## 4 Discussion  
NTS strains commonly cause self-limiting illness with a low lethal rate [35]. However, certain NTS servovars, including \(S\). Typhimurium, \(S\). Enteritidis, and \(S\). Dublin, are occasionally invasive and disseminate to deeper tissues, causing deadly diseases such as septicemia and meningitis [36; 37]. Furthermore, some highly invasive strains, such as \(S\). Typhimurium ST 313 and ST 34, are resistant to multiple antibiotics recommended as the first-line treatment for these infections [37; 38]. According to the Global Burden of Disease (GBD) Study, the burden of invasive NTS diseases was estimated to be 535,000 cases, including 77,500 deaths in 2017, with the most vulnerable populations being young children, the elderly, and immunocompromised individuals [39]. High case fatality rates and emerging multidrug resistance underscore the urgent need to develop proficient vaccines against invasive NTS infection. Currently, although there is no licensed vaccine against invasive NTS, several vaccine candidates are under clinical field trials, including live attenuated vaccines, protein-polysaccharide vaccines (glyoconjugate vaccines), protein vaccines, and Outer Membrane Vesicle (OMV) -based vaccines [10]. Regarding toxicity, immunogenicity, longevity, immunotherapeutic spectrum, and production cost, each vaccine strategy has advantages and disadvantages. Live attenuated vaccines deliver a wide repertoire of antigens on a temporal and spatial basis in the interaction with the host immune system and are expected to elicit successfully both cellular and humoral responses [40]. The cost-effectiveness of both the production and administration of vaccines is also a considerable advantage of live attenuated vaccines. However, live attenuated vaccines, which are generally constructed by deleting genes that are essential for virulence and proliferation in vivo, have the possibility of genetic reversion to a WT phenotype via the re-acquisition of the deleted genes, thus imposing a safety risk. Of course, this possibility can be debilitated by mutating multiple loci localized separately in the genome [41]. In addition, live attenuated vaccines are prone to be excreted in the stool over a long period. For example, the WT05 candidate, which is defective in aromatic acid biosynthesis and SPI-2-associated virulence because of the lack of _aroC_ and _ssaV_, respectively, failed at the clinical stage because of long-lasting fecal shedding (for more than 3 weeks) [42]. Prolonged stool excretion is problematic considering the possibility of _Salmonella_ transmission through food and water contamination. To date, many _Salmonella_ strains have been suggested as candidates for live attenuated vaccines against invasive NTS; _Salmonella_ strains devoid of global transcriptional regulators such as PhoP [43], strains defective in signal transduction via small signaling molecules such as ppGpp [44], and strains unable to biosynthesize aromatic compounds and purines [45] In spite of the protection activity in animal models, the superiority as a live attenuated vaccine in human model cannot be guaranteed unless the dynamic interaction between vaccines and host immune system is fully understood. Unfortunately, there is no live vaccine applicable to humans yet. In an effort to develop a potent live attenuated vaccine against invasive NTS, we compared three \(S\). Typhimurium mutant strains, i.e., _AfruR_, _sssrAB_, and _alftg_, in regard to virulence, immunogenicity, and prophylactic activity. The three genes of _fruR_, _ssrAB_, and _hftg_ encode transcriptional or post-transcriptional regulators, and their deletions are expected to modulate the expression of multiple genes directly or indirectly, leading to extensive alterations in bacterial behavior and physiology, such as decreased SPI-2 expression in an acidic minimal medium mimicking the intracellular environment [20].