Evaluation of Nanodiamond-in-Oil Emulsion with Snake Venom to Enhance Potent Antibody Induction in Mice and Rabbits

Abstract

Nanodiamonds (NDs) are an innovative material in biomedical applications based on their excellent biocompatibility, nanoscale dimensions, and high surface area. In this study, we evaluated the potential of ND-in-oil emulsion to induce potent antibody responses in animals immunized with cobra venom. NDs demonstrated the capacity to bind complex venom proteins as stable conjugates, well dispersed in aqueous solution. Immunization of mice with cobra venom incorporated with ND-in-oil emulsion adjuvant (ND/venom) elicited strong venom-specific antibody responses with titers comparable to those induced by venom formulation with conventional Freund's adjuvants (FA/venom). IgG subclass analysis revealed that ND- and FA-based formulations induced a Th2-biased immune response in mice. Moreover, antibodies elicited by ND/venom or FA/venom immunization specifically recognized the epitopes of the lethal component of short-chain neurotoxin and conferred full protection against lethal cobra venom challenge (3LD₅₀). Further, ND/venom hyperimmunization was capable of inducing high levels of neutralizing antibodies in larger animals, rabbits, highlighting the potential for antivenom manufacturing. Notably, there were no obvious lesions at the injection sites of animals that received ND/venom, in contrast to those that received FA/venom. These findings indicated NDs as an effective and safe additive in venom formulation for antivenom production.

Keywords: antivenom; cobra venom; lesion effect; nanodiamond-in-oil formulation; nanodiamonds.

Figure 1

Characterization of bare NDs and the mixture of NDs with venom proteins. SDS-PAGE analysis on (a) the precipitates and (b) the supernatants from the mixtures of NDs with different amounts of venom after centrifugation. M indicates protein marker. Lane 1 represents the cobra venom. Lanes 2–6 represent the precipitates from the mixtures of NDs mixing with 0, 20, 40, 80, or 160 μg of cobra venoms after sonication and centrifugation. (c) Overlay of size distribution of bare NDs (Blue) and ND/venom mixture (Orange) in DI water.

Figure 2

Immunogenicity evaluation of venom formulations with NDs or FAs. (a) Schematic representation of the immunization regimen. Mice were immunized at weeks 0, 2, and 4 with the prepared dosages of ND/venom or FA/venom immunogens. Black triangle represent individual data point for each mouse. (b) Images of mouse skin after injecting with either FA/venom or ND/venom immunogen. The red arrow indicates the lesion area. The mouse sera at week 6 were collected to measure the titers of (c) total IgG antibodies, (d) IgG1, and (e) IgG2a by ELISA precoated with cobra venom. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (NS, non-significant, * p < 0.05, *** p < 0.001).

Figure 3

Analysis of specific antibody responses and the protection efficacy induced by cobra venom formulations. (a) Endpoint titers of anti-sNTX antibodies in mouse sera collected 2 weeks after final immunization by ELISA. Black dot represent individual data point for each mouse. (b) Measurement of mouse sera reactivity to sNTX-derived peptides. The peptide number indicates the amino acid locations of sNTX. A plate well without coating peptide was taken as a blank control (Ctr) in the assay. (c) Survival rate of immunized mice for 48 h following the injection of a lethal dosage (3LD₅₀) of cobra venom. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (NS, non-significant, * p < 0.05, *** p < 0.001).

Figure 4

Analysis of specific antibody responses in rabbits following hyperimmunization. (a) New Zealand rabbits were immunized with cobra venom adjuvanted with NDs or FA in a five-dose hyperimmunization regimen at a 2-week interval. (b) Representative images of injection sites showing skin conditions post immunization with FA/venom (left) and ND/venom (right). Serum antibody titers were determined by ELISA against (c) cobra venom and (d) sNTX. (e) Measurement of rabbit sera reactivity to sNTX-derived peptides. The peptide number indicates the amino acid residues of sNTX. A plate well without coating peptide was taken as a blank control (Ctr) in the assay. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (*** p < 0.001).