immunization with an SFD AHc dry powder vaccine with i

immunization with an SFD AHc dry powder vaccine with i.t. to be performed to confirm the functional and essential roles of AHc-specific SIgA antibodies in the enhanced protection against BoNT/A, such as evaluating the efficacy of recombinant AHc subunit Naftopidil (Flivas) vaccine via i.t. inoculation in WT and IgA-deficient (IgA (?/?)) mice8. Pulmonary delivery of dry powder has become increasingly attractive in recent years45,46. In this study, Naftopidil (Flivas) the SFD technology was used to prepare AHc dry powder, which could effectively reduce the crystallization of formulation ingredients, enhance the amorphous glassy state and protein activity of powder formulation than that prepared by spray drying35,47,48. Then the protection efficacy of AHc dry powder was compared with that of AHc liquid vaccine. Our results demonstrated that i.t. delivery of a dry powder formulation of AHc confers protection against a lethal aerosolized BoNT challenge that is at least as strong as the protection Rabbit Polyclonal to BTK conferred by i.t. delivery of a liquid formulation. Moreover, there are several potential advantages of dry powder over liquid vaccines. First, the addition of excipients could increase the total amount of dry powder used, which is beneficial for the administration of high quantities. Second, the powder formulation may have increased storage stability and reduced transportation costs, which is becoming even more important for the development of vaccines. Finally, effective mucosal immunization could improve the bioavailability of powder49. The present study implies that the AHc dry powder vaccine made up of the mucosal adjuvant CpG prepared by SFD with i.t. inoculation is usually feasible, and that it could be developed as an effective alternative to improve the existing BoNT vaccination. In summary, we demonstrated that i.t. immunization with an SFD AHc dry powder vaccine with i.t. inoculation was feasible, which could increase storage stability and reduced transportation costs of AHc vaccines. However, compared to liquid formulation and reconstituted powder formulation, i.t. immunization of AHc dry powder was not the most suitable for nebulization. To achieve the optimum immune efficacy, the AHc could be prepared into dry powder formulation by SFD for storage stability increasing as well as transportation costs reducing, and be dispersed in liquid before i.t. immunization. Methods Animals and ethics statement Six-week-old female BALB/c mice (SPF) were obtained from Charles River Laboratories (Beijing, China). All infectious experiments were conducted in the animal biosafety level-3 laboratory. This study was performed with the permission of Institute of Animal Care and Use Committee (IACUC) at the Academy of Military Medical Science, and the ethical approval number was IACUC-DWZX-2020-049. Preparation of BoNT/A The BoNT/A complex (including type A neurotoxin and six or seven nontoxic proteins6) was isolated from the bacterial type A Naftopidil (Flivas) strain 62A, based on a modified version of a previously described extraction method50,51. Bacteria were cultured in PYG medium for 4 days, and the protein complex was isolated from the bacterial culture by acid precipitation, ion-exchange chromatography, and gel filtration. The BoNT/A complex was stored at ?80?C until use. Expression and purification of AHc The pTIG-Trx-Hc plasmid, which expresses recombinant AHc, was kindly provided by Dr. Yu from the Beijing Institute of Biotechnology as a generous gift. AHc was expressed in strain BL21 (DE3) (Agilent Technologies, Santa Clara, CA, USA) and purified by affinity chromatography as described previously52. The purified AHc was analyzed by SDS-PAGE and western blot using hyperimmune horse toxin A antiserum (from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). The gels and blots that use the same reagents and that are presented in the same panel derive from the same experiment.