Laser mimicking mosquito bites for skin delivery of malaria sporozoite vaccines

Abstract

Immunization with radiation-attenuated sporozoites (RAS) via mosquito bites has been shown to induce sterile immunity against malaria in humans, but this route of vaccination is neither practical nor ethical. The importance of delivering RAS to the liver through circulation in eliciting immunity against this parasite has been recently verified by human studies showing that high-level protection was achieved only by intravenous (IV) administration of RAS, not by intradermal (ID) or subcutaneous (SC) vaccination. Here, we report in a murine model that ID inoculation of RAS into laser-illuminated skin confers immune protection against malarial infection almost as effectively as IV immunization. Brief illumination of the inoculation site with a low power 532 nm Nd:YAG laser enhanced the permeability of the capillary beneath the skin, owing to hemoglobin-specific absorbance of the light. The increased blood vessel permeability appeared to facilitate an association of RAS with blood vessel walls by an as-yet-unknown mechanism, ultimately promoting a 7-fold increase in RAS entering circulation and reaching the liver over ID administration. Accordingly, ID immunization of RAS at a laser-treated site stimulated much stronger sporozoite-specific antibody and CD8(+)IFN-γ(+) T cell responses than ID vaccination and provided nearly full protection against malarial infection, whereas ID immunization alone was ineffective. This novel, safe, and convenient strategy to augment efficacy of ID sporozoite-based vaccines warrants further investigation in large animals and in humans.

Keywords: Delivery; Laser; Malaria; Sporozoites; Vaccine.

Figure 1

Laser illumination enhances blood vessel permeability. (A) Representative images showing blood vessel leakage induced by laser. Blood vessels were marked by FITC-dextran IV injected, after which the dorsal skin of the mice was treated by different lasers and examined under an intravital confocal microscope within 30 min. (B) Representative histological examination of laser-treated skins. (C) Alterations of blood vessels induced by lasers. Bar=100 μm in A and B or 10 μm in C. Arrows indicate blood vessels. n=5.

Figure 2

Laser enhances the delivery of sporozoites from the skin to liver. (A) A standard curve of liver parasite loads after IV injection of indicated numbers of sporozoites. (B) Effects of laser skin illumination on skin-to-liver delivery of sporozoites. Mice were injected with 4,000 sporozoites either by an IV route or through ID injection into laser-treated or un-treated dorsal skin (ID). The liver parasite loads were determined by RT-qPCR and estimated using the standard curve 2A. (C and D). GFP⁺ cells were measured by flow cytometry 42 hrs after freshly isolated PyGFP sporozoites were injected by IV or ID in the presence or absence of laser treatment (C). Liver parasite loads were quantified by RT-qPCR in the animals (D). Results are expressed as means ± standard deviation (SD). The experiment was repeated twice with similar results. n=8, *p<0.05, **p<0.01, ***p<0.001, and ns, not significant.

Figure 3

Confocal microscopy of sporozoites in the skin. (A) Blood vessels were marked by Texas red-dextran (MW 70,000). (B and C) Representative images of CFSE-stained sporozoites in un-treated (B) or laser-treated skin (C). Bar =10 μm. (D) Percentages of sporozoites in association with vessel walls or inside the vessels. Data are shown as means ± SD. n=10, ***p<0.001.

Figure 4

Peripheral immune responses against sporozoites. Mice were immunized with three doses of RAS each with 10,000 sporozoites at an interval of two weeks. Geometric mean titers (GMT) of anti-sporozoite antibody were determined by immunofluorescence assay (A) and CD8⁺ T cells in PBMCs were analyzed by flow cytometry (B) 7 days after the final immunization. The results are expressed as means ± SD. N=8, *p<0.05, **p<0.01, and ***p<0.001.

Figure 5

Frequencies of sporozoite-specific CD8⁺ T cells in the liver and spleen. Mice were immunized with three doses of RAS each with 10,000 sporozoites at an interval of two weeks. Representative flow profiles of sporozoite-experienced CD11a^hi CD8α^lo cells in the liver and spleen are shown in (A). Mean frequencies ± SD of CD11a^hi CD8α^lo cells (B) and IFN-γ⁺-producing CD8⁺ T cells (C) were attained in the liver and spleen by flow cytometry 7 days after the final immunization. n=8, **p<0.01, ***p<0.001 and ns, not significant.

Figure 6

Protection against malarial challenge. Mice were immunized with three doses of RAS each with 2,000 sporozoites at an interval of two weeks. All animals were challenged by IV injection of 200 live P. yoelii sporozoites 7 days after the final immunization. Parasitemia were monitored at indicated days post-challenge by blood smear (A) and blood parasite burdens were determined by RT-qPCR on day 10 post challenge (B). The results are expressed as Mean ± SD. n=8, **p<0.01 and ***p<0.001 between ID and Laser+ID group in A or ***p<0.001 and ns, not significant in B.