Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2

Abstract

Biolistic vaccination using gene gun is developed as a safer tool for delivery of DNA vaccines, a technique that combines high vaccine efficiency with lower antigen dosage and lower cost per vaccine dose. In this study, we compared the protective responses in mice after delivering the Brugia malayi abundant larval transcript-2 (BmALT-2) DNA vaccine using the conventional intradermal approach or with the needleless gene gun delivery approach. BmALT-2 is a leading vaccine candidate against B. malayi, a lymphatic filarial parasite of human. After optimizing the DNA dose and gene gun parameters for delivery into mouse skin, groups of mice were biolistically vaccinated with 5 μg of BmALT-2pVAX. Groups of mice vaccinated intradermally with 5 μg or 100 μg of BmALT-2pVAX was used for comparison of vaccine efficacy. Results demonstrated that gene gun vaccination with 5 μg of BmALT-2pVAX conferred significant protection against challenge infection that was comparable to the degree of protection conferred by intradermal vaccination with 100 μg of BmALT-2pVAX. This observation was further supported by an in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Analysis of the immune response showed that the gene gun vaccination predominantly induced an IgG1 antibody response and significantly high Th2 cytokine response (IL-4) from spleen cells compared to intradermal BmALT-2 DNA delivery that induced predominantly an IgG2a and Th1 cytokine response (IFN-γ, IL-12 and TNF-α). These findings show that host protective responses could be achieved with 20 fold decrease in DNA dose using a gene gun and could prove to be an efficient delivery method in BmALT-2 DNA vaccination against lymphatic filariasis.

Figure 1

GFP expression in COS-1 cells (A–D) and mouse skin (E–H) after 48hrs of transfection with BmALT-2GFPpVAX plasmid. COS-1 cells were transfected with 1µg of BmALT-2GFPpVAX using gene gun at 150 Psi (B) and 100 Psi(C) helium pressures. Empty pVAX plasmid was used as negative control (D) while transfection using Lipofectamine was used as positive control (A). GFP expression was observed in skin sections of mice transfected with 5µg BmALT-2GFPpVAX using gene gun at 200psi (F), 300psi (G) and 400psi (H) helium pressure after 48 hours, while normal skin had no fluorescence (E).

Figure 2

Confirmation of mRNA (A) and protein (B) expression of BmALT-2 in the skin and lymph nodes of BmALT-2pVAX gene gun vaccinated mice. Lanes 1 and 2 represent the skin and node samples respectively from BmALT-2pVAX gene gun vaccinated mice while lanes 3 and 4 represent the skin and node samples respectively from control pVAX gene gun vaccinated mice.

Figure 3

BmALT-2 specific IgG levels in the serum of Balb/c mice vaccinated with 2.5, 5, 7.5 and 15µg of BmALT-2pVAX plasmid using a gene gun. The IgG levels were determined by indirect ELISA from serum obtained after two gene gun vaccinations. IgG levels remained significantly high (P<0.01) at all dilutions in mice vaccinated with 5, 7.5 and 15µg of DNA vaccine against the control given 5µg of pVAX vaccination using gene gun. N=5.

Figure 4

BmALT-2 specific IgG1, IgG2a, IgG2b and IgG3 antibody isotype profiles in the serum of mice vaccinated with BmALT-2pVAX using gene gun and intradermal delivery methods. The bars represent the mean optical density at 492nm of five mice determined by indirect ELISA with a serum dilution of 1:200. ***P<0.001.

Figure 5

Cytokine profile of spleen cells from mice vaccinated with BmALT-2pVAX using gene gun or intradermal delivery methods. A cytokine bead array was used to determine the level of each cytokine in the culture supernatants of 1 × 10⁵ spleen cells stimulated with 1 µg/ml of rBmALT-2. The supernatants were incubated with equal volumes of capture beads and PE detection solution after which the florescence was read in a BD™ FACS Calibur instrument. ***P<0.001.