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non-radioactive approaches for the early detection of tumors. These engineered bacteria are referred to as
[125]
bacterial whole-cell biosensors (BWCBs) . In the context of disease detection, BWCBs offer advantages
such as reduced reliance on expensive equipment, faster detection, greater specificity, and lower costs,
which facilitate the early diagnosis of certain diseases. Furthermore, engineered bacteria can be designed as
imaging vectors to improve the sensitivity and specificity of medical imaging. Additionally, integrated
diagnostic and therapeutic engineered bacteria are being developed to concurrently release therapeutic
drugs while diagnosing disease markers .
[116]

Potential application of mucosal vaccines developed based on engineered bacteria in disease
prevention
Mucosal surfaces in the respiratory, digestive, and genitourinary systems serve as crucial entry points for
pathogens from the environment. The mucosal immune system plays a pivotal role as the body’s initial
defense barrier against these pathogens. Therefore, effective vaccines that stimulate immune responses at
mucosal sites are vital.

Research dating back to the early 1990s has underscored the potential of Lactobacillus casei in enhancing
mucosal immune responses. For example, studies have shown that Lactobacillus casei can protect against
[126]
Salmonella typhimurium infection by boosting the production of sIgA in intestinal mucosal secretions .
This highlights the ability of natural Lactobacillus casei to serve as an immune adjuvant for preventing
intestinal infections.

Recent advancements in probiotic genomics and synthetic biology have significantly advanced the design of
engineered bacteria. These probiotics are engineered to express antigenic components from various
pathogens such as bacteria , viruses , and parasites . Administering these engineered bacteria
[128]
[129]
[127]
mucosally can stimulate immune responses within the mucosal immune system, thereby exerting anti-
infective effects targeted against specific pathogens.
Several recent animal studies have demonstrated the superior immunoprotective effects of
engineered bacteria against pathogens such as Streptococcus pneumoniae , Trichinella spiralis ,
[127]
[129]
Helicobacter pylori [130,131] , Avian influenza A virus , and COVID-19 . Various factors influence the
[133]
[132]
immunization efficacy of engineered bacteria, including chassis type, antigen expression site, administration
route, and target antigen type. A study examined the impact of varying antigen anchoring positions on
immunization efficacy. They introduced two plasmids, pPG1 (surface display) and pPG2 (secretion), into
the probiotic strain Lactobacillus casei CC16 for oral administration to carp, aiming to stimulate mucosal
immune responses against Aeromonas hydrophilia. They discovered that surface-displayed Lc-pPG1-Aha1
induced higher levels of specific antibodies and enhanced leukocyte phagocytosis . Additionally, related
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studies have explored different administration routes. For instance, in a study by Zhang et al., the peptide
hormone IP-673 promoted the use of recombinant probiotic strain L. plantarum strains expressing the HA1
protein, which were administered both orally and intranasally. Mice challenged with a lethal dose of the
H1N1 virus showed improved immunization when the administration was intranasal . Huynh et al.
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demonstrated HA1 subunit and Bacillus subtilis poly gamma-glutamate synthetase A (pgsA) fusion proteins
on the surface of Lactobacillus casei L525 and they compared immune response intensities between
intranasal and oral administration routes, finding both routes provided similar immune protection, with
intranasal administration proving more effective. Several factors, including genetic diversity among
[136]
Lactobacilli , antigen characteristics, and mucosal immunization sites, complicate the interpretation of
immune outcomes and hinder comparative studies. Table 1 provides a summary of selected probiotics
engineered for use as mucosal vaccines [Table 1].