biostudies-literature00470Stefanetti GEuropean Union&apos;s Horizon 2020 Research and Innovation Programme under Marie Skłodowska Curie Grant AgreementNIAID, NIHNIAID NIH HHS7062-7070https://www.ebi.ac.uk/biostudies/studies/S-EPMC6452683biostudies-literatureUnknown116(14)<i>Francisella tularensis</i> is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of <i>F. tularensis</i> has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When <i>F. tularensis</i> OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an <i>F. tularensis</i> live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing <i>in F. tularensis</i> a heterologous chain-length regulator gene (<i>wzz</i>) from the related species <i>Francisella novicida</i> Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism.Proceedings of the National Academy of Sciences of the United States of AmericaGlycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to <i>Francisella tularensis</i>.PMC64526835R01AI089915661138R01 AI0899155U19AI109764U19 AI109764Gardner EStefanetti GKasper DLOkan NFink A47falseGlycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to <i>Francisella tularensis</i>.<i>Francisella tularensis</i> is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of <i>F. tularensis</i> has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When <i>F. tularensis</i> OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an <i>F. tularensis</i> live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing <i>in F. tularensis</i> a heterologous chain-length regulator gene (<i>wzz</i>) from the related species <i>Francisella novicida</i> Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism.2019-01-01T00:00:00Z2019 Apr2024-02-14T20:13:44.614Z2019-08-13T07:01:42ZS-EPMC64526833087247110.1073/pnas.1900144116