DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

Abstract

Background: Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis.

Objectives: We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection.

Methods: An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge.

Findings: rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05).

Main conclusions: NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Fig. 1:. evaluation of cytotoxic effect of HNTs and NH₂-MWCNTs in CHO cells by MTT assay. The inhibition rate was expressed as the optical density of treated cells compared to the negative control cells (only medium). The positive control cells were treated with 1% dimethyl sulphoxide. The data are expressed as mean ± SEM of three independent experiments. Asterisks indicate significant differences (p < 0.001) compared to the positive control.

Fig. 2:. indirect immunofluorescent microscopy of CHO cells transfected with pTARGET, HNT-pTARGET/lemA, NH₂-MWCNT-pTARGET/lemA, and pTARGET/lemA with and without lipofectamine (Invitrogen). The results were expressed as the percentage of cells with green fluorescence. Values are presented as means ± SEM of two independent experiments. Asterisks represent a difference between groups in comparison with pTARGET/lemA (*p < 0.05 and **p < 0.0001). The samples were analysed in triplicate.

Fig. 3:. survival of hamsters immunised with rLemA-based DNA nanovaccines after challenge. Percent survival conferred by NH₂-MWCNTs-pTARGET/lemA, HNT-pTARGET/lemA, and bacterin against lethal challenge was significant (p < 0.05*) in comparison to negative control groups. Survival curves were compared using the log rank (Mantel Cox test) analysis.

Fig. 4:. IgG antibody response in hamsters immunised with rLemA DNA nanovaccines. The specific IgG responses stimulated by the different immunogens were determined by an ELISA of the hamster serum diluted at 1:50, using rLemA produced in Escherichia coli as the antigen. Values are presented as means ± SEM of two independent experiments. Asterisk represent a difference compared to control groups (p < 0.05). The samples were analysed in triplicate.