Atypical pneumonia testing in transplant recipients.

BACKGROUND: The incidence of atypical pneumonia among immunocompromised patients is not well characterized. Establishing a diagnosis of atypical pneumonia is challenging as positive tests must be carefully interpreted. We aimed to assess the test positivity rate and incidence of atypical pneumonia in transplant recipients. METHODS: A retrospective cohort study was conducted at the Yale New Haven Health System in Connecticut. Adults with solid organ transplant, hematopoietic stem cell transplant (HSCT), or chimeric antigen receptor T-cell, who underwent testing for atypical pathogens of pneumonia (Legionella pneumophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella pertussis) between January 2016 and August 2022 were included. Positive results were adjudicated in a clinical context using pre-defined criteria. A cost analysis of diagnostic testing was performed. RESULTS: Note that, 1021 unique tests for atypical pathogens of pneumonia were performed among 481 transplant recipients. The testing positivity rate was 0.7% (n = 7). After clinical adjudication, there were three cases of proven Legionella and one case of possible Mycoplasma infection. All cases of legionellosis were in transplant recipients within 1-year post-transplantation with recently augmented immunosuppression and lymphopenia. The possible case of Mycoplasma infection was in an HSCT recipient with augmented immunosuppression. The cost of all tests ordered was $50,797.73. CONCLUSION: The positivity rate of tests for atypical pneumonia was very low in this transplant cohort. An algorithmic approach that targets testing for those with compatible host, clinical, radiographic, and epidemiologic factors, and provides guidance on test selection and test interpretation, may improve the diagnostic yield and lead to substantial cost savings.

Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) Contributes to Bordetella pertussis Inflammatory Pathology.

Whooping cough (pertussis) is a severe pulmonary infectious disease caused by the bacteria Bordetella pertussis. Pertussis infects an estimated 24 million people annually, resulting in >150,000 deaths. The NIH placed pertussis on the list of emerging pathogens in 2015. Antibiotics are ineffective unless administered before the onset of the disease characteristic cough. Therefore, there is an urgent need for novel pertussis therapeutics. We have shown that sphingosine-1-phosphate receptor (S1PR) agonists reduce pertussis inflammation without increasing bacterial burden. Transcriptomic studies were performed to identify this mechanism and allow for the development of pertussis therapeutics that specifically target problematic inflammation without sacrificing bacterial control. These data suggested a role for triggering receptor expressed on myeloid cells-1 (TREM-1). TREM-1 cell surface receptor functions as an amplifier of inflammatory responses. Expression of TREM-1 is increased in response to bacterial infection of mucosal surfaces. In mice, B. pertussis infection results in Toll-like receptor 9 (TLR9)-dependent increased expression of TREM-1 and its associated cytokines. Interestingly, S1PR agonists dampen pulmonary inflammation and TREM-1 expression. Mice challenged intranasally with B. pertussis and treated with ligand-dependent (LP17) and ligand-independent (GF9) TREM-1 inhibitors showed no differences in bacterial burden and significantly reduced tumor necrosis factor-α (TNF-α) and C-C motif chemokine ligand 2 (CCL-2) expression compared to controls. Mice receiving TREM-1 inhibitors showed reduced pulmonary inflammation compared to controls, indicating that TREM-1 promotes inflammatory pathology, but not bacterial control, during pertussis infection. This implicates TREM-1 as a potential therapeutic target for the treatment of pertussis.

Does Bordetella pertussis vaccine offer any cross-protection against Bordetella bronchiseptica? Implications for pet owners with cystic fibrosis.

WHAT IS KNOWN AND OBJECTIVE: The Gram-negative bacterium, Bordetella bronchiseptica, causes lower airway respiratory disease in people with cystic fibrosis (CF), as well as in companion animals, especially dogs. Presently, there are several acellular vaccines available for B. pertussis but no vaccine available for B. bronchiseptica. However given the shared protein homology between these two closely related species, we wished to explore whether pertussis vaccines may offer some cross-protection against B. bronchiseptica. COMMENT: Bordetella pertussis and B. bronchiseptica are closely related phylogenetically, as well as sharing protein homology in several pertussis vaccine components, including (i) pertussis toxin (PT), (ii) filamentous haemagglutinin (FHA), (iii) pertactin and (iv) fimbriae (types 2 and 3). Given that pertussis vaccine contains cross-reactive antigens with B. bronchiseptica, licensed pertussis vaccines may therefore offer cross-protection against B. bronchiseptica. WHAT IS NEW AND CONCLUSION: Cystic fibrosis pet owners should ensure that they have an up-to-date vaccination record relating to their pertussis vaccine. Although no monovalent human pertussis vaccines are currently available, licensed non-live booster vaccines for B. pertussis are available for individuals in the age range >10 years old. People with CF should ensure that they are adequately and currently protected against pertussis, to avoid whooping cough, which may also offer some cross-protection against B. bronchiseptica and therefore help further mitigate the risk of zoonotic infection of this organism from pets to their owners.

Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis.

Outer Membrane Vesicles (OMVs) derived from different Gram-negative bacteria have been proposed as an attractive vaccine platform because of their own immunogenic adjuvant properties. Pertussis or whooping cough is a highly contagious vaccine-preventable respiratory disease that resurged during the last decades in many countries. In response to the epidemiological situation, new boosters have been incorporated into vaccination schedules worldwide and new vaccine candidates have started to be designed. Particularly, our group designed a new pertussis vaccine candidate based on OMVs derived from Bordetella pertussis (BpOMVs). To continue with the characterization of the immune response induced by our OMV based vaccine candidate, this work aimed to investigate the ability of OMVs to activate the inflammasome pathway in macrophages. We observed that NLRP3, caspase-1/11, and gasdermin-D (GSDMD) are involved in inflammasome activation by BpOMVs. Moreover, we demonstrated that BpOMVs as well as transfected B. pertussis lipooligosaccharide (BpLOS) induce caspase-11 (Casp11) and guanylate-binding proteins (GBPs) dependent non-canonical inflammasome activation. Our results elucidate the mechanism by which BpOMVs trigger one central pathway of the innate response activation that is expected to skew the adaptive immune response elicited by BpOMVs vaccination.

Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages.

Pertussis is a highly contagious respiratory infection caused by the bacterium Bordetella pertussis. Humans are the only known natural reservoir of B. pertussis. In mice, macrophages and NK cells have a key role in confining B. pertussis to the respiratory tract. However, the mechanisms underlying this process, particularly during human infections, remain unclear. Here we characterized the activation of human macrophages and NK cells in response to B. pertussis and unraveled the role of inflammasomes in this process. NLRP3 inflammasome activation by B. pertussis in human macrophage-like THP-1 cells and primary monocyte-derived macrophages (mo-MΦ) was shown by the visualization of ASC-speck formation, pyroptosis, and the secretion of caspase-mediated IL-1ß and IL-18. In contrast to macrophages, stimulation of human CD56+CD3- NK cells by B. pertussis alone did not result in activation of these cells. However, co-culture of B. pertussis-stimulated mo-MΦ and autologous NK cells resulted in high amounts of IFNγ secretion and an increased frequency of IL-2Rα+ and HLA-DR+ NK cells, indicating NK cell activation. This activation was significantly reduced upon inhibition of inflammasome activity or blocking of IL-18 in the mo-MΦ/NK cell co-culture. Furthermore, we observed increased secretion of proinflammatory cytokines in the B. pertussis-stimulated mo-MΦ/NK co-culture compared to the mo-MΦ single culture. Our results demonstrate that B. pertussis induces inflammasome activation in human macrophages and that the IL-18 produced by these cells is required for the activation of human NK cells, which in turn enhances the pro-inflammatory response to this pathogen. Our data provides a better understanding of the underlying mechanisms involved in the induction of innate immune responses against B. pertussis. These findings contribute to the knowledge required for the development of improved intervention strategies to control this highly contagious disease.

Serosurveillance for vaccine-preventable diseases: A look inside the pertussis experience / Serovigilancia de enfermedades prevenibles por vacunación: una mirada desde la experiencia con la tosferina

Abstract Introduction: Serological surveillance (serosurveillance) provides the most direct measure of herd immunity of vaccine-preventable diseases. Little is known about the opportunities and challenges of serosurveillance experiences, particularly pertussis. Objective: To describe the process of serosurveillance for vaccine-preventable diseases with an emphasis on the experience of pertussis in the metropolitan area of Antioquia (Valle de Aburrá) in 2015 and 2016 and analyze the contributions and challenges for its sustainability. Materials and methods: We described the planning and conduction of serosurveillance of pertussis antibodies of mothers and in the umbilical cord at the time of delivery in eight hospitals based on random sampling and their capacity to advance the serosurveillance periodically. We compared the contributions and the challenges of this experience with other probabilistic and non-probabilistic programs. Results: We achieved the participation of hospitals and mothers respecting the delivery care process. We established a serum bank following ethical and technical guidelines. This program based on the random selection of hospitals and mothers has enabled the estimation of antibodies prevalence in mothers and in the umbilical cord, which has been possible given the high coverage of hospital care during childbirth at a lower cost and fewer risks than a population-based survey in conflictive areas. The main challenges for the sustainability of this program are the creation of stable jobs and access to funding and legal and methodological long-term frameworks. Conclusions: Hospital serosurveillance as described is an option to monitor the impact of vaccination on the population. Our experience could be reproduced in other regions under similar conditions if the above-mentioned challenges are solved.

Resumen Introducción. La vigilancia serológica es la forma más directa de medir la inmunidad de rebaño frente a las enfermedades prevenibles por vacunación. Poco se sabe acerca de las oportunidades y los desafíos de las experiencias de serovigilancia, en general y, específicamente, la de la tosferina. Objetivo. Describir el proceso de serovigilancia de enfermedades prevenibles por vacunación con énfasis en la experiencia en el caso de la tosferina en el área metropolitana de Antioquia (Valle de Aburrá) en el 2015 y el 2016 y analizar lo que dicha experiencia ha aportado y los desafíos que persisten para su sostenibilidad. Materiales y métodos. Se describió el proceso de planeación y el desarrollo de la serovigilancia de tosferina en el momento del parto en ocho hospitales seleccionados al azar, así como la capacidad para adelantar el programa de manera periódica. Se compararon los aportes y los desafíos en el curso de esta experiencia con los de otros programas poblacionales probabilistas e institucionales no probabilistas. Resultados. Se logró la participación de los hospitales y de las madres con pleno respeto del proceso de atención del parto, y se conformó un banco de sueros siguiendo lineamientos éticos y técnicos. El programa permitió estimar la prevalencia de anticuerpos en la madre y en el cordón umbilical, lo que se facilitó por la alta cobertura de atención hospitalaria del parto, a un menor costo y menos riesgos que los programas poblacionales en zonas conflictivas. Los principales desafíos para la sostenibilidad del programa son la estabilidad laboral del personal de salud, así como normas y una financiación de largo plazo. Conclusiones. La serovigilancia hospitalaria es una opción para monitorizar el impacto poblacional de la vacunación. Esta experiencia se podría extender a otras regiones en condiciones similares si se resuelven los retos mencionados.

Probiotics and the immunological response to infant vaccinations; a double-blind randomized controlled trial.

OBJECTIVES: To examine the effect of a combination of probiotics on the antibody response to pneumococcal and pertussis vaccination in healthy Danish children, aged 8-14 months, at the time of starting day care. Moreover, the cytokine response to lipopolysaccharide of whole blood was assessed. METHODS: A total of 290 children were randomly allocated to receive a combination of Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG daily for a 6-month intervention period, and blood samples were drawn at the start and end of the study. Specific antibody response towards Streptococcus pneumoniae serotypes and Bordetella pertussis toxin, as well as endotoxin-induced interleukin-6 (IL-6) and interferon-γ (IFN-γ) production in blood were analysed by Luminex and ELISA. RESULTS: There was no significant difference between the average individual changes from baseline to end of study in antibody concentrations for S. pneumoniae for both the probiotics (340.4% ± 11.2%) and the placebo group (382.9% ± 10.4%) (p 0.525), nor for B. pertussis toxin in the two groups (probiotics 190.1% ± 12.6% versus placebo 238.8% ± 1.1%, p 0.340). The average individual change in IL-6 concentration was significantly lower in the probiotics versus the placebo group (2.9% ± 10.3% versus 33.7% ± 9.0%, p 0.024), whereas there was no difference in IFN-γ concentration (0.0% ± 0.2% versus -0.2% ± 0.1%, p 0.279). CONCLUSIONS: The probiotic intervention did not affect the antibody response against S. pneumoniae and B. pertussis toxin in healthy Danish children.

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.

Lower Sustained Diphtheria and Pertussis Antibody Concentrations in Inflammatory Bowel Disease Patients.

BACKGROUND: Patients with inflammatory bowel disease (IBD) are often immunosuppressed, and those patients receiving anti-tumor necrosis factor α (TNF) therapy can have lower antibody responses to vaccines. Pertussis cases are at their highest levels in the post-vaccine era. There is little data regarding responses to the Tdap (tetanus, diphtheria, and acellular pertussis) vaccine in IBD patients. AIMS: The aim of this study was to compare sustained vaccine-induced Tdap antibody concentrations in a cohort of IBD patients stratified by medication regimens with healthy controls (HC) who had received an adult Tdap booster. METHODS: We performed a cross-sectional study evaluating antibody responses to Tdap vaccine among IBD patients compared to HC. Our study consisted of three patient groups: adults with IBD stratified by maintenance medication regimen: (1) thiopurine monotherapy; (2) anti-TNF monotherapy; and (3) combination therapy (anti-TNF and immunomodulator (thiopurine or methotrexate)). RESULTS: Ninety IBD patients and 20 HC participated. Pertussis pertactin antibody concentrations were significantly lower in IBD patients (p = 0.021) compared to HC, and those on anti-TNF agents (monotherapy or combination) had lower antibody concentrations compared to those on thiopurine monotherapy (p = 0.028). Diphtheria antibody concentrations were also lower in IBD patients (p < 0.001), and those on anti-TNF agents (monotherapy or combination) had lower antibody concentrations compared to the thiopurine monotherapy group (p < 0.001). CONCLUSION: IBD patients on anti-TNF agents had lower antibody concentrations to diphtheria and pertussis. These findings suggest a need for different Tdap booster schedules for IBD patients on anti-TNF therapy. Clinical Trials Registry NCT02434133.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis.

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

Seroprevalencia contra Bordetella pertussis en embarazadas vacunadas y no vacunadas y neonatos en un hospital universitario de la provincia de Buenos Aires / Seroprevalence of Bordetella pertussis among vaccinated and unvaccinated pregnant women and newborn infants in a university hospital of Buenos Aires

Introducción. La tos convulsa es una enfermedad altamente contagiosa causada por Bordetella pertussis. Tiene una alta tasa de morbilidad y mortalidad, especialmente, en los lactantes menores de seis meses de edad. En la Argentina, la incidencia y la mortalidad se han encontrado en aumento en las últimas 3 décadas. Objetivo. Determinar anticuerpos contra Bordetella pertussis en las mujeres embarazadas en el tercer trimestre de la gestación y en el recién nacido, medidos en la sangre del cordón. Métodos. Se disenó un estudio observacional, transversal. El estudio se inició en 2011 cuando la vacunación contra pertussis en la embarazada no estaba incluida en el Calendario Nacional de Vacunación y era opcional. Los anticuerpos se midieron en las madres en el tercer trimestre del embarazo y en la sangre del cordón umbilical al nacer. Las determinaciones de anticuerpos se realizaron con el kit de ELISA humano para IgG toxina pertussis ABCAMR. Se utilizó la prueba de chi² para comparar la prevalencia. Resultados. Se incluyó a 111 madres y a sus bebés, 35 hijos de madres no vacunadas (antes de la implementación de la vacuna en embarazadas) y 76 hijos de madres vacunadas. Los bebés de madres vacunadas presentaron anticuerpos IgG positivos en el 92% (70/76), mientras que los bebés de madres no vacunadas fueron negativos para anticuerpos IgG en el 100% (35/35) con una p < 0,001. Conclusión. En la población de vacunadas del estudio, se observó que sus hijos presentaron anticuerpos IgG positivos en el 92%. Este estudio apoya la necesidad de la inmunización materna contra Bordetella pertussis para proteger al recién nacido.

Introduction. Pertussis is a highly contagious disease caused by Bordetella pertussis. It poses a high morbidity and mortality rate, especially among infants younger than 6 months old. In Argentina, pertussis incidence and mortality have increased over the past three decades. Objective. To establish Bordetella pertussis antibody titers among pregnant women in their third trimester and among newborn infants, as measured in cord blood. Methods. This was an observational, crosssectional study. The study started in 2011; at that time, pertussis vaccination was not mandatory for pregnant women as per the national immunization schedule, only optional. Maternal antibodies were measured in the last trimester of pregnancy for women and in cord blood for newborn infants. Antibody titers were determined using Abcam's anti-Bordetella pertussis toxin (PT) IgG in vitro ELISA kit. The X2 test was used to compare prevalence rates. Results. The study included 111 mother-newborn infant dyads; 35 infants from unvaccinated mothers (before the introduction of the vaccine) and 76 from vaccinated mothers. Positive IgG antibodies were found in 92% (70/76) of infants born from vaccinated mothers whereas 100% (35/35) of infants born from unvaccinated mothers had negative results for antibodies; p < 0.001. Conclusion. In the vaccinated population of this study, 92% of infants had positive IgG antibodies. This study supports the need for maternal immunization against Bordetella pertussis to provide protection to newborn infants.

Influence of Hesperidin on the Systemic and Intestinal Rat Immune Response.

Polyphenols, widely found in edible plants, influence the immune system. Nevertheless, the immunomodulatory properties of hesperidin, the predominant flavanone in oranges, have not been deeply studied. To establish the effect of hesperidin on in vivo immune response, two different conditions of immune system stimulations in Lewis rats were applied. In the first experimental design, rats were intraperitoneally immunized with ovalbumin (OVA) plus Bordetella pertussis toxin and alum as the adjuvants, and orally given 100 or 200 mg/kg hesperidin. In the second experimental design, rats were orally sensitized with OVA together with cholera toxin and fed a diet containing 0.5% hesperidin. In the first approach, hesperidin administration changed mesenteric lymph node lymphocyte (MLNL) composition, increasing the TCRαß+ cell percentage and decreasing that of B lymphocytes. Furthermore, hesperidin enhanced the interferon (IFN)-γ production in stimulated MLNL. In the second approach, hesperidin intake modified the lymphocyte composition in the intestinal epithelium (TCRγδ+ cells) and the lamina propria (TCRγδ+, CD45RA+, natural killer, natural killer T, TCRαß+CD4+, and TCRαß+CD8+ cells). Nevertheless, hesperidin did not modify the level of serum anti-OVA antibodies in either study. In conclusion, hesperidin does possess immunoregulatory properties in the intestinal immune response, but this effect is not able to influence the synthesis of specific antibodies.

Protective Role of Passively Transferred Maternal Cytokines against Bordetella pertussis Infection in Newborn Piglets.

Maternal vaccination represents a potential strategy to protect both the mother and the offspring against life-threatening infections. This protective role has mainly been associated with antibodies, but the role of cell-mediated immunity, in particular passively transferred cytokines, is not well understood. Here, using a pertussis model, we have demonstrated that immunization of pregnant sows with heat-inactivated bacteria leads to induction of a wide range of cytokines (e.g., tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], interleukin-6 [IL-6], IL-8, and IL-12/IL-23p40) in addition to pertussis-specific antibodies. These cytokines can be detected in the sera and colostrum/milk of vaccinated sows and subsequently were detected at significant levels in the serum and bronchoalveolar lavage fluid of piglets born to vaccinated sows together with pertussis-specific antibodies. In contrast, active vaccination of newborn piglets with heat-inactivated bacteria induced high levels of specific IgG and IgA but no cytokines. Although the levels of antibodies in vaccinated piglets were comparable to those of passively transferred antibodies, no protection against Bordetella pertussis infection was observed. Thus, our results demonstrate that a combination of passively transferred cytokines and antibodies is crucial for disease protection. The presence of passively transferred cytokines/antibodies influences the cytokine secretion ability of splenocytes in the neonate, which provides novel evidence that maternal immunization can influence the newborn's cytokine milieu and may impact immune cell differentiation (e.g., Th1/Th2 phenotype). Therefore, these maternally derived cytokines may play an essential role both as mediators of early defense against infections and possibly as modulators of the immune repertoire of the offspring.

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity.

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in immune evasion and virulence of the whooping cough agent Bordetella pertussis. CyaA penetrates the complement receptor 3-expressing phagocytes and ablates their bactericidal capacities by catalyzing unregulated conversion of cytosolic ATP to the key second messenger molecule cAMP. We show that signaling of CyaA-generated cAMP blocks the oxidative burst capacity of neutrophils by two converging mechanisms. One involves cAMP/protein kinase A-mediated activation of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and limits the activation of MAPK ERK and p38 that are required for assembly of the NADPH oxidase complex. In parallel, activation of the exchange protein directly activated by cAMP (Epac) provokes inhibition of the phospholipase C by an as yet unknown mechanism. Indeed, selective activation of Epac by the cell-permeable analog 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate counteracted the direct activation of phospholipase C by 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide. Hence, by inhibiting production of the protein kinase C-activating lipid, diacylglycerol, cAMP/Epac signaling blocks the bottleneck step of the converging pathways of oxidative burst triggering. Manipulation of neutrophil membrane composition by CyaA-produced signaling of cAMP thus enables B. pertussis to evade the key innate host defense mechanism of reactive oxygen species-mediated killing of bacteria by neutrophils.

Evaluation of Commercial Assays for Single-Point Diagnosis of Pertussis in the US.

BACKGROUND: Pertussis serodiagnosis is increasingly being used in the United States despite the lack of a US Food and Drug Administration-approved, commercially available assay. To better understand the utility of these assays in diagnosing pertussis, serology assays were evaluated for analytical parameters and clinical accuracy. METHODS: Forty-three antigen-antibody combinations were evaluated for single-point diagnosis of pertussis. Serum panels included sera from laboratory-confirmed cases, an international reference standard, and healthy donors. Phase I panel (n = 20) of sera was used to assess precision, linearity, and accuracy; Phase II panel (n = 226) followed with positive percent agreement (PPA) and negative percent agreement (NPA) estimates. Analytical analyses included coefficients of variation (CV) and concordance correlation coefficients (rc). RESULTS: Intra-analyst variability was found to be relatively low among samples per assay, with only 6% (78 of 1240) having CV >20%, primarily with the highly concentrated immunoglobulin (Ig)G anti-pertussis toxin (PT) specimens and IgM assays. The rc measurements to assess linearity ranged between 0.282 and 0.994, 0.332 and 0.999, and -0.056 and 0.482 for IgA, IgG, and IgM, respectively. Analytical accuracy for calibrated IgG anti-PT assays was 86%-115%. The PPA and NPA varied greatly for all assays; PPA/NPA ranges for IgA, IgG, and IgM assays, with culture and/or polymerase chain reaction positivity as control, were 29-90/13-100, 26-96/27-100, and 0-73/42-100, respectively. In IgG assays, mixing filamentous hemagglutinin antigen with PT increased PPA but decreased NPA. CONCLUSIONS: Seroassays varied substantially under both analytical and clinical parameters; however, those that were calibrated to a reference standard were highly accurate. Our findings support incorporation of calibrated pertussis seroassays to the pertussis case definition for improved diagnosis and surveillance.

Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis.

Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

Seroprevalence of Bordetella pertussis specific Immunoglobulin G antibody levels among asymptomatic individuals aged 4 to 24 years: a descriptive cross sectional study from Sri Lanka.

BACKGROUND: In Sri Lanka pertussis continues to circulate in the community and cases among adolescents and adults have been reported despite 95% coverage of the four dose pertussis vaccination during early childhood. Waning of immunity following natural infection or immunization may contribute to the persistent circulation. An adolescent booster dose is not included in the national immunization schedule of Sri Lanka, although this is routine practice in many countries. Therefore information on immunity to pertussis in the adolescent group is needed prior to considering vaccination schedule changes. METHODS: The quantitative determination of specific Immunoglobulin G antibodies to Bordetella pertussis toxin was done using a commercially available validated ELISA method. The antibody values were categorized into groups according to the interpretive criteria provided by the manufacturer. The values were <55 IU/mL, negative; 55-<60 IU/mL, borderline; 60-125 IU/mL, positive; >125, strongly positive respectively. Sera of 385 asymptomatic individuals aged 4 to 24 years admitted to surgical units of Lady Ridgeway Hospital, Colombo and Colombo South Teaching Hospital were used for the study. Mann-Whitney U and Kruskal-Wallis tests were used in analysis of results and p ≤0.05 was considered as statistically significant. Details of epidemiological variables were collected using a questionnaire and correlation with significant levels of pertussis antibodies was determined. RESULTS: Median age of the study population was 12 years with 212 (55.1%) females. The median anti PT antibody level was 3.31 IU/mL and 352 (91%) had anti PT levels ≤55 IU/mL. Median of anti PT levels were 3.18 IU/mL for 4-7 years, 1.43 IU/mL (IQR 0.336-6.27) for 8-11 years, 4.28 IU/mL (IQR 0.978-13.39) for 12-15 years, 6.14 IU/mL for 16-19 years and 4.89 IU/mL for 20-24 years and the differences were statistically significant (p = 0.000). Females (p < 0.003) and those having a sibling aged ≥12 years (p = 0.017) had significantly higher anti PT levels. CONCLUSIONS: The majority of the study population, especially 8 to 11 year age group had low anti PT IgG levels. The higher antibody titers in the 12-15 year age group seem to indicate infection in early adolescence. A booster dose of acellular pertussis vaccine need to be considered.

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN-γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post-challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD-MPLA than in those that had received rFeSOD-Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD-MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific-IFN-γ responses were significantly stronger in the group vaccinated with rFeSOD-MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component.

PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model.

Poly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response.

Synergistic Neutralization of Pertussis Toxin by a Bispecific Antibody In Vitro and In Vivo.

Bispecific antibodies are a rapidly growing class of therapeutic molecules, originally developed for the treatment of cancer but recently explored for the treatment of autoimmune and infectious diseases. Bordetella pertussis is a reemerging pathogen, and several of the key symptoms of infection are caused by the pertussis toxin (PTx). Two humanized antibodies, hu1B7 and hu11E6, bind distinct epitopes on PTx and, when coadministered, mitigate disease severity in murine and baboon models of infection. Here we describe the generation of a bispecific human IgG1 molecule combining the hu1B7 and hu11E6 binding sites via a knobs-in-holes design. The bispecific antibody showed binding activity equivalent to that of the antibody mixture in a competition enzyme-linked immunosorbent assay (ELISA). A CHO cell neutralization assay provided preliminary evidence for synergy between the two antibodies, while a murine model of PTx-induced leukocytosis definitively showed synergistic neutralization. Notably, the bispecific antibody retained the synergy observed for the antibody mixture, supporting the conclusion that synergy is due to simultaneous blockade of both the catalytic and receptor binding activities of pertussis toxin. These data suggest that a hu1B7/hu11E6 bispecific antibody is a viable alternative to an antibody mixture for pertussis treatment.