedly boosted antigen-specific T-cell responses in the airway lumen while also augmenting immune activation in the systemic compartment, after AdAg85A priming. This translated into significantly better protective efficacy against pulmonary challenge with M.tb than either vaccine used alone. Our study therefore suggests that VSV as a vector system is a promising candidate to be used in a heterologous viral prime-boost immunization regimen against intracellular bacterial infection.

Figure 1

Design and characterization of VSVAg85A. (a) Diagram of the VSV genome and location for insertion of the foreign transgene. A549 cells were infected with VSVAg85A and AdAg85A and cell lysates were harvested 24 and 48 hours after infection, respectively. Equal volumes of the sample were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with (b) a monoclonal Ag85A antibody, or (c) polyclonal VSV antibodies. These data are representative of two independent experiments. G, glycoprotein; kb, kilobase; L, large protein; M, matrix protein; N, nucleoprotein; P, phosphoprotein; tPA, tissue plasminogen activator; VSV, vesicular stomatitis virus.

Figure 2

Determination of an appropriate dose for immunization with VSVAg85A, and verification of its safety for mucosal administration. (a) Balb/c mice were immunized intramuscularly with increasing doses of VSVAg85A. One week later, the mice were killed and peptide-stimulated splenocytes were analyzed using enzyme-linked immunosorbent spot. The data are shown as mean values ± SEM of three mice per group. (b) Other mice were immunized intranasally (IN) with 1 × 10⁷ plaque forming units (PFU) of VSVAg85A, and 1 week later the naive mice as well as the immunized mice were examined for lung pathology. For this purpose, the mice were killed, the lungs were sectioned and hematoxylin and eosin stained, and representative images were presented. Original magnification: top images—×10; bottom images—×20. NS, not statistically significant as compared to the other two doses; SFU, spot forming units; VSV, vesicular stomatitis virus.

Figure 3

Kinetics of tetramer+ CD8 T-cell responses following intramuscular (IM) and intranasal (IN) immunization with VSVAg85A. Balb/c mice were immunized with 1 × 10⁷ plaque forming units of VSVAg85A, either IM or IN. At 1, 2, and 3 weeks after immunization, the mice were killed and the total tetramer+ CD8 T cells in (a) the airway lumen, (b) lung, and (c) spleen were determined using immunostaining and fluorescence-activated cell sorting analysis. The data are shown as mean values ± SEM of three mice per group. ^§P ≤ 0.01 as compared to time-points of week 2 and week 3; ^*P ≤ 0.05 as compared to week 3 in b, and as compared to week 1 or week 3 in c. VSV, vesicular stomatitis virus.

Figure 4

Kinetics of interferon-γ+ (IFN-γ+) CD4 and CD8 T-cell responses following intramuscular (IM) and intranasal (IN) immunization with VSVAg85A. Balb/c mice were immunized with 1 × 10⁷ plaque forming units (PFU) of VSVAg85A, either IM or IN. At 1, 2, and 3 weeks after immunization the mice were killed, and peptide-stimulated cells were analyzed using intracellular cytokine staining in order to determine the number of IFN-γ+ CD4 and CD8 T cells in (a) the airway lumen, (b) lung, and (c) spleen. The data are shown as mean values ± SEM of three mice per group. ^§P ≤ 0.01 as compared to week 2 CD4 or CD8 T-cell data; ^*P ≤ 0.05 as compared to week 3 CD4 T-cell data in b, and as compared to week 3 CD8 T-cell data in c; NS, not statistically significant as compared to week 3 data. VSV, vesicular stomatitis virus.

Figure 5

Immune protection against pulmonary Mycobacterium tuberculosis (M.tb) challenge. A group of Balb/c mice were immunized subcutaneously with 1 × 10⁵ colony forming units (CFU) of bacille Calmette-Guérin (BCG). Three weeks later, other groups of mice were immunized with intramuscular (IM) administration of AdAg85A, or IM administration of VSVAg85A, or intranasal (IN) delivery of VSVAg85A, each of them alone, and all at a dose of 1 × 10⁷ plaque forming units. At (a) 2 weeks or (b) 4 weeks after immunization, the mice were challenged through the airway with 15,000 CFU of the M.tb H37Rv strain. At 4 weeks after the challenge, the mice were killed and the lungs were analyzed for CFU counts. The data are shown as mean values ± SEM of five to seven mice per group. ^*P ≤ 0.05 as compared to naive controls, IM administered adenovirus (Ad), or IM administered VSV; ^†P ≤ 0.001 as compared to naive controls, IM administered Ad or IM administered VSV in a, and as compared to naive controls or IN administered VSV in b.VSV, vesicular stomatitis virus.

Figure 6

Enhanced tetramer+ T cells by prime-and-boost regimen of intramuscular (IM) AdAg85A (prime) and intranasal (IN) VSVAg85A (boost). Two groups of Balb/c mice were immunized with IM administration of 5 × 10⁷ plaque forming units (PFU) of AdAg85A. Three weeks later, one of these two groups received a boost with IN administration of 1 × 10⁷ PFU of VSVAg85A while the other did not. At the same time-point, a fresh group of mice was immunized with IN administration of 1 × 10⁷ PFU VSVAg85A alone. Two weeks later, all the mice were killed and the total number of tetramer+ CD8 T cells was determined using immunostaining and fluorescence-activated cell sorting analysis. (a) Representative dot plots are presented for the corresponding treatment and tissue. Data from (b) the airway lumen, (c) lung interstitium, and (d) spleen. Values in the left corner of the dot plots represent the percentage of tetramer+ CD8 T cells within the total CD8 T-cell population. The data are shown as mean values ± SEM of three mice per group, representative of two independent experiments. ^*P ≤ 0.05 as compared to IM AdAg85A or IN VSVAg85A alone; ^§P ≤ 0.01 as compared to IM AdAg85A or IN VSVAg85A alone. VSV, vesicular stomatitis virus.

Figure 7

Enhanced T-cell activation by intramuscular (IM) AdAg85A–intranasal (IN) VSVAg85A prime-and-boost regimen. The mice were prepared and treated as explained in the caption for Figure 6. The total numbers of interferon-γ+ (IFN-γ+) CD4 and IFN-γ+ CD8 T cells were determined using intracellular cytokine staining and fluorescence-activated cell sorting analysis after in vitro peptide stimulation of the cells isolated from (a) the airway lumen, (b) lung, and (c) spleen. The data are shown as mean values ± SEM of three mice per group, representative of two independent experiments. ^*P ≤ 0.05 as compared to CD8 T-cell count data after IM AdAg85A or IN VSVAg85A alone; ^†P ≤ 0.001 as compared to CD4 T-cell count data after IM AdAg85A or IN VSVAg85A alone; NS, not statistically significant as compared to IM AdAg85A or IN VSVAg85A alone. VSV, vesicular stomatitis virus.

Figure 8

Enhanced immune protection conferred by intramuscular (IM) AdAg85A–intranasal (IN) VSVAg85A prime-and-boost regimen. Female Balb/c mice were immunized either subcutaneously with 1 × 10⁵ colony forming units (CFU) bacille Calmette-Guérin (BCG) or IM with 1 × 10⁷ plaque forming units (PFU) AdAg85A. Three weeks later, the latter group was boosted IN with 1 × 10⁷ PFU VSVAg85A. At the same time-point, a fresh group was immunized IN with 1 × 10⁷ PFU VSVAg85A alone. At 2 weeks [(a)/(b)] or at 4 weeks [(c)/(d)] after immunization, the mice were challenged with 15,000 CFU of the Mycobacterium tuberculosis H37Rv strain. At 4 weeks after the challenge, the mice were killed, and (a)/(b) lungs and (c)/(d) spleens were analyzed for CFU counts. The data are shown as mean values ± SEM of five to seven mice per group. ^*P ≤ 0.05; ^§P ≤ 0.01; ^†P ≤ 0.001. VSV, vesicular stomatitis virus.