## 4 Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy
### Vaccine Development  
ClyA is useful in vaccine development because surface-exposed or secreted antigens are often more immunogenic than cytoplasmic antigens, which is an important factor in developing effective vaccines to protect against pathogens. [PERSON] et al. successfully engineered a recombinant protein comprising ClyA fused to the _Bacillus anthracis_ protective antigen (domain 4 moiety) and demonstrated that this fusion protein was exported and exhibited immunostimulatory activity in mice [90]. Their findings showed that ClyA could be an attractive export system for displaying foreign (heterologous) antigens in attenuated bacterial strains, which paved the way for exploring this feature of ClyA in numerous subsequent studies.  
Importantly, ClyA is secreted by OMVs, and is therefore a good fusion partner and an ideal scaffold for delivering exogenous proteins or antigens to OMVs, which are then presented on the surface of released OMVs. [PERSON] et al. demonstrated that fully functional, recombinant beta lactamase, GFP, and anti-digoxin antibody fused to the \(C\)-terminus of ClyA were secreted in OMVs. This work highlighted the flexibility of recombinant protein display on the OMV surface [91]. A subsequent study showed that an antibody response directed against GFP was induced in mice immunized with OMVs expressing recombinant GFP fused to ClyA [92]. These seminal studies opened a new research avenue for the development of next-generation therapies that take advantage of the genetic flexibility offered by the ClyA-OMV system.  
The engineered ClyA-OMV system has been recently applied in the development of vaccines against various pathogens, including SARS-CoV-2 [93]. For example, it has been reported that the successful expression and localization of ClyA-M2e fusion proteins in OMVs led to protection against a lethal dose of H1N1 influenza A virus [94]. Another successful example of the use of ClyA in vaccine development was a study in which a complete _Acinetobacter baumannii_ Omp22 protein fused to ClyA was displayed on the surface of _E. coli_ OMVs. Immunization with the engineered OMVs induced a strong antigen-specific humoral immunity response in mice and protected mice from a lethal challenge with a clinical _A. baumannii_ isolate [95]. During the ongoing COVID-19 pandemic, messenger RNA has been widely used to generate immunogens in SARS-CoV-2 vaccines, which have been very effective in preventing COVID-19 [96; 97; 98; 99]. However, the current vaccines against SARS-CoV-2 have several issues, including low yields, transportation difficulties, high manufacturing costs, and complex and delicate manufacturing processes. In recent work, [PERSON] et al. reported the development of bacterial biomimetic vesicles (BBVs)loaded with a fusion protein comprising ClyA and the SARS-CoV-2 spike protein receptor binding domain (RBD), called RBD-BBV [100]. Combining this strategy with high-pressure homogenization technology further enhanced the presentation of the RBD on BBVs and the yields of RBD-BBVs. When injected subcutaneously, these RBD-BBVs accumulated in lymph nodes, promoted antigen uptake and processing, and elicited SARS-CoV-2-specific humoral and cellular immune responses in mice. Thus, it is expected that the ClyA-OMV system could be used in the development of vaccines against emerging pathogens, such as SARS-CoV-2.