[Where we stand with asthma: optimized medical care and biologic therapy for children]. / Le point sur l'asthme en pédiatrie : optimisation de la prise en charge et thérapies biologiques.

Asthma recommendations are in constant evolution. Salbutamol exclusivity as the historical reference treatment for asthma exacerbations is now questioned. In case of light to moderate crisis, budesonide/formoterol, combining an inhaled corticosteroid and a fast acting long lasting beta2 agonist, can now be proposed as first line as needed treatment, for adolescents older than 12 years old. In addition, progress in fundamental research revealed the heterogeneous nature of asthma and allowed for the emergence of new-targeted therapies.

La prise en charge de l'asthme est en constante évolution. L'exclusivité du salbutamol, traitement historique de référence des exacerbations aiguës, même légères, est remise en question depuis plusieurs années. En cas de symptômes ou crise légère à modérée, le budésonide/formotérol, combiné de corticostéroïde inhalé et bronchodilatateur (ß2-agoniste) d'action rapide et prolongée, peut dorénavant être proposé en première intention, à la demande, y compris en pédiatrie, notamment à partir de 12 ans. De plus, les progrès en recherche fondamentale ont permis de révéler la nature hétérogène de l'asthme et de faire émerger de nouvelles cibles thérapeutiques. En particulier, les patients asthmatiques peuvent bénéficier de l'entrée des biologiques dans l'arsenal thérapeutique.

Tocilizumab for Severe Worsening COVID-19 Pneumonia: a Propensity Score Analysis.

BACKGROUND: High levels of serum interleukin-6 (IL-6) correlate with disease severity in COVID-19. We hypothesized that tocilizumab (a recombinant humanized anti-IL-6 receptor) could improve outcomes in selected patients with severe worsening COVID-19 pneumonia and high inflammatory parameters. METHODS: The TOCICOVID study included a prospective cohort of patients aged 16-80 years with severe (requiring > 6 L/min of oxygen therapy to obtain Sp02 > 94%) rapidly deteriorating (increase by ≥ 3 L/min of oxygen flow within the previous 12 h) COVID-19 pneumonia with ≥ 5 days of symptoms and C-reactive protein levels > 40 mg/L. They entered a compassionate use program of treatment with intravenous tocilizumab (8 mg/kg with a maximum of 800 mg per infusion; and if needed a second infusion 24 to 72 h later). A control group was retrospectively selected with the same inclusion criteria. Outcomes were assessed at D28 using inverse probability of treatment weighted (IPTW) methodology. RESULTS: Among the 96 patients included (81% male, mean (SD) age: 60 (12.5) years), underlying conditions, baseline disease severity, and concomitant medications were broadly similar between the tocilizumab (n = 49) and the control (n = 47) groups. In the IPTW analysis, treatment with tocilizumab was associated with a reduced need for overall ventilatory support (49 vs. 89%, wHR: 0.39 [0.25-0.56]; p < 0.001). Albeit lacking statistical significance, there was a substantial trend towards a reduction of mechanical ventilation (31% vs. 45%; wHR: 0.58 [0.36-0.94]; p = 0.026). However, tocilizumab did not improve overall survival (wHR = 0.68 [0.31-1.748], p = 0.338). Among the 85 (89%) patients still alive at D28, patients treated with tocilizumab had a higher rate of oxygen withdrawal (82% vs. 73.5%, wHR = 1.66 [1.17-2.37], p = 0.005), with a shorter delay before being weaned of oxygen therapy (mean 11 vs. 16 days; p < 0.001). At D28, the rate of patients discharged from hospital was higher in the tocilizumab group (70% vs. 40%, wHR = 1.82 [1.22-2.75]; p = 0.003). The levels of CRP and fibrinogen post therapy (p < 0.001 for both variables) were significantly lower in the tocilizumab group (interaction test, mixed model). Rates of neutropenia (35% vs. 0%; p < 0.001) were higher in the tocilizumab group, yet rates of infections (22% vs. 38%, p = 0.089) including ventilator-acquired pneumonia (8% vs. 26%, p = 0.022) were higher in the control group. CONCLUSION: These data could be helpful for the design of future trials aiming to counter COVID-19-induced inflammation, especially before patients require admission to the intensive care unit.

COVID-19 pneumonia: high diagnostic accuracy of chest CT in patients with intermediate clinical probability.

OBJECTIVES: To assess inter-reader agreements and diagnostic accuracy of chest CT to identify COVID-19 pneumonia in patients with intermediate clinical probability during an acute disease outbreak. METHODS: From March 20 to April 8, 319 patients (mean age 62.3 years old) consecutive patients with an intermediate clinical probability of COVID-19 pneumonia underwent a chest CT scan. Two independent chest radiologists blinded to clinical information and RT-PCR results retrospectively reviewed and classified images on a 1-5 confidence level scale for COVID-19 pneumonia. Agreements between radiologists were assessed with kappa statistics. Diagnostic accuracy of chest CT compared with RT-PCR assay and patient outcomes was measured using receiver operating characteristics (ROC). Positive predictive value (PPV) and negative predictive value (NPV) for COVID-19 pneumonia were calculated. RESULTS: Inter-observer agreement for highly probable (kappa: 0.83 [p < .001]) and highly probable or probable (kappa: 0.82 [p < .001]) diagnosis of COVID-19 pneumonia was very good. RT-PCR tests performed in 307 patients were positive in 174 and negative in 133. The areas under the curve (AUC) were 0.94 and 0.92 respectively. With a disease prevalence of 61.2%, PPV were 95.9% and 94.3%, and NPV 84.4% and 77.1%. CONCLUSION: During acute COVID-19 outbreak, chest CT scan may be used for triage of patients with intermediate clinical probability with very good inter-observer agreements and diagnostic accuracy. KEY POINTS: • Concordances between two chest radiologists to diagnose or exclude a COVID-19 pneumonia in 319 consecutive patients with intermediate clinical probability were very good (kappa: 0.82; p < .001). • When compared with RT-PCR results and patient outcomes, the diagnostic accuracy of CT to identify COVID-19 pneumonia was high for both radiologists (AUC: 0.94 and 0.92). • With a disease prevalence of 61.2% in the studied population, the positive predictive values of CT for diagnosing COVID-19 pneumonia were 95.9% and 94.3% with negative predictive values of 84.4% and 77.1%.

A low level of CD16pos monocytes in SARS-CoV-2 infected patients is a marker of severity.

OBJECTIVES: Severe forms of coronavirus disease 2019 (COVID-19) are characterized by an excessive production of inflammatory cytokines. Activated monocytes secrete high levels of cytokines. Human monocytes are divided into three major populations: conventional (CD14posCD16neg), non-classical (CD14dimCD16pos), and intermediate (CD14posCD16pos) monocytes. The aim of this study was to analyze whether the distribution of conventional (CD16neg) and CD16pos monocytes is different in patients with COVID-19 and whether the variations could be predictive of the outcome of the disease. METHODS: We performed a prospective study on 390 consecutive patients referred to the Emergency Unit, with a proven diagnosis of SARS-CoV 2 infection by RT-PCR. Using the CytoDiff™ reagent, an automated routine leukocyte differential, we quantified CD16neg and CD16pos monocytes. RESULTS: In the entire population, median CD16neg and CD16pos monocyte levels (0.398 and 0.054×109/L, respectively) were in the normal range [(0.3-0.7×109/L) and (0.015-0.065×109/L), respectively], but the 35 patients in the intensive care unit (ICU) had a significantly (p<0.001) lower CD16pos monocyte count (0.018 × 109/L) in comparison to the 70 patients who were discharged (0.064 × 109/L) or were hospitalized in conventional units (0.058 × 109/L). By ROC curve analysis, the ratio [absolute neutrophil count/CD16pos monocyte count] was highly discriminant to identify patients requiring ICU hospitalization: with a cut-off 193.1, the sensitivity and the specificity were 74.3 and 81.8%, respectively (area under the curve=0.817). CONCLUSIONS: Quantification of CD16pos monocytes and the ratio [absolute neutrophil count/CD16pos monocyte count] could constitute a marker of the severity of disease in COVID-19 patients.

Improved performances of the second generation of the ID NOW influenza A&B 2® and comparison with the GeneXpert®.

Due to the congestion of emergency wards, a rapid and accurate method for flu diagnosis is required. However, the first evaluations of the ID NOW® influenza A&B performances were heterogeneous. We aimed to (i) assess the performance of the second generation of the ID NOW® influenza A&B 2 and (ii) compare the ID NOW® to the GeneXpert® use when performed within a central laboratory. (i) Analytical performances of ID NOW® were assessed using a collection of 112 clinical samples in comparison with a reference multiplex PCR (Seegene®); (ii) Invalid rate per operator and time to result were calculated for the GeneXpert® Xpress Flu/RSV and the ID NOW® influenza A&B 2® during 2017-2018 and 2018-2019 flu outbreaks respectively. ID NOW® reaches an overall sensitivity and specificity of 96.6% and 96.1% respectively. Most of the false-negative display a CT > 37. For both instruments, a single operator was involved in about a half of all invalid results. Excluding this operator involved in most invalid result, the invalid rate was about 1% for both instruments. Time to result from sampling was significantly shorter in the ID NOW® versus the GeneXpert® group (33 vs 97 min, P < 0.01). The second generation of the ID NOW® influenza A&B 2 displays high performances, comparable with conventional PCR method. In order to prevent invalid results, we highlight the need for adequate training of operators. Also, when implemented in a central laboratory, the location of the instrument could have a strong impact on the time-to-results.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 µg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Prognostic value of cellular population data in patients with COVID-19.

Background and aims: Beckman Coulter hematology analysers identify leukocytes by their volume (V), conductivity (C) and scatter (S) of a laser beam at different angles. Each leukocyte sub-population [neutrophils (NE), lymphocytes (LY), monocytes (MO)] is characterized by the mean (MN) and the standard deviation (SD) of 7 measurements called "cellular population data" (@CPD), corresponding to morphological analysis of the leukocytes. As severe forms of infections to SARS-CoV-2 are characterized by a functional activation of mononuclear cells, leading to a cytokine storm, we evaluated whether CPD variations are correlated to the inflammation state, oxygen requirement and lung damage and whether CPD analysis could be useful for a triage of patients with COVID-19 in the Emergency Department (ED) and could help to identify patients with a high risk of worsening. Materials and method: The CPD of 825 consecutive patients with proven COVID-19 presenting to the ED were recorded and compared to classical biochemical parameters, the need for hospitalization in the ward or ICU, the need for oxygen, or lung injury on CT-scan. Results: 40 of the 42 CPD were significantly modified in COVID-19 patients in comparison to 245 controls. @MN-V-MO and @SD-V-MO were highly correlated with C-reactive protein, procalcitonin, ferritin and D-dimers. SD-UMALS-LY > 21.45 and > 23.92 identified, respectively, patients with critical lung injuries (>75%) and requiring tracheal intubation. @SD-V-MO > 25.03 and @SD-V-NE > 19.4 identified patients required immediate ICU admission, whereas a @MN-V-MO < 183 suggested that the patient could be immediately discharged. Using logistic regression, the combination of 8 CPD with platelet and basophil counts and the existence of diabetes or obesity could identify patients requiring ICU after a first stay in conventional wards (area under the curve = 0.843). Conclusion: CPD analysis constitutes an easy and inexpensive tool for triage and prognosis of COVID-19 patients in the ED.

Stratification of COVID-19 Severity Using SeptiCyte RAPID, a Novel Host Immune Response Test.

SeptiCyte® RAPID is a gene expression assay measuring the relative expression levels of host response genes PLA2G7 and PLAC8, indicative of a dysregulated immune response during sepsis. As severe forms of COVID-19 may be considered viral sepsis, we evaluated SeptiCyte RAPID in a series of 94 patients admitted to Foch Hospital (Suresnes, France) with proven SARS-CoV-2 infection. EDTA blood was collected in the emergency department (ED) in 67 cases, in the intensive care unit (ICU) in 23 cases and in conventional units in 4 cases. SeptiScore (0-15 scale) increased with COVID-19 severity. Patients in ICU had the highest SeptiScores, producing values comparable to 8 patients with culture-confirmed bacterial sepsis. Receiver operating characteristic (ROC) curve analysis had an area under the curve (AUC) of 0.81 for discriminating patients requiring ICU admission from patients who were immediately discharged or from patients requiring hospitalization in conventional units. SeptiScores increased with the extent of the lung injury. For 68 patients, a chest computed tomography (CT) scan was performed within 24 h of COVID-19 diagnosis. SeptiScore >7 suggested lung injury ≥50% (AUC = 0.86). SeptiCyte RAPID was compared to other biomarkers for discriminating Critical + Severe COVID-19 in ICU, versus Moderate + Mild COVID-19 not in ICU. The mean AUC for SeptiCyte RAPID was superior to that of any individual biomarker or combination thereof. In contrast to C-reactive protein (CRP), correlation of SeptiScore with lung injury was not impacted by treatment with anti-inflammatory agents. SeptiCyte RAPID can be a useful tool to identify patients with severe forms of COVID-19 in ED, as well as during follow-up.

Interest of the cellular population data analysis as an aid in the early diagnosis of SARS-CoV-2 infection.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is characterized by a high contagiousness requiring isolation measures. At this time, diagnosis is based on the positivity of specific RT-PCR and/or chest computed tomography scan, which are time-consuming and may delay diagnosis. Complete blood count (CBC) can potentially contribute to the diagnosis of COVID-19. We studied whether the analysis of cellular population data (CPD), provided as part of CBC-Diff analysis by the DxH 800 analyzers (Beckman Coulter), can help to identify SARS-CoV-2 infection. METHODS: Cellular population data of the different leukocyte subpopulations were analyzed in 137 controls, 322 patients with proven COVID-19 (COVID+), and 285 patients for whom investigations were negative for SARS-CoV-2 infection (COVID-). When CPD of COVID+ were different from controls and COVID- patients, we used receiver operating characteristic analysis to test the discriminating capacity of the individual parameters. Using a random forest classifier, we developed the algorithm based on the combination of 4 monocyte CPD to discriminate COVID+ from COVID- patients. This algorithm was tested prospectively in a series of 222 patients referred to the emergency unit. RESULTS: Among the 222 patients, 86 were diagnosed as COVID-19 and 60.5% were correctly identified using the discriminating protocol. Among the 136 COVID- patients, 10.3% were misclassified (specificity 89.7%, sensitivity 60.5%). False negatives were observed mainly in patients with a low inflammatory state whereas false positives were mainly seen in patients with sepsis. CONCLUSION: Consideration of CPD could constitute a first step and potentially aid in the early diagnosis of COVID-19.

Automated AI-Driven CT Quantification of Lung Disease Predicts Adverse Outcomes in Patients Hospitalized for COVID-19 Pneumonia.

The purpose of our work was to assess the independent and incremental value of AI-derived quantitative determination of lung lesions extent on initial CT scan for the prediction of clinical deterioration or death in patients hospitalized with COVID-19 pneumonia. 323 consecutive patients (mean age 65 ± 15 years, 192 men), with laboratory-confirmed COVID-19 and an abnormal chest CT scan, were admitted to the hospital between March and December 2020. The extent of consolidation and all lung opacities were quantified on an initial CT scan using a 3D automatic AI-based software. The outcome was known for all these patients. 85 (26.3%) patients died or experienced clinical deterioration, defined as intensive care unit admission. In multivariate regression based on clinical, biological and CT parameters, the extent of all opacities, and extent of consolidation were independent predictors of adverse outcomes, as were diabetes, heart disease, C-reactive protein, and neutrophils/lymphocytes ratio. The association of CT-derived measures with clinical and biological parameters significantly improved the risk prediction (p = 0.049). Automated quantification of lung disease at CT in COVID-19 pneumonia is useful to predict clinical deterioration or in-hospital death. Its combination with clinical and biological data improves risk prediction.

Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing.

Dendritic cells (DCs) that drain the gut and skin are known to favor the establishment of T cell populations that home to the original site of DC-antigen (Ag) encounter by providing soluble "imprinting" signals to T cells in the lymph node (LN). To study the induction of lung T cell-trafficking, we used a protein-adjuvant murine intranasal and intramuscular immunization model to compare in vivo-activated Ag+ DCs in the lung and muscle-draining LNs. Higher frequencies of Ag+ CD11b+ DCs were observed in lung-draining mediastinal LNs (MedLN) compared to muscle-draining inguinal LNs (ILN). Ag+ CD11b+ MedLN DCs were qualitatively superior at priming CD4+ T cells, which then expressed CD49a and CXCR3, and preferentially trafficked into the lung parenchyma. CD11b+ DCs from the MedLN expressed higher levels of surface podoplanin, Trem4, GL7, and the known co-stimulatory molecules CD80, CD86, and CD24. Blockade of specific MedLN DC molecules or the use of sorted DC and T cell co-cultures demonstrated that DC surface phenotype influences the ability to prime T cells that then home to the lung. Thus, the density of dLN Ag+ DCs, and DC surface molecule signatures are factors that can influence the output and differentiation of lung-homing CD4+ T cells.

Corrigendum: Site-Specific DC Surface Signatures Influence CD4+ T Cell Co-stimulation and Lung-Homing.

[This corrects the article DOI: 10.3389/fimmu.2019.01650.].

Reactivating Immunity Primed by Acellular Pertussis Vaccines in the Absence of Circulating Antibodies: Enhanced Bacterial Control by TLR9 Rather Than TLR4 Agonist-Including Formulation.

Pertussis is still observed in many countries despite of high vaccine coverage. Acellular pertussis (aP) vaccination is widely implemented in many countries as primary series in infants and as boosters in school-entry/adolescents/adults (including pregnant women in some). One novel strategy to improve the reactivation of aP-vaccine primed immunity could be to include genetically- detoxified pertussis toxin and novel adjuvants in aP vaccine boosters. Their preclinical evaluation is not straightforward, as it requires mimicking the human situation where T and B memory cells may persist longer than vaccine-induced circulating antibodies. Toward this objective, we developed a novel murine model including two consecutive adoptive transfers of the memory cells induced by priming and boosting, respectively. Using this model, we assessed the capacity of three novel aP vaccine candidates including genetically-detoxified pertussis toxin, pertactin, filamentous hemagglutinin, and fimbriae adsorbed to aluminum hydroxide, supplemented-or not-with Toll-Like-Receptor 4 or 9 agonists (TLR4A, TLR9A), to reactivate aP vaccine-induced immune memory and protection, reflected by bacterial clearance. In the conventional murine immunization model, TLR4A- and TLR9A-containing aP formulations induced similar aP-specific IgG antibody responses and protection against bacterial lung colonization as current aP vaccines, despite IL-5 down-modulation by both TLR4A and TLR9A and IL-17 up-modulation by TLR4A. In the absence of serum antibodies at time of boosting or exposure, TLR4A- and TLR9A-containing formulations both enhanced vaccine antibody recall compared to current aP formulations. Unexpectedly, however, protection was only increased by the TLR9A-containing vaccine, through both earlier bacterial control and accelerated clearance. This suggests that TLR9A-containing aP vaccines may better reactivate aP vaccine-primed pertussis memory and enhance protection than current or TLR4A-adjuvanted aP vaccines.

Nanoparticle conjugation enhances the immunomodulatory effects of intranasally delivered CpG in house dust mite-allergic mice.

An emerging strategy in preventing and treating airway allergy consists of modulating the immune response induced against allergens in the lungs. CpG oligodeoxynucleotides have been investigated in airway allergy studies, but even if promising, efficacy requires further substantiation. We investigated the effect of pulmonary delivery of nanoparticle (NP)-conjugated CpG on lung immunity and found that NP-CpG led to enhanced recruitment of activated dendritic cells and to Th1 immunity compared to free CpG. We then evaluated if pulmonary delivery of NP-CpG could prevent and treat house dust mite-induced allergy by modulating immunity directly in lungs. When CpG was administered as immunomodulatory therapy prior to allergen sensitization, we found that NP-CpG significantly reduced eosinophilia, IgE levels, mucus production and Th2 cytokines, while free CpG had only a moderate effect on these parameters. In a therapeutic setting where CpG was administered after allergen sensitization, we found that although both free CpG and NP-CpG reduced eosinophilia and IgE levels to the same extent, NP conjugation of CpG significantly enhanced reduction of Th2 cytokines in lungs of allergic mice. Taken together, these data highlight benefits of NP conjugation and the relevance of NP-CpG as allergen-free therapy to modulate lung immunity and treat airway allergy.

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes.

The sentinel or tumor-draining lymph node (tdLN) serves as a metastatic niche for many solid tumors and is altered via tumor-derived factors that support tumor progression and metastasis. tdLNs are often removed surgically, and therapeutic vaccines against tumor antigens are typically administered systemically or in non-tumor-associated sites. Although the tdLN is immune-suppressed, it is also antigen experienced through drainage of tumor-associated antigens (TAA), so we asked whether therapeutic vaccines targeting the tdLN would be more or less effective than those targeting the non-tdLN. Using LN-targeting nanoparticle (NP)-conjugate vaccines consisting of TAA-NP and CpG-NP, we compared delivery to the tdLN versus non-tdLN in two different cancer models, E.G7-OVA lymphoma (expressing the nonendogenous TAA ovalbumin) and B16-F10 melanoma. Surprisingly, despite the immune-suppressed state of the tdLN, tdLN-targeting vaccination induced substantially stronger cytotoxic CD8+ T-cell responses, both locally and systemically, than non-tdLN-targeting vaccination, leading to enhanced tumor regression and host survival. This improved tumor regression correlated with a shift in the tumor-infiltrating leukocyte repertoire toward a less suppressive and more immunogenic balance. Nanoparticle coupling of adjuvant and antigen was required for effective tdLN targeting, as nanoparticle coupling dramatically increased the delivery of antigen and adjuvant to LN-resident antigen-presenting cells, thereby increasing therapeutic efficacy. This work highlights the tdLN as a target for cancer immunotherapy and shows how its antigen-experienced but immune-suppressed state can be reprogrammed with a targeted vaccine yielding antitumor immunity.