Self-destructing attenuated adjuvant Salmonella serves as a safe and potent live vaccine adjuvant

self-destructing-attenuated-adjuvant-salmonella-serves-as-a-safe-and-potent-live-vaccine-adjuvant
Self-destructing attenuated adjuvant Salmonella serves as a safe and potent live vaccine adjuvant

References

  1. Moni, S. S. et al. Advancements in vaccine adjuvants: the journey from alum to nano formulations. Vaccines 11, https://doi.org/10.3390/vaccines11111704 (2023).

  2. Facciolà, A., Visalli, G., Laganà, A. & Di Pietro, A. An overview of vaccine adjuvants: current evidence and future perspectives. Vaccines 10, https://doi.org/10.3390/vaccines10050819 (2022).

  3. Lavelle, E. C. & McEntee, C. P. Vaccine adjuvants: tailoring innate recognition to send the right message. Immunity 57, 772–789 (2024).

    Google Scholar 

  4. Mata-Haro, V. et al. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 316, 1628–1632 (2007).

    Google Scholar 

  5. Facchini, F. A. et al. Synthetic glycolipids as molecular vaccine adjuvants: mechanism of action in human cells and in vivo activity. J. Med. Chem. 64, 12261–12272 (2021).

    Google Scholar 

  6. Kong, Q. et al. Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J. Immunol 187, 412–423 (2011).

    Google Scholar 

  7. Swain, B. & Miryala, K. R. NOD-like receptors in fish: evolution, structure, immune signaling, and targeting for aquaculture vaccine adjuvants. Front. Immunol. 16, 1665071 (2025).

    Google Scholar 

  8. Chen, R. et al. Pattern recognition receptors: function, regulation and therapeutic potential. Signal Transduct. Target Ther. 10, 216 (2025).

    Google Scholar 

  9. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen Recognition and Innate Immunity. Cell 124, 783–801 (2006).

    Google Scholar 

  10. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010).

    Google Scholar 

  11. Li, D. & Wu, M. Pattern recognition receptors in health and diseases. Signal Transduct. Target. Ther. 6, 291 (2021).

    Google Scholar 

  12. Zhao, T. et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct. Target. Ther. 8, 283 (2023).

    Google Scholar 

  13. Guo, Q. et al. NF-κB in biology and targeted therapy: new insights and translational implications. Signal Transduct. Target. Ther. 9, 53 (2024).

    Google Scholar 

  14. P.ulendran, B., P., S. A. & O’Hagan, D. T. Emerging concepts in the science of vaccine adjuvants. Nat. Rev. Drug Discov. 20, 454–475 (2021).

  15. Sinani, G. & Şenel, S. Advances in vaccine adjuvant development and future perspectives. Drug Deliv. 32, 2517137 (2025).

    Google Scholar 

  16. Kool, M. et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J. Exp. Med. 205, 869–882 (2008).

    Google Scholar 

  17. McKee, A. S. et al. Alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity. J. Immunol. 183, 4403–4414 (2009).

    Google Scholar 

  18. Kong, W., Brovold, M., Koeneman, B. A., Clark-Curtiss, J. & Curtiss, R. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform. Proc. Natl. Acad. Sci. 109, 19414–19419 (2012).

    Google Scholar 

  19. Swain, B., Campodonico, V. A. & Curtiss, R., 3rd. Recombinant Attenuated Edwardsiella piscicida Vaccine Displaying Regulated Lysis to Confer Biological Containment and Protect Catfish against Edwardsiellosis. Vaccines 11, 1470 (2023).

  20. Wang, S., Kong, Q. & Curtiss, R. New technologies in developing recombinant attenuated Salmonella vaccine vectors. Microb. Pathog. 58, 17–28 (2013).

    Google Scholar 

  21. Swain, B., Powell, C. T. & Curtiss, R. 3rd. Construction and evaluation of recombinant attenuated edwardsiella piscicida vaccine (RAEV) vector system encoding ichthyophthirius multifiliis (Ich) antigen IAG52B. Front. Immunol. 12, 802760 (2021).

    Google Scholar 

  22. Wei, Y. et al. Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans. Int. J. Oral. Sci. 8, 231–238 (2016).

    Google Scholar 

  23. Xin, W. et al. The Asd+-DadB+ dual-plasmid system offers a novel means to deliver multiple protective antigens by a recombinant attenuated salmonella vaccine. Infect. Immun. 80, 3621–3633 (2012).

    Google Scholar 

  24. Kong, Q. et al. Salmonella synthesizing 1-monophosphorylated lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J. Immunol. 187, 412–423 (2011).

    Google Scholar 

  25. Kawasaki, K., Ernst, R. K. & Miller, S. I. 3-O-deacylation of lipid A by PagL, a PhoP/PhoQ-regulated deacylase of Salmonella typhimurium, modulates signaling through Toll-like receptor 4. J. Biol. Chem. 279, 20044–20048 (2004).

    Google Scholar 

  26. Gunn, J. S. et al. PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance. Mol. Microbiol. 27, 1171–1182 (1998).

    Google Scholar 

  27. Needham, B. D. & Trent, M. S. Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis. Nat. Rev. Microbiol. 11, 467–481 (2013).

    Google Scholar 

  28. Kong, Q. et al. Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity. Infect. Immun. 80, 3215–3224 (2012).

    Google Scholar 

  29. Cunningham, A. F. et al. Responses to the soluble flagellar protein FliC are Th2, while those to FliC on Salmonella are Th1. Eur. J. Immunol. 34, 2986–2995 (2004).

    Google Scholar 

  30. Macnab, R. M. Genetics and biogenesis of bacterial flagella. Annu. Rev. Genet. 26, 131–158 (1992).

    Google Scholar 

  31. Silverman, M. & Simon, M. Phase variation: genetic analysis of switching mutants. Cell 19, 845–854 (1980).

    Google Scholar 

  32. Beuzón, C. R. et al. Salmonella maintains the integrity of its intracellular vacuole through the action of SifA. EMBO J. 19, 3235–3249 (2000).

    Google Scholar 

  33. Brawn, L. C., Hayward, R. D. & Koronakis, V. Salmonella SPI1 effector SipA persists after entry and cooperates with a SPI2 effector to regulate phagosome maturation and intracellular replication. Cell Host Microbe 1, 63–75 (2007).

    Google Scholar 

  34. Raetz, C. R. H. & Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 (2002).

    Google Scholar 

  35. Hur, J., Lee, J. & John, H. Enhancement of immune responses by an attenuated salmonella enterica serovar typhimurium strain secreting an Escherichia coli heat-labile enterotoxin b subunit protein as an adjuvant for a live salmonella vaccine candidate. Clin. Vaccin. Immunol. 18, 203–209 (2011).

    Google Scholar 

  36. Galen, J. E., Wahid, R. & Buskirk, A. D. Strategies for enhancement of live-attenuated salmonella-based carrier vaccine immunogenicity. Vaccines 9, 162 (2021).

    Google Scholar 

  37. Sirard, J. C., Niedergang, F. & Kraehenbuhl, J. P. Live attenuated Salmonella: a paradigm of mucosal vaccines. Immunol. Rev. 171, 5–26 (1999).

    Google Scholar 

  38. Kong, Q. et al. Effect of deletion of genes involved in lipopolysaccharide core and O-antigen synthesis on virulence and immunogenicity of salmonella enterica serovar typhimurium. Infect. Immun. 79, 4227–4239 (2011).

    Google Scholar 

  39. Hassan, J. O. & Curtiss, R. Virulent Salmonella typhimurium-induced lymphocyte depletion and immunosuppression in chickens. Infect. Immun. 62, 2027–2036 (1994).

    Google Scholar 

  40. Kennedy, M. J. et al. Attenuation and immunogenicity of Δcya Δcrp derivatives of salmonella choleraesuis in pigs. Infect. Immun. 67, 4628–4636 (1999).

    Google Scholar 

  41. Kong, W. et al. Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment. Proc. Natl. Acad. Sci. USA 105, 9361–9366 (2008).

    Google Scholar 

  42. Kong, W. et al. Mucosal delivery of a self-destructing salmonella-based vaccine inducing immunity against eimeria. Avian Dis. 64, 254–268 (2020).

    Google Scholar 

  43. Wang, S. et al. A triple-sugar regulated Salmonella vaccine protects against Clostridium perfringens-induced necrotic enteritis in broiler chickens. Poult. Sci. 101, 101592 (2022).

    Google Scholar 

  44. Matsuda, S., Haneda, T., Saito, H., Miki, T. & Okada, N. Salmonella enterica Effectors SifA, SpvB, SseF, SseJ, and SteA contribute to type III secretion system 1-independent inflammation in a streptomycin-pretreated mouse model of colitis. Infect. Immun. 87, https://doi.org/10.1128/iai.00872-18 (2019).

  45. Yang, L. et al. Deficiency in the msbB gene reduced the salmonella typhimurium virulence through mechanisms beyond LPS modification. Microorganisms 13, 2510 (2025).

    Google Scholar 

  46. Stepien, T. A. et al. Nuclear factor kappa B-dependent persistence of Salmonella Typhi and Paratyphi in human macrophages. mBio 15, e0045424 (2024).

    Google Scholar 

  47. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449, 819–826 (2007).

    Google Scholar 

  48. Graham, S. V. Human papillomavirus: gene expression, regulation and prospects for novel diagnostic methods and antiviral therapies. Future Microbiol. 5, 1493–1506 (2010).

    Google Scholar 

  49. Hajam, I. A., Dar, P. A., Shahnawaz, I., Jaume, J. C. & Lee, J. H. Bacterial flagellin—a potent immunomodulatory agent. Exp. Mol. Med. 49, e373–e373 (2017).

    Google Scholar 

  50. Cui, B. et al. Flagellin as a vaccine adjuvant. Expert Rev. Vaccines 17, 335–349 (2018).

    Google Scholar 

  51. Pasetti, M. F., Simon, J. K., Sztein, M. B. & Levine, M. M. Immunology of gut mucosal vaccines. Immunol. Rev. 239, 125–148 (2011).

    Google Scholar 

  52. Holmgren, J. & Svennerholm, A.-M. Vaccines against mucosal infections. Curr. Opin. Immunol. 24, 343–353 (2012).

    Google Scholar 

  53. Iwasaki, A. & Medzhitov, R. Regulation of adaptive immunity by the innate immune system. Science 327, 291–295 (2010).

    Google Scholar 

  54. Moyle, P. M. & Toth, I. Modern subunit vaccines: development, components, and research opportunities. ChemMedChem 8, 360–376 (2013).

    Google Scholar 

  55. Coffman, R. L., Sher, A. & Seder, R. A. Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492–503 (2010).

    Google Scholar 

  56. Rizzo, C. et al. Cross-reactive antibody responses to the 2009 A/H1N1v influenza virus in the Italian population in the pre-pandemic period. Vaccine 28, 3558–3562 (2010).

    Google Scholar 

  57. Zhu, Q. et al. Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: Implications for vaccines. Proc. Natl. Acad. Sci. 105, 16260–16265 (2008).

    Google Scholar 

  58. In brief. Nat. Rev. Immunol. 10, 677–677 (2010).

  59. Kasturi, S. P. et al. Programming the magnitude and persistence of antibody responses with innate immunity. Nature 470, 543–547 (2011).

    Google Scholar 

  60. Philpott, D. J., Sorbara, M. T., Robertson, S. J., Croitoru, K. & Girardin, S. E. NOD proteins: regulators of inflammation in health and disease. Nat. Rev. Immunol. 14, 9–23 (2014).

    Google Scholar 

  61. Reed, S. G., Orr, M. T. & Fox, C. B. Key roles of adjuvants in modern vaccines. Nat. Med. 19, 1597–1608 (2013).

    Google Scholar 

  62. Chen, J. et al. Trained immunity modulators: a new frontier in immunoregulation and disease intervention. J. Adv. Res. https://doi.org/10.1016/j.jare.2025.09.029 (2025).

  63. Netea, M. G. et al. Trained immunity: aprogram of innate immune memory in health and disease. Science 352, aaf1098 (2016).

    Google Scholar 

  64. Moliva, J. I., Turner, J. & Torrelles, J. B. Immune responses to bacillus calmette-guérin vaccination: why do they fail to protect against Mycobacterium tuberculosis?. Front. Immunol. 8, 407 (2017).

    Google Scholar 

  65. Chen, J. et al. BCG-induced trained immunity: history, mechanisms and potential applications. J. Transl. Med. 21, 106 (2023).

    Google Scholar 

  66. Sallusto, F., Lenig, D., Förster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999).

    Google Scholar 

  67. Mosmann, T. R. & Sad, S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. today 17, 138–146 (1996).

    Google Scholar 

  68. Du, J. & Sun, H. Co-expression network analysis identifies innate immune signatures for Albizia julibrissin saponin active fraction-adjuvanted avian influenza vaccine. Int. Immunopharmacol. 93, 107417 (2021).

    Google Scholar 

  69. Kazmin, D. et al. Memory-like innate response to booster vaccination with MF-59 adjuvanted influenza vaccine in children. npj Vaccines 8, 100 (2023).

    Google Scholar 

  70. Zhou, R., Sun, K., Xie, X., Yin, F. & Galindo-Villegas, J. Integrated transcriptomic and immune enzymatic analyses uncover coordinated immunometabolic responses in large yellow croaker (Larimichthys crocea) to Metanophrys sp. infection. Front. Immunol. ume 16, 2025 (2025).

    Google Scholar 

  71. Gram, A. M. et al. Salmonella flagellin activates NAIP/NLRC4 and canonical NLRP3 inflammasomes in human macrophages. J. Immunol. 206, 631–640 (2021).

    Google Scholar 

  72. Li, Y. et al. Salmonella-NLRP3 inflammasome crosstalk: host defense activation versus bacterial immune evasion strategies. J. Inflamm. Res. 18, 5133–5148 (2025).

    Google Scholar 

  73. Maisonneuve, C., Bertholet, S., Philpott, D. J. & De Gregorio, E. Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants. Proc. Natl. Acad. Sci. USA 111, 12294–12299 (2014).

    Google Scholar 

  74. Caruso, R., Warner, N., Inohara, N. & Núñez, G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity 41, 898–908 (2014).

    Google Scholar 

  75. Swain, B. & Miryala, K. R. NOD-like receptors in fish: evolution, structure, immune signaling, and targeting for aquaculture vaccine adjuvants. Front. Immunol. ume 16, 2025 (2025).

    Google Scholar 

  76. Curtiss, R. 3rd & Hassan, J. O. Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet. Immunol. Immunopathol. 54, 365–372 (1996).

    Google Scholar 

  77. Swain, B., Powell, C. T. & Curtiss, R. Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish. Fish. Shellfish Immunol. 107, 497–510 (2020).

    Google Scholar 

  78. Swain, B., Powell, C. T. & Curtiss, R. 3rd. Virulence, immunogenicity and live vaccine potential of aroA and phoP mutants of Edwardsiella piscicida in zebrafish. Micro Pathog. 162, 105355 (2022).

    Google Scholar 

  79. Roland, K., Curtiss, R. 3rd & Sizemore, D. Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis. 43, 429–441 (1999).

    Google Scholar 

  80. Swain, B., Campodonico, V. A. & Curtiss, R. Recombinant attenuated edwardsiella piscicida vaccine displaying regulated lysis to confer biological containment and protect catfish against edwardsiellosis. Vaccines 11, 1470 (2023).

    Google Scholar 

  81. Hitchcock, P. J. & Brown, T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269–277 (1983).

    Google Scholar 

  82. Swain, B., Powell, C. T. & Curtiss, R. Virulence, immunogenicity and live vaccine potential of aroA and phoP mutants of Edwardsiella piscicida in zebrafish. Microb. Pathog. 162, 105355 (2022).

    Google Scholar 

  83. Nakayama, K., Kelly, S. M. & Curtiss III, R. Construction of an Asd+ expression-cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. BioTechnol. 6, 693–697 (1988).

    Google Scholar 

  84. Galan, J. E., Nakayama, K. & Curtiss, R., 3rd. Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene 94, 29–35 (1990).

  85. Xin, W. et al. The Asd(+)-DadB(+) dual-plasmid system offers a novel means to deliver multiple protective antigens by a recombinant attenuated Salmonella vaccine. Infect. Immun. 80, 3621–3633 (2012).

    Google Scholar 

  86. Wasserman, S. A., Walsh, C. T. & Botstein, D. Two alanine racemase genes in Salmonella typhimurium that differ in structure and function. J. Bacteriol. 153, 1439–1450 (1983).

    Google Scholar 

  87. Nempont, C. et al. Deletion of flagellin’s hypervariable region abrogates antibody-mediated neutralization and systemic activation of TLR5-dependent immunity. J. Immunol. 181, 2036–2043 (2008).

    Google Scholar 

  88. Wang, X., Karbarz, M. J., McGrath, S. C., Cotter, R. J. & Raetz, C. R. MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of Francisella novicida LpxE expressed in Escherichia coli. J. Biol. Chem. 279, 49470–49478 (2004).

    Google Scholar 

  89. Raetz, C. R., Reynolds, C. M., Trent, M. S. & Bishop, R. E. Lipid A modification systems in gram-negative bacteria. Annu. Rev. Biochem. 76, 295–329 (2007).

    Google Scholar 

  90. Bonifield, H. R. & Hughes, K. T. Flagellar phase variation in Salmonella enterica is mediated by a posttranscriptional control mechanism. J. Bacteriol. 185, 3567–3574 (2003).

    Google Scholar 

  91. Kong, Q. et al. Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant. Infect. Immun. 79, 5027–5038 (2011).

    Google Scholar 

  92. Mullally, C. et al. Novel small molecules that increase the susceptibility of Neisseria gonorrhoeae to cationic antimicrobial peptides by inhibiting lipid A phosphoethanolamine transferase. J. Antimicrob. Chemother. 77, 2441–2447 (2022).

    Google Scholar 

  93. Ohlson, M. B. et al. Structure and function of Salmonella SifA indicate that its interactions with SKIP, SseJ, and RhoA family GTPases induce endosomal tubulation. Cell Host Microbe 4, 434–446 (2008).

    Google Scholar 

  94. Kong, Q., Liu, Q., Jansen, A. M. & Curtiss, R. 3rd. Regulated delayed expression of rfc enhances the immunogenicity and protective efficacy of a heterologous antigen delivered by live attenuated Salmonella enterica vaccines. Vaccine 28, 6094–6103 (2010).

    Google Scholar 

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