Reinvestigating the Coughing Rat Model of Pertussis To Understand Bordetella pertussis Pathogenesis.

Bordetella pertussis is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP, DTaP, and Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the reemergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. The emergence of genetically diverging strains, combined with waning aP vaccine efficacy, calls for reevaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged 7-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of B. pertussis infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole-body plethysmography. Onset of cough occurred between 2 and 4 days after B. pertussis challenge, averaging five coughs per 15 min, with peak coughing occurring at day 8 postinfection, averaging upward of 13 coughs per 15 min. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days postchallenge. The rats exhibited increased bronchial restriction following B. pertussis infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-B. pertussis IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between B. pertussis strains.

Intranasal Immunization with Acellular Pertussis Vaccines Results in Long-Term Immunity to Bordetella pertussis in Mice.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

Innate and Adaptive Immune Responses against Bordetella pertussis and Pseudomonas aeruginosa in a Murine Model of Mucosal Vaccination against Respiratory Infection.

Whole cell vaccines are frequently the first generation of vaccines tested for pathogens and can inform the design of subsequent acellular or subunit vaccines. For respiratory pathogens, administration of vaccines at the mucosal surface can facilitate the generation of a localized mucosal immune response. Here, we examined the innate and vaccine-induced immune responses to infection by two respiratory pathogens: Bordetella pertussis and Pseudomonas aeruginosa. In a model of intranasal administration of whole cell vaccines (WCVs) with the adjuvant curdlan, we examined local and systemic immune responses following infection. These studies showed that intranasal vaccination with a WCV led to a reduction of the bacterial burden in the airways of animals infected with the respective pathogen. However, there were unique changes in the cytokines produced, cells recruited, and inflammation at the site of infection. Both mucosal vaccinations induced antibodies that bind the target pathogen, but linear regression and principal component analysis revealed that protection from these pathogens is not solely related to antibody titer. Protection from P. aeruginosa correlated to a reduction in lung weight, blood lymphocytes and neutrophils, and the cytokines IL-6, TNF-α, KC/GRO, and IL-10, and promotion of serum IgG antibodies and the cytokine IFN-γ in the lung. Protection from B. pertussis infection correlated strongly with increased anti-B-pertussis serum IgG antibodies. These findings reveal valuable correlates of protection for mucosal vaccination that can be used for further development of both B. pertussis and P. aeruginosa vaccines.

Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia.

Intranasal acellular pertussis vaccine provides mucosal immunity and protects mice from Bordetella pertussis.

Current acellular pertussis vaccines fall short of optimal protection against the human respiratory pathogen Bordetella pertussis resulting in increased incidence of a previously controlled vaccine- preventable disease. Natural infection is known to induce a protective mucosal immunity. Therefore, in this study, we aimed to use acellular pertussis vaccines to recapitulate these mucosal immune responses. We utilized a murine immunization and challenge model to characterize the efficacy of intranasal immunization (IN) with DTaP vaccine or DTaP vaccine supplemented with curdlan, a known Th1/Th17 promoting adjuvant. Protection from IN delivered DTaP was compared to protection mediated by intraperitoneal injection of DTaP and whole-cell pertussis vaccines. We tracked fluorescently labeled DTaP after immunization and detected that DTaP localized preferentially in the lungs while DTaP with curdlan was predominantly in the nasal turbinates. IN immunization with DTaP, with or without curdlan adjuvant, resulted in anti-B. pertussis and anti-pertussis toxin IgG titers at the same level as intraperitoneally administered DTaP. IN immunization was able to protect against B. pertussis challenge and we observed decreased pulmonary pro-inflammatory cytokines, neutrophil infiltrates in the lung, and bacterial burden in the upper and lower respiratory tract at day 3 post challenge. Furthermore, IN immunization with DTaP triggered mucosal immune responses such as production of B. pertussis-specific IgA, and increased IL-17A. Together, the induction of a mucosal immune response and humoral antibody-mediated protection associated with an IN administered DTaP and curdlan adjuvant warrant further exploration as a pertussis vaccine candidate formulation.

Analysis of the In Vivo Transcriptome of Bordetella pertussis during Infection of Mice.

Bordetella pertussis causes the disease whooping cough through coordinated control of virulence factors by the Bordetella virulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describe in vitro gene expression profiles of B. pertussis and other pathogens. In previous studies, we have analyzed the in vitro gene expression profiles of B. pertussis, and we hypothesize that the infection transcriptome profile in vivo is significantly different from that under laboratory growth conditions. To study the infection transcriptome of B. pertussis, we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-µm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing the in vitro and in vivo gene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical for B. pertussis survival in vivo Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile of B. pertussis during infection, and this method will facilitate efforts to understand how this pathogen causes infection.IMPORTANCEIn vitro growth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the "infection transcriptome" of B. pertussis in the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection.

Bordetella pertussis Whole Cell Immunization, Unlike Acellular Immunization, Mimics Naïve Infection by Driving Hematopoietic Stem and Progenitor Cell Expansion in Mice.

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis (Bp), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220+ cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naïve infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response.

Evaluation of Adenylate Cyclase Toxoid Antigen in Acellular Pertussis Vaccines by Using a Bordetella pertussis Challenge Model in Mice.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions.

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.