UNLABELLED: The adenylate cyclase toxin (ACT, AC-Hly, or CyaA) plays a key role in airway infections by Bordetella pertussis and ablates the oxidative burst and opsonophagocytic capacity of sentinel phagocytes. CyaA fragments eliciting toxin-neutralizing antibodies are considered prime antigen candidates for improved acellular pertussis (aP) vaccines but their contribution to aP-mediated protection against B. pertussis infection awaits demonstration. We explored whether hybrid antigens inducing simultaneously CyaA-neutralizing and anti-Prn opsonizing antibody responses can enhance aP-elicited protection of mouse airways from infection. Fusion to the N-terminus of an RTX908 antigen derived from CyaA enabled an accelerated folding of the pertactin passenger domain (rPrn) in function of calcium loading of the RTX908 moiety and conferred on the rPrn-RTX908 fusion antigen a superior capacity to induce functional anti-Prn IgG antibodies. The rPrn-RTX908 fusion antigen also elicited CyaA neutralizing anti-RTX antibodies that relieved the toxin-imposed inhibition of oxidative burst and opsonophagocytic uptake of B. pertussis bacteria by HL-60 cells exposed to physiological concentrations of the CyaA toxin. Intranasal immunization of mice with the rPrn-RTX908 antigen admixed into a PT and FHA-based aP vaccine elicited specific sIgA responses in mucosal secretions (saliva) and conferred a significantly enhanced protection of mouse lung and nose mucosa against B. pertussis infection, yielding a significantly accelerated clearance of bacteria from the infected lungs within a single day from infection. These results demonstrate the added value of anti-CyaA antibodies elicited by intranasal application of the rPrn-RTX908 fusion antigen in the protection of the airway against B. pertussis infection. IMPORTANCE: Despite high vaccine coverage, unexpectedly massive whooping cough outbreaks are currently resurging in the most developed countries using the acellular pertussis (aP) vaccine. Accelerated development of improved aP vaccines, conferring a more complete and longer-lasting protection of the airway from Bordetella pertussis infection, is sorely needed. The highly immunosuppressive RTX adenylate cyclase toxin (CyaA) was proposed as a prime antigen candidate for inclusion into improved aP vaccines. We show here that a soluble RTX-derived antigen fused to the major opsonizing antibody target pertactin (rPrn-RTX908 hybrid) elicits opsonizing and toxin-neutralizing antibody responses that relieve the CyaA-imposed block of bactericidal opsonophagocytic uptake capacities of sentinel phagocytes. Intranasal immunization with the rPrn-RTX908 hybrid antigen then enables a significantly accelerated clearance of B. pertussis bacteria from mouse lungs and superior protection of mouse nasal mucosa from bacterial infection. These results unravel the added value of RTX antigen inclusion into the next generation of aP vaccines.

• Klíčová slova
• Bordetella pertussis, adenylate cyclase toxin, pertactin, pertussis, protection, protein folding, whooping cough,
• MeSH
• adenylátcyklasový toxin * imunologie   genetika   aplikace a dávkování   MeSH
• antigeny bakteriální * imunologie   genetika   aplikace a dávkování   MeSH
• aplikace intranazální MeSH
• Bordetella pertussis * imunologie   genetika   MeSH
• faktory virulence rodu Bordetella * imunologie   genetika   aplikace a dávkování   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• neutralizující protilátky krev   imunologie   MeSH
• pertuse * prevence a kontrola   imunologie   mikrobiologie   MeSH
• pertusová vakcína * imunologie   aplikace a dávkování   genetika   MeSH
• proteiny vnější bakteriální membrány * imunologie   genetika   aplikace a dávkování   MeSH
• protilátky bakteriální krev   imunologie   MeSH
• rekombinantní fúzní proteiny imunologie   genetika   aplikace a dávkování   MeSH
• respirační sliznice * imunologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny bakteriální * MeSH
• faktory virulence rodu Bordetella * MeSH
• neutralizující protilátky MeSH
• pertactin MeSH Prohlížeč
• pertusová vakcína * MeSH
• proteiny vnější bakteriální membrány * MeSH
• protilátky bakteriální MeSH
• rekombinantní fúzní proteiny MeSH

Pertussis resurged over the last decade in most countries that replaced the traditional whole-cell pertussis vaccines (wP) by the less reactogenic acellular pertussis vaccines (aP). The aP vaccines induce a Th2-polarized immune response and by a yet unknown mechanism hamper the clearance of Bordetella pertussis from infected nasopharyngeal mucosa. The aP-induced pertussis toxin-neutralizing antibodies effectively prevent the life-threatening pertussis pneumonia in infants, but aP-elicited immunity fails to prevent infection of nasopharyngeal mucosa and transmission of B. pertussis. In contrast, the more reactogenic traditional wP vaccines, alike natural infection, elicit a broad antibody response and trigger a Th1/Th17-polarized T cell immunity. We tackled here the reactogenicity of the conventional wP vaccines by genetic modification of the Fim2 and Fim3-producing B. pertussis strains used for wP vaccine manufacturing. Mutations were introduced into the genomes of vaccine strains (i) to reduce the TLR4 signaling potency of the lipid A of B. pertussis lipooligosaccharide (ΔlgmB), (ii) eliminate the enzymatic (immunosuppressive) activity of the pertussis toxin (PtxS1-R9K/E129G), and (iii) ablate the production of the dermonecrotic toxin (Δdnt). Experimental alum-adjuvanted wP vaccines prepared from such triply modified bacteria exhibited a reduced pyrogenicity in rabbits and a reduced systemic toxicity in mice, while conferring a comparable protection from B. pertussis infection as the unmodified wP vaccine.IMPORTANCEThe occasionally severe adverse reactions associated with some lots of the whole-cell pertussis vaccine (wP) led the industrialized nations to switch to the use of less reactogenic acellular pertussis vaccines that confer shorter-lasting protection. This yielded whooping cough resurgence and large whooping cough outbreaks are currently sweeping throughout European countries, calling for the replacement of the pertussis vaccine component of pediatric hexavaccines by an improved wP vaccine. We show that genetic detoxification of the Bordetella pertussis bacteria used for wP preparation yields a reduced reactogenicity wP vaccine that exhibits a reduced systemic toxicity in mice and reduced pyrogenicity in rabbits, while retaining high immunogenicity and protective potency in the mouse model of pneumonic infection by B. pertussis. This result has now been confirmed in a nonhuman primate model of B. pertussis infection of olive baboons, paving the way for the development of the next generation of pertussis vaccines.

• Klíčová slova
• Bordetella pertussis, dermonecrotic toxin, immunogenicity, lipooligosaccharide, pertussis toxin, protection, reactogenicity, whole-cell vaccine, whooping cough,
• MeSH
• Bordetella pertussis * imunologie   genetika   MeSH
• faktory virulence rodu Bordetella genetika   imunologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• pertuse * prevence a kontrola   imunologie   MeSH
• pertusová vakcína * imunologie   genetika   aplikace a dávkování   MeSH
• proteiny fimbrií genetika   imunologie   MeSH
• protilátky bakteriální krev   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny bakteriální MeSH
• faktory virulence rodu Bordetella MeSH
• fim2 protein, Bordetella MeSH Prohlížeč
• fim3 protein, Bordetella MeSH Prohlížeč
• pertusová vakcína * MeSH
• proteiny fimbrií MeSH
• protilátky bakteriální MeSH

The adenylate cyclase (ACT) and the pertussis (PT) toxins of Bordetella pertussis exert potent immunomodulatory activities that synergize to suppress host defense in the course of whooping cough pathogenesis. We compared the mouse lung infection capacities of B. pertussis (Bp) mutants (Bp AC- or Bp PT-) producing enzymatically inactive toxoids and confirm that ACT action is required for maximal bacterial proliferation in the first days of infection, whereas PT action is crucial for persistence of B. pertussis in mouse lungs. Despite accelerated and near complete clearance from the lungs by day 14 of infection, the PT- bacteria accumulated within the lymphoid tissue of lung-draining mediastinal lymph nodes (mLNs). In contrast, the wild type or AC- bacteria colonized the lungs but did not enter into mLNs. Lung infection by the PT- mutant triggered an early arrival of migratory conventional dendritic cells with associated bacteria into mLNs, where the PT- bacteria entered the T cell-rich paracortex of mLNs by day 5 and proliferated in clusters within the B-cell zone (cortex) of mLNs by day 14, being eventually phagocytosed by infiltrating neutrophils. Finally, only infection by the PT- bacteria triggered an early production of anti-B. pertussis serum IgG antibodies already within 14 days of infection. These results reveal that action of the pertussis toxin blocks DC-mediated delivery of B. pertussis bacteria into mLNs and prevents bacterial colonization of mLNs, thus hampering early adaptive immune response to B. pertussis infection.

• MeSH
• Bordetella pertussis * MeSH
• dendritické buňky patologie   MeSH
• lymfatické uzliny patologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• pertuse * MeSH
• pertusový toxin MeSH
• plíce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pertusový toxin MeSH

Pertussis (whooping cough) is a highly transmissible human respiratory disease caused by Bordetella pertussis, a human-restricted pathogen. Animal models generally involve pneumonic infections induced by depositing large numbers of bacteria in the lungs of mice. These models have informed us about the molecular pathogenesis of pertussis and guided development of vaccines that successfully protect against severe disease. However, they bypass the catarrhal stage of the disease, when bacteria first colonize and initially grow in the upper respiratory tract. This is a critical and highly transmissible stage of the infection that current vaccines do not prevent. Here, we demonstrate a model system in which B. pertussis robustly and persistently infects the nasopharynx of TLR4-deficient mice, inducing localized inflammation, neutrophil recruitment and mucus production as well as persistent shedding and occasional transmission to cage mates. This novel experimental system will allow the study of the contributions of bacterial factors to colonization of and shedding from the nasopharynx, as occurs during the catarrhal stage of pertussis, and interventions that might better control the ongoing circulation of pertussis.

• Klíčová slova
• Bordetella pertussis, Catarrhal stage, Mouse, Shedding, TLR4 receptor,
• MeSH
• Bordetella pertussis MeSH
• infekce dýchací soustavy * MeSH
• myši MeSH
• pertuse * mikrobiologie   prevence a kontrola   MeSH
• pertusová vakcína MeSH
• plíce mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• pertusová vakcína MeSH

Pulmonary infections caused by Bordetella pertussis used to be the prime cause of infant mortality in the pre-vaccine era and mouse models of pertussis pneumonia served in characterization of B. pertussis virulence mechanisms. However, the biologically most relevant catarrhal disease stage and B. pertussis transmission has not been adequately reproduced in adult mice due to limited proliferation of the human-adapted pathogen on murine nasopharyngeal mucosa. We used immunodeficient C57BL/6J MyD88 KO mice to achieve B. pertussis proliferation to human-like high counts of 108 viable bacteria per nasal cavity to elicit rhinosinusitis accompanied by robust shedding and transmission of B. pertussis bacteria to adult co-housed MyD88 KO mice. Experiments with a comprehensive set of B. pertussis mutants revealed that pertussis toxin, adenylate cyclase toxin-hemolysin, the T3SS effector BteA/BopC and several other known virulence factors were dispensable for nasal cavity infection and B. pertussis transmission in the immunocompromised MyD88 KO mice. In contrast, mutants lacking the filamentous hemagglutinin (FhaB) or fimbriae (Fim) adhesins infected the nasal cavity poorly, shed at low levels and failed to productively infect co-housed MyD88 KO or C57BL/6J mice. FhaB and fimbriae thus appear to play a critical role in B. pertussis transmission. The here-described novel murine model of B. pertussis-induced nasal catarrh opens the way to genetic dissection of host mechanisms involved in B. pertussis shedding and to validation of key bacterial transmission factors that ought to be targeted by future pertussis vaccines.

• MeSH
• adenylátcyklasový toxin MeSH
• bakteriální adheziny * metabolismus   MeSH
• Bordetella pertussis * genetika   MeSH
• faktory virulence rodu Bordetella genetika   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myeloidní diferenciační faktor 88 MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nosní dutina mikrobiologie   MeSH
• pertuse * přenos   MeSH
• pertusová vakcína MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• bakteriální adheziny * MeSH
• faktory virulence rodu Bordetella MeSH
• myeloidní diferenciační faktor 88 MeSH
• pertusová vakcína MeSH