Pertactin-deficient Bordetella pertussis isolates: evidence of increased circulation in Europe, 1998 to 2015.

IntroductionPertussis outbreaks have occurred in several industrialised countries using acellular pertussis vaccines (ACVs) since the 1990s. High prevalence of pertactin (PRN)-deficient Bordetella pertussis isolates has been found in these countries.AimsTo evaluate in Europe: (i) whether proportions of PRN-deficient strains increased in consecutive collections of B. pertussis clinical isolates; (ii) if the frequency of PRN-deficient strains in countries correlated with the time since ACV introduction; (iii) the presence of pertussis toxin (PT)-, filamentous haemagglutinin (FHA)- or fimbriae (Fim)-deficient isolates.MethodsB. pertussis clinical isolates were obtained from different European countries during four periods (EUpert I-IV studies): 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), 2007 to 2009 (n = 140) and 2012 to 2015 (n = 265). The isolates' selection criteria remained unchanged in all periods. PRN, PT, FHA and Fim2 and Fim3 expression were assessed by ELISA.ResultsIn each period 1.0% (1/102), 1.9% (3/154), 6.4% (9/140) and 24.9% (66/265) of isolates were PRN-deficient. In EUpert IV, PRN-deficient isolates occurred in all countries sampled and in six countries their frequency was higher than in EUpert III (for Sweden and the United Kingdom, p < 0.0001 and p = 0.0155, respectively). Sweden and Italy which used ACVs since the mid 1990s had the highest frequencies (69%; 20/29 and 55%; 11/20, respectively) while Finland, where primary immunisations with ACV containing PRN dated from 2009 had the lowest (3.6%). Throughout the study, no PT- or FHA-deficient isolate and one Fim2/3-deficient was detected.ConclusionResults suggest that the longer the period since the introduction of ACVs containing PRN, the higher the frequency of circulating PRN-deficient isolates.

Surveillance of Circulating Bordetella pertussis Strains in Europe during 1998 to 2015.

One reason for increased pertussis incidence is the adaptation of Bordetella pertussis to vaccine-induced immunity by modulating its genomic structure. This study, EUpert IV, includes 265 isolates collected from nine European countries during 2012 to 2015 (n = 265) and compares the results to previous EUpert I to III studies (1998 to 2009). The analyses included genotyping, serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus variable-number tandem-repeat analysis (MLVA). Genotyping results showed only small variations among the common virulence genes of B. pertussis The frequencies of serotypes Fim2 and Fim3 varied among the four collections. Genomic analyses showed that MLVA type 27 increased to 80% between the periods of 1998 to 2001 and 2012 to 2015. Two PFGE profiles, BpSR3 (29.4%) and BpSR10 (27.2%), constituted more than 50% of the circulating isolates in the present collection. Our study indicates that the European B. pertussis population is changing and became more homogenous after the introduction of acellular pertussis vaccines.

Pertussis seroprevalence among adults of reproductive age (20-39 years) in fourteen European countries.

The reported incidence of pertussis in European countries varies considerably. We aimed to study specific Bordetella pertussis seroprevalence in Europe by measuring serum IgG antibody levels to pertussis toxin (anti-PT IgG). Fourteen national laboratories participated in this study including Belgium, Denmark, Finland, Greece, Hungary, Italy, Lithuania, Malta, Norway, Poland, Portugal, Romania, Spain, and Sweden. Each country collected approximately 250 samples (N = 7903) from the age groups 20-29 years (N = 3976) and 30-39 years (N = 3927) during 2010-2013. Samples were anonymous residual sera from diagnostic laboratories and were analyzed at the national laboratories by a Swedish reference method, a commercial ELISA kit, or were sent to Sweden for analysis. The median anti-PT IgG concentrations ranged from 4 to 13.6 IU/mL. The proportion of samples with anti-PT IgG ≥100 IU/mL, indicating a recent infection ranged from 0.2% (Hungary) to 5.7% (Portugal). The highest proportion of sera with anti-PT IgG levels between 50 and <100 IU/mL, indicating an infection within the last few years, was found in Portugal (12.3%) and Italy (13.9%). This study shows that the circulation of B. pertussis is quite extensive in adults, aged 20-39 years, despite well-established vaccination programs in Europe.

Circulation of pertussis and poor protection against diphtheria among middle-aged adults in 18 European countries.

Reported incidence of pertussis in the European Union (EU) and the European Economic Area (EEA) varies and may not reflect the real situation, while vaccine-induced protection against diphtheria and tetanus seems sufficient. We aimed to determine the seroprevalence of DTP antibodies in EU/EEA countries within the age groups of 40-49 and 50-59 years. Eighteen countries collected around 500 samples between 2015 and 2018 (N = 10,302) which were analysed for IgG-DTP specific antibodies. The proportion of sera with pertussis toxin antibody levels ≥100 IU/mL, indicative of recent exposure to pertussis was comparable for 13/18 countries, ranging between 2.7-5.8%. For diphtheria the proportion of sera lacking the protective level (<0.1 IU/mL) varied between 22.8-82.0%. For tetanus the protection was sufficient. Here, we report that the seroprevalence of pertussis in these age groups indicates circulation of B. pertussis across EU/EEA while the lack of vaccine-induced seroprotection against diphtheria is of concern and deserves further attention.

High content cellular immune profiling reveals differences between rhesus monkeys and men.

A better understanding of similarities and differences in the composition of the cellular immune system in non-human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8alpha, CD8beta, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin-7 receptor alpha-chain (IL-7Ralpha) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti-CD3, -CD4, -CD25, -FoxP3, and -IL-7Ralpha monoclonal antibodies. Responsiveness to IL-7 was gauged in a signal transducer and activation of transcription 5 (STAT-5) phosphorylation assay. Human and NHP PBMCs showed a similar T-cell composition pattern with some remarkable differences. Similarities: human and NHP CD4(+) and CD8(+) cells showed a similar STAT-5 phosphorylation pattern in response to IL-7. Multicolour flow cytometric analysis identified a CD4(+) CD8alphaalpha(+) CD8alphabeta(+) T-cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4(+) T-cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5.16% in CD3(+) T cells) of CD8alphaalpha(+) T cells when compared with human donors (1.22% in CD3(+) T cells). NHP CD8alphaalpha(+) T cells produced tumour necrosis factor-alpha / interferon-gamma (TNF-alpha/IFN-gamma) or TNF-alpha, whereas human CD8alphaalpha(+) T cells produced simultaneously TNF-alpha/IFN-gamma and IL-2. A minor percentage of human CD8(+) T cells expressed CD25(bright) and FoxP3 (0.01%). In contrast, 0.07% of NHP CD8(+) T cells exhibited the CD25(bright) FoxP3(+) phenotype. PBMCs from NHPs showed less IL-7Ralpha-positive events in all T-cell subsets including CD4(+) Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long-lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

Reduced intracellular oxygen radical production in whole blood leukocytes from COPD patients and asymptomatic smokers.

RATIONALE AND OBJECTIVES: COPD is characterized by irreversible airflow obstruction. It has, however, become clear that COPD also is a systemic disease. In the present study, we sought to investigate its impact on different peripheral leukocyte subpopulations that are recognized as important effector cells in the lung tissue. METHODS: We enrolled 20 patients with stable, moderate COPD (FEV1, 33 to 69%). Ten asymptomatic smokers and 10 nonsmokers served as control groups. Flow cytometry and whole blood analysis were used to minimize unwanted ex vivo modulation. Oxidative burst and adhesion molecule mobilization were analyzed on freshly drawn cells and after in vitro activation. MEASUREMENTS AND MAIN RESULTS: We found reduced oxidative burst in neutrophils, monocytes, and eosinophils after in vitro stimulation with tumor necrosis factor (TNF) and the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in both COPD patients and asymptomatic smokers as compared to nonsmoking control subjects. Vascular involvement was determined as increased soluble intercellular adhesion molecule-1 (sICAM-1) in the COPD group. There were no differences in adhesion molecule expression among the three groups. However, in COPD patients who had smoked the same morning prior to blood sampling, we found a reduced ability to mobilize adhesion molecule CD11b after TNF plus fMLP activation in all investigated cell types. "Acute" smoking did not significantly alter respiratory burst measurements. CONCLUSIONS: Both COPD patients and asymptomatic smokers have increased levels of sICAM-1 and a reduced intracellular oxidative burst in vitro, indicating a vascular endothelial activation and a possible state of refractoriness in circulating phagocytes in COPD. Although expression and mobilization of adhesion molecules were similar between groups, the acute smoke effect on CD11b points out the value of information on smoking behavior when analyzing function of peripheral inflammatory cells in a smoking population.

Activation of complement and leukocyte receptors during on- and off pump coronary artery bypass surgery.

OBJECTIVE: The aim of this prospective, randomised study was to investigate the influence of extracorporeal circulation on the inflammatory response, our hypothesis being that off pump coronary artery bypass grafting (OFFCAB) procedures would generate less activation than on pump procedures (ONCAB). METHODS: Patients admitted for elective CABG were randomised to either ONCAB or OFFCAB surgery and blood samples were taken during and up to 24 h after the operation. We measured complement factors C5a and the terminal complement complex (TCC, C59-b), and the interleukins IL-6 and IL-8. Leukocytes were studied for cellular counts and adhesion molecules (CD11b, CD35 and CD62L) by flow cytometry. We included a combination of activity markers with different aspects of neutrophil function and combined these with in vitro activation. RESULTS: The complement factors C5a and TCC showed a more rapid (P=0.02, P<0.001) and TCC a more profound (P<0.001) increase in the ONCAB group than in the OFFCAB group during the operation, after that there were no inter-group differences. Cellular markers, cell counts and interleukin levels were activated by surgery but with no difference between groups. CONCLUSION: This prospective, randomised study showed less complement activation in low risk OFFCAB, compared to ONCAB patients.

B-cell responses after intranasal vaccination with the novel attenuated Bordetella pertussis vaccine strain BPZE1 in a randomized phase I clinical trial.

Despite high vaccination coverage, pertussis is still a global concern in infant morbidity and mortality, and improved pertussis vaccines are needed. A live attenuated Bordetella pertussis strain, named BPZE1, was designed as an intranasal vaccine candidate and has recently been tested in man in a phase I clinical trial. Here, we report the evaluation of the B-cell responses after vaccination with BPZE1. Forty-eight healthy males with no previous pertussis-vaccination were randomized into one of three dose-escalating groups or into a placebo group. Plasma blast- and memory B-cell responses were evaluated by ELISpot against three different pertussis antigens: pertussis toxin, filamentous haemagglutinin and pertactin. Seven out of the 36 subjects who had received the vaccine were colonized by BPZE1, and significant increases in the memory B-cell response were detected against all three tested antigens in the culture-positive subjects between days 0 and 28 post-vaccination. The culture-positive subjects also mounted a significant increase in the filamentous haemagglutinin-specific plasma blast response between days 7 and 14 post-vaccination. No response could be detected in the culture-negatives or in the placebo group post-vaccination. These data show that BPZE1 is immunogenic in humans and is therefore a promising candidate for a novel pertussis vaccine. This trial is registered at ClinicalTrials.gov (NCT01188512).

A phase I clinical study of a live attenuated Bordetella pertussis vaccine--BPZE1; a single centre, double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally to healthy adult male volunteers.

BACKGROUND: Acellular pertussis vaccines do not control pertussis. A new approach to offer protection to infants is necessary. BPZE1, a genetically modified Bordetella pertussis strain, was developed as a live attenuated nasal pertussis vaccine by genetically eliminating or detoxifying 3 toxins. METHODS: We performed a double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally for the first time to human volunteers, the first trial of a live attenuated bacterial vaccine specifically designed for the respiratory tract. 12 subjects per dose group received 10³, 105 or 107 colony-forming units as droplets with half of the dose in each nostril. 12 controls received the diluent. Local and systemic safety and immune responses were assessed during 6 months, and nasopharyngeal colonization with BPZE1 was determined with repeated cultures during the first 4 weeks after vaccination. RESULTS: Colonization was seen in one subject in the low dose, one in the medium dose and five in the high dose group. Significant increases in immune responses against pertussis antigens were seen in all colonized subjects. There was one serious adverse event not related to the vaccine. Other adverse events were trivial and occurred with similar frequency in the placebo and vaccine groups. CONCLUSIONS: BPZE1 is safe in healthy adults and able to transiently colonize the nasopharynx. It induces immune responses in all colonized individuals. BPZE1 can thus undergo further clinical development, including dose optimization and trials in younger age groups. TRIAL REGISTRATION: ClinicalTrials.gov NCT01188512.

rBCG induces strong antigen-specific T cell responses in rhesus macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector.

BACKGROUND: BCG vaccination, combined with adenoviral-delivered boosts, represents a reasonable strategy to augment, broaden and prolong immune protection against tuberculosis (TB). We tested BCG (SSI1331) (in 6 animals, delivered intradermally) and a recombinant (rBCG) AFRO-1 expressing perfringolysin (in 6 animals) followed by two boosts (delivered intramuscullary) with non-replicating adenovirus 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4, for the capacity to induce antigen-specific cellular immune responses in rhesus macaques (Macaca mulatta). Control animals received diluent (3 animals). METHODS AND FINDINGS: Cellular immune responses were analyzed longitudinally (12 blood draws for each animal) using intracellular cytokine staining (TNF-alpha, IL-2 and IFN-gamma), T cell proliferation was measured in CD4(+), CD8alpha/beta(+), and CD8alpha/alpha(+) T cell subsets and IFN-gamma production was tested in 7 day PBMC cultures (whole blood cell assay, WBA) using Ag85A, Ag85B, TB10.4 recombinant proteins, PPD or BCG as stimuli. Animals primed with AFRO-1 showed i) increased Ag85B-specific IFN-gamma production in the WBA assay (median >400 pg/ml for 6 animals) one week after the first boost with adenoviral-delivered TB-antigens as compared to animals primed with BCG (<200 pg/ml), ii) stronger T cell proliferation in the CD8alpha/alpha(+) T cell subset (proliferative index 17%) as compared to BCG-primed animals (proliferative index 5% in CD8alpha/alpha(+) T cells). Polyfunctional T cells, defined by IFN-gamma, TNF-alpha and IL-2 production were detected in 2/6 animals primed with AFRO-1 directed against Ag85A/b and TB10.4; 4/6 animals primed with BCG showed a Ag85A/b responses, yet only a single animal exhibited Ag85A/b and TB10.4 reactivity. CONCLUSION: AFRO-1 induces qualitatively and quantitatively different cellular immune responses as compared with BCG in rhesus macaques. Increased IFN-gamma-responses and antigen-specific T cell proliferation in the CD8alpha/alpha+ T cell subset represents a valuable marker for vaccine-take in BCG-based TB vaccine trials.

Peripheral blood monocyte activation during coronary artery bypass grafting with or without cardiopulmonary bypass.

OBJECTIVE: The aim of this prospective, randomized study was to investigate the impact of coronary artery bypass grafting (CABG) on peripheral monocytes and to evaluate the additional effect of cardiopulmonary bypass (CPB). DESIGN: Twenty patients admitted for elective CABG were randomized to either on-pump (ONCAB, n = 9) or off-pump (OFFCAB, n = 11) surgery and blood samples were drawn before, during and 24 h after the operation. The total number of monocytes and the proportion of the more mature CD16+/CD14+ monocytes were measured. Expression of activation markers (CD11b, CD35 and CD62L) and oxidative burst were determined using flow cytometry on both resting and in vitro stimulated cells. Serum concentrations of soluble CD14 and monocytes/macrophage chemotactic protein 1 (MCP-1) were analysed. RESULTS: During surgery there was a selective decrease in the proportion of CD16+/CD14+ monocytes compared to total monocytes. These had returned to preoperative values 24 h after surgery while the total number of monocytes had increased more than 100%. Intracellular production of oxygen free radical H2O2 was increased in the ONCAB group during surgery compared to OFFCAB. Monocyte expression and in vitro mobilization of complement receptors, CD11b and CD35, were similar in both study groups during and after surgery as was the expression of CD62L. Serum levels of MCP-1 decreased during surgery as did soluble CD14, both with increased levels again the day after surgery. CONCLUSION: It is concluded that the circulating monocyte population is activated during and as a consequence of CABG. There were few apparent additional effects of CPB found in this study. In this setting the inflammation caused by the surgery procedure per se probably surpasses the impact of the CPB on circulating blood monocytes.

beta2-Integrin and lipid modifications indicate a non-antioxidant mechanism for the anti-atherogenic effect of dietary coenzyme Q10.

Dietary supplementation with coenzyme Q (CoQ) has been proposed to have anti-atherogenic effects by virtue of its antioxidant capacity. To investigate this question, the leukocyte status of 5 males and 5 females (52-68 years) was evaluated before and after supplementation with 200mg CoQ/day for 5 and 10 weeks. CoQ was selectively taken up by mononuclear cells and alpha-tocopherol increased in polynuclear and mononuclear cells. The expression of beta2-integrin CD11b and complement receptor CD35 on the plasma membrane of resting and stimulated monocytes was significantly decreased upon dietary CoQ. Fatty acid and aldehyde analysis revealed that there was a selective increase of arachidonic acid and plasmalogens in only mononuclear cells. These selective lipid changes are not consistent with a general improvement in antioxidant status and indicate that CoQ most likely inhibits a phospholipase A2. Thus, these results strongly suggest that the anti-atherogenic effects of CoQ be mediated by other mechanisms beside its antioxidant protection.