Potential of Polyethyleneimine as an Adjuvant To Prepare Long-Term and Potent Antifungal Nanovaccine

Abstract

Background: Candida albicans infections are particularly prevalent in immunocompromised patients. Even with appropriate treatment with current antifungal drugs, the mortality rate of invasive candidiasis remains high. Many positive results have been achieved in the current vaccine development. There are also issues such as the vaccine's protective effect is not persistent. Considering the functionality and cost of the vaccine, it is important to develop safe and efficient new vaccines with long-term effects. In this paper, an antifungal nanovaccine with Polyethyleneimine (PEI) as adjuvant was constructed, which could elicit more effective and long-term immunity via stimulating B cells to differentiate into long-lived plasma cells.

Materials and methods: Hsp90-CTD is an important target for protective antibodies during disseminated candidiasis. Hsp90-CTD was used as the antigen, then introduced SDS to "charge" the protein and added PEI to form the nanovaccine. Dynamic light scattering and transmission electron microscope were conducted to identify the size distribution, zeta potential, and morphology of nanovaccine. The antibody titers in mice immunized with the nanovaccine were measured by ELISA. The activation and maturation of long-lived plasma cells in bone marrow by nanovaccine were also investigated via flow cytometry. Finally, the kidney of mice infected with Candida albicans was stained with H&E and PAS to evaluate the protective effect of antibody in serum produced by immunized mice.

Results: Nanoparticles (NP) formed by Hsp90-CTD and PEI are small, uniform, and stable. NP had an average size of 116.2 nm with a PDI of 0.13. After immunizing mice with the nanovaccine, it was found that the nano-group produced antibodies faster and for a longer time. After 12 months of immunization, mice still had high and low levels of antibodies in their bodies. Results showed that the nanovaccine could promote the differentiation of B cells into long-lived plasma cells and maintain the long-term existence of antibodies in vivo. After immunization, the antibodies in mice could protect the mice infected by C. albicans.

Conclusion: As an adjuvant, PEI can promote the differentiation of B cells into long-lived plasma cells to maintain long-term antibodies in vivo. This strategy can be adapted for the future design of vaccines.

Keywords: fungal infections; long-lived plasma cell; long-term protection; nanoparticles; polyethylenimine.

Figure 1

Design and expression of antigen. (A) The amino acid sequences of the recombinant protein Hsp90-CTD. (B) The His-tagged recombinant proteins were purified by Ni-affinity chromatography and analyzed by SDS-PAGE. Red arrow shows monomers of Hsp90-CTD. The other band was from the supernatant that contained soluble proteins.

Figure 2

Synthesis and characterization of the particles. (A) Size of different proportions (PEI: Hsp90-CTD) nanoparticles after treating with SDS. (B) PDI of NP@PEI+Hsp90-CTD nanoparticles after treating with SDS. (C) Surface zeta potential of nanoparticles. The nanoparticles’ stability of (D) size, and (E) PDI stored at 4°C for a month. (F) Comparable results between pure protein and SDS-modified protein were obtained by circular dichroism (CD) spectroscopy. (G) TEM images of NP@PEI+Hsp90-CTD and particles without SDS (Hsp90-CTD+PEI).

Figure 3

Formation mechanism of Hsp90-CTD nanovaccine. (A) The putative binding site of SDS on protein Hsp90-CTD as obtained from Autodock, shows the hydrophobic interaction between SDS and Hsp90-CTD. Molecule SDS forms 2 hydrogen bonds with amino acid LYS65, acting at distances of 2.3 Å and 2.1 Å (B) DLS detects the solution potential value. Changes in potential of Hsp90-CTD protein combined with different volumes of SDS solution. (C) Native-PAGE: The migration efficiency of protein Hsp90-CTD combined with different volumes of SDS solution was analyzed on 10% Native-PAGE. (D) Schematic illustration of the process for synthesizing nanoparticles. Hsp90-CTD and SDS were combined with hydrophobic force. Then the polymer PEI was introduced to combine with the mixture by electrostatic force. Finally, well-characterized nanoparticles were formed.

Figure 4

Hsp90-CTD (1 µg/ml) was coated into 96-well ELISA plates at 4°C overnight. Blood from 4 groups (control, Hsp90-CTD, Hsp90-CTD+AL, and NP) of mice was collected at different time points, 3 mice/group. Sera were obtained after centrifugation at 9000 rpm for 10 min. The sera were diluted 500 times and the antibody levels were then measured by ELISA. (A) 48 h (B) 120 h (C) 144 h (D) 168 h (E) 240 h (F) 360 h.

Figure 5

Hsp90-CTD (1 µg/ml) was coated into 96-well ELISA plates at 4°C overnight. Blood from 4 groups (control, Hsp90-CTD, Hsp90-CTD+AL, and NP) of mice was collected at different time points, 3 mice/group. Sera were obtained after centrifugation at 9000 rpm for 10 min. The sera were diluted 500 times and the antibody levels were then measured by ELISA. (A) 28 days. (B) 2 months. (C) 3 months. (D) 4 months. (E) 5 months. (F) 12 months.

Figure 6

Levels of LLPCs in each group of mice detected by flow cytometry. CD44/CD138 gating signify plasma cells, and CD44/MHC II gating signify long-lived plasma cells. 1*104 cells per condition were analyzed by flow cytometry. The proportion in the red box represents the proportion of LLPCs. (A) LLPCs of different groups mice. The proportion of LLPCs in the NP@Hsp90-CTD+PEI group was significantly higher than that in other groups, especially in the Hsp90-CTD group. *P < 0.05; ***P < 0 .001. (B) LLPCs of group control mice. (C) LLPCs of group pure protein mice. (D) LLPCs of group Hsp90-CTD+PEI mice. (E) LLPCs of group Al adjuvants mice. (F) LLPCs of group NP mice.

Figure 7

The infected kidneys were protected by the serum of nanovaccine-immunized mice. (A) Kidneys of mice were protected with serum of the control group and the nanovaccine group, respectively. The kidneys of unprotected mice were infested with C. albicans SC5314 and the surface was already covered with it. In contrast, the kidneys of antibody-protected mice had a very smooth surface. (B) Representative H&E staining of kidneys from infected mice with indicated treatment at 48 h post-infection. (C) Representative PAS staining of kidneys from infected mice with indicated treatment at 48 h post-infection. The darker areas framed by red lines were the locations of fungal infestation inside the kidney.

Figure 8

Detection of nanoparticle toxicity. (A) Cell survival was measured with CCK8 assay after nanovaccine injected. (B) Cell survival was measured with CTL assay after nanovaccine injected. No significant difference between the experimental group and the control group. P > 0.05.

Figure 9

Schematic representation of Hsp90-based antigen delivery system and its role in eliciting cellular immunity response. Nanovaccine in mice caused immune response, so that the body quickly produces antibodies and plays a protective role. At the same time, the vaccine can promote the differentiation of B cells into LLPCs, so that the body has long-time protective antibodies.