Alum Pickering Emulsion as Effective Adjuvant to Improve Malaria Vaccine Efficacy

Abstract

Malaria is a life-threatening global epidemic disease and has caused more than 400,000 deaths in 2019. To control and prevent malaria, the development of a vaccine is a potential method. An effective malaria vaccine should either combine antigens from all stages of the malaria parasite's life cycle, or epitopes of multiple key antigens due to the complexity of the Plasmodium parasite. Malaria's random constructed antigen-1 (M.RCAg-1) is one of the recombinant vaccines, which was selected from a DNA library containing thousands of diverse multi-epitope chimeric antigen genes. Moreover, besides selecting an antigen, using an adjuvant is another important procedure for most vaccine development procedures. Freund's adjuvant is considered an effective vaccine adjuvant for malaria vaccine, but it cannot be used in clinical settings because of its serious side effects. Traditional adjuvants, such as alum adjuvant, are limited by their unsatisfactory immune effects in malaria vaccines, hence there is an urgent need to develop a novel, safe and efficient adjuvant. In recent years, Pickering emulsions have attracted increasing attention as novel adjuvant. In contrast to classical emulsions, Pickering emulsions are stabilized by solid particles instead of surfactant, having pliability and lateral mobility. In this study, we selected aluminum hydroxide gel (termed as "alum") as a stabilizer to prepare alum-stabilized Pickering emulsions (ALPE) as a malaria vaccine adjuvant. In addition, monophosphoryl lipid A (MPLA) as an immunostimulant was incorporated into the Pickering emulsion (ALMPE) to further enhance the immune response. In vitro tests showed that, compared with alum, ALPE and ALMPE showed higher antigen load rates and could be effectively endocytosed by J774a.1 cells. In vivo studies indicated that ALMPE could induce as high antibody titers as Freund's adjuvant. The biocompatibility study also proved ALMPE with excellent biocompatibility. These results suggest that ALMPE is a potential adjuvant for a malaria vaccine.

Keywords: M.RCAg-1; adjuvant; alum stabilized Pickering emulsion; immune response; malaria vaccine.

Figure 1

The schematic diagram of vaccination regimens and blood collection in animal immunization. (a) Immunization of OVA as antigen. (b) Immunization of M.RCAg-1 as antigen.

Figure 2

Optical micrographs and the appearance of Pickering emulsion prepared with different particle concentration. Scale bar = 10 μm.

Figure 3

Optical micrograph of Pickering emulsions prepared with different buffer type of the aqueous phase. Scale bar = 10 μm.

Figure 4

Pickering emulsion varies with pH of the aqueous phase. (a) Size and size distribution of the droplet. (b) The appearance of the emulsion.

Figure 5

Characteristics of ALPE for adjuvant application. (a) Size distribution of ALPE. (b) Confocal image of ALPE droplets. Alum particles was labeled by lumogallion (blue). (c) Hyperspectral image of ALPE droplets. (d) Antigen adsorption efficiency in different times. (e) Confocal image of antigen-adsorbed ALPE droplets. Alum particles and OVA were labeled by lumogallion (red) and Cy5 (green), respectively. Scale bar = 5 μm. Data are expressed as mean ± s.e.m. (n = 3).

Figure 6

Characteristics of ALMPE for adjuvant application. (a) Size distribution of ALMPE. (b) Confocal image of ALMPE droplets. Alum particles was labeled by lumogallion (green). (c) Hyperspectral image of ALMPE droplets. (d) Antigen obsorption efficiency in different times. (e) Confocal image of antigen-adsorbed ALMPE droplets. Alum particles and OVA were labeled by lumogallion (red) and Cy5 (green), respectively. Scale bar = 5 μm. Data are expressed as mean ± s.e.m. (n = 3).

Figure 7

FRAP analysis on the lateral mobility of OVA antigens on the surface of ALPE (a) and ALMPE (b) droplets. Alum particles and OVA were labeled by lumogallion (red) and Cy5 (green), respectively. Scale bar = 2 μm.

Figure 8

Confocal images of endocytosis. DC nucleus were stained by DAPI (blue). DC membrane and alum particles were labeled with TRITC-phalloidin (red) and Lumogallion (green), respectively. Scale bar = 10 μm.

Figure 9

Confocal images of lysosomal escape on BMDCs after 24 h treatment. Lysotracker and OVA antigen were labeled with Lyso Tracker (red) and Cy5 (green). Scale bar = 10 μm.

Figure 10

Systemic immunity in vivo. Production of OVA antigen-specific antibodies in the serum at day 28 (a) and day 38 (b). ELISPOT assay on IFN-γ spot-forming cells among splenocytes (c,d). “Ag” and “Al” represent the individual antigen group and alum group respectively. Data are expressed as mean ± s.e.m. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 11

M.RCAg-1 vaccination. Production of M.RCAg-1 antigen-specific antibodies in the serum at day 28 (a) and day 38 (b). ELISPOT assay on IFN-γ spot-forming cells among splenocytes (c,d). “Ag” represents the individual antigen group. Data are expressed as mean ± s.e.m. (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 12

Biocompatibility evaluations via histological analysis of major organs from C57BL/6 mice after 14 day of immunization.