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.

• Keywords
• Bordetella pertussis, dermonecrotic toxin, immunogenicity, lipooligosaccharide, pertussis toxin, protection, reactogenicity, whole-cell vaccine, whooping cough,
• MeSH
• Antigens, Bacterial MeSH
• Bordetella pertussis * immunology   genetics   MeSH
• Virulence Factors, Bordetella genetics   immunology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Whooping Cough * prevention & control   immunology   MeSH
• Pertussis Vaccine * immunology   genetics   administration & dosage   MeSH
• Fimbriae Proteins genetics   immunology   MeSH
• Antibodies, Bacterial blood   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antigens, Bacterial MeSH
• Virulence Factors, Bordetella MeSH
• fim2 protein, Bordetella MeSH Browser
• fim3 protein, Bordetella MeSH Browser
• Pertussis Vaccine * MeSH
• Fimbriae Proteins MeSH
• Antibodies, Bacterial MeSH

Bordetella pertussis infects human upper airways and deploys an array of immunosuppressive virulence factors, among which the adenylate cyclase toxin (CyaA) plays a prominent role in disarming host phagocytes. CyaA binds the complement receptor-3 (CR3 aka αMβ2 integrin CD11b/CD18 or Mac-1) of myeloid cells and delivers into their cytosol an adenylyl cyclase enzyme that hijacks cellular signaling through unregulated conversion of cytosolic ATP to cAMP. We found that the action of as little CyaA as 22 pM (4 ng/mL) blocks macrophage colony-stimulating factor (M-CSF)-driven transition of migratory human CD14+ monocytes into macrophages. Global transcriptional profiling (RNAseq) revealed that exposure of monocytes to 22 pM CyaA for 40 hours in culture with 20 ng/mL of M-CSF led to upregulation of genes that exert negative control of monocyte to macrophage differentiation (e.g., SERPINB2, DLL1, and CSNK1E). The sustained CyaA action yielded downregulation of numerous genes involved in processes crucial for host defense, such as myeloid cell differentiation, chemotaxis of inflammatory cells, antigen presentation, phagocytosis, and bactericidal activities. CyaA-elicited signaling also promoted deacetylation and trimethylation of lysines 9 and 27 of histone 3 (H3K9me3 and H3K27me3) and triggered the formation of transcriptionally repressive heterochromatin patches in the nuclei of CyaA-exposed monocytes. These effects were partly reversed by the G9a methyltransferase inhibitor UNC 0631 and by the pleiotropic HDAC inhibitor Trichostatin-A, revealing that CyaA-elicited epigenetic alterations mediate transcriptional reprogramming of monocytes and play a role in CyaA-triggered block of monocyte differentiation into bactericidal macrophage cells.IMPORTANCETo proliferate on host airway mucosa and evade elimination by patrolling sentinel cells, the whooping cough agent Bordetella pertussis produces a potently immunosubversive adenylate cyclase toxin (CyaA) that blocks opsonophagocytic killing of bacteria by phagocytes like neutrophils and macrophages. Indeed, chemotactic migration of CD14+ monocytes to the infection site and their transition into bactericidal macrophages, thus replenishing the exhausted mucosa-patrolling macrophages, represents one of the key mechanisms of innate immune defense to infection. We show that the cAMP signaling action of CyaA already at a very low toxin concentration triggers massive transcriptional reprogramming of monocytes that is accompanied by chromatin remodeling and epigenetic histone modifications, which block the transition of migratory monocytes into bactericidal macrophage cells. This reveals a novel layer of toxin action-mediated hijacking of functional differentiation of innate immune cells for the sake of mucosal pathogen proliferation and transmission to new hosts.

• Keywords
• Bordetella pertussis, RTX toxins, cyclic AMP, differentiation, epigenetics, macrophages, monocytes,
• MeSH
• Adenylate Cyclase Toxin * metabolism   MeSH
• Bordetella pertussis * pathogenicity   enzymology   MeSH
• Cell Differentiation * drug effects   MeSH
• Macrophage Colony-Stimulating Factor MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Macrophages * drug effects   cytology   MeSH
• Monocytes * drug effects   cytology   physiology   MeSH
• Cellular Reprogramming * MeSH
• Chromatin Assembly and Disassembly * drug effects   MeSH
• Signal Transduction MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenylate Cyclase Toxin * MeSH
• Macrophage Colony-Stimulating Factor 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.

• Keywords
• Bordetella pertussis, Catarrhal stage, Mouse, Shedding, TLR4 receptor,
• MeSH
• Bordetella pertussis MeSH
• Respiratory Tract Infections * MeSH
• Mice MeSH
• Whooping Cough * microbiology   prevention & control   MeSH
• Pertussis Vaccine MeSH
• Lung microbiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Pertussis Vaccine MeSH