Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein

Abstract

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.

Figure 1

(A) In vivo expression of LUC on day 3 post vaccination. C57Bl/6 mice received a single IN vaccination of naked or virosome-encapsulated DNA encoding LUC. Luminescence was detected by the IVIS live imager and quantified by Living Image software. (a) Empty pVAX vector, (b) 100 μg pVax LUC, (c) 20 μg pVAX LUC, (d) Liposome plus 20 μg pVAX LUC, E) Virosome plus HA plus 20 μg pVAX LUC. (B)The graph shows mean photons s⁻¹ (±s.e.m.) and each point represents a single mouse.

Figure 2

Systemic and mucosal T-cell responses to LUC. IFN-γ ELIspot was performed with cells restimulated with a single immunodominant LUC peptide. SFU for cells from (a) spleens and (b) vaginal-draining lymph nodes from vaccinated mice. Graphs show mean SFU per 10⁶ cells (±s.e.m.), and each point represents a single mouse. Significant differences were determined by the Kruskal–Wallis multiple comparison test.

Figure 3

A schematic diagram of the plasmid constructs. The constructs were organised into three groups as noted in the figure. (a) TK constructs, (b) cytolytic gene constructs in which the cytolytic gene expression was controlled by the SV40 promoter and (c) cytolytic gene constructs in which the cytolytic gene expression was controlled by the CMV promoter.

Figure 4

The induction of HEK293T cell death by cytolytic gene expression as determined by LUC expression. The cells were transfected with the different DNA constructs as noted in a, b and c, and LUC expression used as a measure of cell viability compared with the control pVAX LUC. c was published originally in reference number 21.

Figure 5

Markers of cell death after transfection of HEK293T cells with the different DNA constructs, as determined by Annexin V and PI staining. (a) The gating strategy for the Annexin V (x axes) and PI (y axes) staining analysis. Two panels are shown for each construct; the left panel represents cells in the dead cell gate and the right panel represents cells in the live cell gate. (b) pVAX LUC, pVAX LUC TK, pVAX LUC+GCV and pVAX LUC TK+GCV, (c) pVAX LUC NSP4, pVAX LUC PRF and pVAX LUC DTa representing cytolytic genes expressed from the SV40 promoter and (d) pVAX PRF LUC and pVAX LUC 2A PRF representing cytolytic genes expressed from the CMV promoter.

Figure 6

In vivo cell death as determined by LUC expression post vaccination. C57Bl/6 mice were vaccinated with 50 μg DNA and luminescence detected by IVIS imaging at intervals post vaccination. (a) pVAX LUC, pVAX LUC TK, pVAX LUC+GCV and pVAX LUC TK+GCV, (b) pVAX LUC NSP4, pVAX LUC PRF and pVAX LUC DTa representing cytolytic genes expressed from the SV40 promoter and (c) pVAX PRF LUC and pVAX LUC 2A PRF representing cytolytic genes expressed from the CMV promoter. Graphs show the mean luminescence±s.e.m. for five mice as measured by the IVIS live imager. c was published originally in reference number 21.

Figure 7

Humoral and cell-mediated responses to LUC in mice vaccinated with the different DNA constructs. IgG titres were determined by enzyme-linked immunosorbent assay in serum from mice vaccinated with (a) the LUC±TK constructs or (b) LUC+the directly cytolytic genes. Graphs show mean reciprocal IgG titre,±s.e.m. IFN-γ-secreting cells after restimulation of splenocytes with an immunodominant epitopes as detected by ELIspot in mice vaccinated with (c) the LUC±TK constructs or (d) LUC+the directly cytolytic genes. Graphs show meant SFU per 10⁶ cells,±s.e.m.