Epsilon toxin (ETX) is an exotoxin produced by type B and D Clostridium perfringens that causes enterotoxemia or necrotic enteritis in animals such as goats, sheep, and cattle. Vaccination is a key method in preventing such diseases. In this study, we developed a new type of dissolving microneedle patch (dMN) with a nanoparticle adjuvant for enhanced immune response to deliver the rETX^Y196E-C protein vaccine. We chose FDA-approved poly(lactic-co-glycolic acid) (PLGA) to prepare nanospheres as the vaccine adjuvant and introduced dimethyldioctadecylammonium bromide (DDAB) to make the surface of PLGA nanoparticles (PLGA NPs) positively charged for antigen adsorption. PLGA NPs with a diameter of 100~200 nm, a surface ZETA potential of approximately +40 mV, and good safety were successfully prepared and could effectively adsorb rETX^Y196E-C protein. Using non-toxic and antibacterial fish gelatin as the microneedle (MN) matrix, we prepared a PLGA-DDAB dMN vaccine with good mechanical properties that successfully penetrated the skin. After immunization of subcutaneous (SC) and dMN, antibody titers of the PLGA and Al adjuvant groups were similar in both two immune ways. However, in vivo neutralization experiments showed that the dMN vaccines had a better protective effect. When challenged with 100 × LD₅₀ GST-ETX, the survival rate of the MN group was 100%, while that of the SC Al group was 80%. However, a 100% protective effect was achieved in both immunization methods using PLGA NPs. In vitro neutralization experiments showed that the serum antibodies from the dMN and SC PLGA NPs groups both protect naive mice from 10 × LD₅₀ GST-ETX attack after being diluted 20 times and could also protect MDCK cells from 20 × CT₅₀ GST-ETX attack. In conclusion, the PLGA-DDAB dMN vaccine we prepared has good mechanical properties, immunogenicity, and protection, and can effectively prevent ETX poisoning. This provides a better way of delivering protein vaccines.

Background: As a Class A bioterrorism agent, botulinum neurotoxin serotype A (BoNT/A) carries the risk of being used by terrorists to cause mass poisoning. The microneedle (MN) patch has a great potential for application as a novel vaccine delivery method. The aim of this study is to develop a thermally stable, dissolving microneedle patch for the delivery of a recombinant protein vaccine using a recombinant C-terminal heavy chain of BoNT/A (Hc of BoNT/A, AHc) to prevent botulism. Methods: Fish gelatin, a natural non-toxic and bacteriostatic material, was selected as the microneedle matrix for the preparation of the dissolving microneedle vaccine. Subsequently, the mechanical performance, bacteriostatic properties, vaccination effect, and stability of the microneedle patches were evaluated using instruments such as the displacement-force test station and optical coherence tomography (OCT) scanner. Results: Fish gelatin matrix at high concentrations has good bacteriostatic properties, and excellent mechanical performance and vaccination effect, meeting the necessities of a vaccine. In both in vivo and in vitro neutralization experiments, MN vaccines containing different antigen doses achieved the same protective efficacy as subcutaneous vaccinations, protecting mice against 10⁶ LD₅₀ of BoNT/A injected intraperitoneally. Thermal stability analysis of the MN vaccines revealed that the fish gelatin matrix protected the AHc vaccine from protein denaturation even after 7 days of storage at 37 °C and enabled the vaccine patches to maintain good immunogenicity and protective efficacy even after 6 months of storage at room temperature. Conclusion: In this study, we successfully prepared a bacteriostatic MN patch using a fish gelatin matrix that not only has a good vaccination effect, but also obviates the need for a cold chain for the AHc vaccine, providing the possibility of rapid, painless, and large-scale vaccination.

Ricin and abrin are phytotoxins that can be easily used as biowarfare and bioterrorism agents. Therefore, developing a rapid detection method for both toxins is of great significance in the field of biosecurity. In this study, a novel nanoforest silicon microstructure was prepared by the micro-electro-mechanical systems (MEMS) technique; particularly, a novel microfluidic sensor chip with a capillary self-driven function and large surface area was designed. Through binding with the double antibodies sandwich immunoassay, the proposed sensor chip is confirmed to be a candidate for sensing the aforementioned toxins. Compared with conventional immunochromatographic test strips, the proposed sensor demonstrates significantly enhanced sensitivity (≤10 pg/mL for both toxins) and high specificity against the interference derived from juice or milk, while maintaining good linearity in the range of 10–6250 pg/mL. Owing to the silicon nanoforest microstructure and improved homogeneity of the color signal, short detection time (within 15 min) is evidenced for the sensor chip, which would be helpful for the rapid tracking of ricin and abrin for the field of biosecurity.