Diversification of the VH3-53 immunoglobulin gene segment by somatic hypermutation results in neutralization of SARS-CoV-2 virus variants.

COVID-19 induces re-circulating long-lived memory B cells (MBC) that, upon re-encounter with the pathogen, are induced to mount immunoglobulin responses. During convalescence, antibodies are subjected to affinity maturation, which enhances the antibody binding strength and generates new specificities that neutralize virus variants. Here, we performed a single-cell RNA sequencing analysis of spike-specific B cells from a SARS-CoV-2 convalescent subject. After COVID-19 vaccination, matured infection-induced MBC underwent recall and differentiated into plasmablasts. Furthermore, the transcriptomic profiles of newly activated B cells transiently shifted toward the ones of atypical and CXCR3+ B cells and several B-cell clonotypes massively expanded. We expressed monoclonal antibodies (mAbs) from all B-cell clones from the largest clonotype that used the VH3-53 gene segment. The in vitro analysis revealed that some somatic hypermutations enhanced the neutralization breadth of mAbs in a putatively stochastic manner. Thus, somatic hypermutation of B-cell clonotypes generates an anticipatory memory that can neutralize new virus variants.

Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

Secreted NS1 proteins of tick-borne encephalitis virus and West Nile virus block dendritic cell activation and effector functions.

The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4+ and CD8+ T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections. IMPORTANCE The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.

Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells.

Dendritic cells (DCs) translate local innate immune responses into long-lasting adaptive immunity by priming antigen-specific T cells. Accordingly, there is an ample interest in exploiting DCs for therapeutic purposes, e.g., in personalized immunotherapies. Despite recent advances in elucidating molecular pathways of antigen processing, in DCs the exact spatial organization of the underlying processes is largely unknown. Here, we unraveled the nanoscale organization of the transporter associated with antigen processing (TAP)-dependent peptide-loading machinery in human monocyte-derived DCs (moDC). We detected an unexpected accumulation of MHC I peptide-loading complexes (PLCs) and TAP-dependent peptide compartmentalization in protrusions of activated DCs. Using single-molecule localization microscopy we revealed that PLCs display homogeneously sized assemblies, independent of the DC activation status or cellular localization. Our data indicate that moDCs show augmentation of subcellular PLC density during DC maturation. We observed a twofold density increase in the cell body, while an even fourfold accumulation was detected in the tips of the protrusions at the mature DC stage in comparison to immature DCs. In these tip regions, PLC assemblies are found along highly compressed tubular ER networks. These findings provide novel insights into nanoscale organization of the antigen presentation machinery, and open new perspectives on the T cell stimulatory capacity of DCs.

Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells.

Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.

Anopheline and human drivers of malaria risk in northern coastal, Ecuador: a pilot study.

BACKGROUND: Understanding local anopheline vector species and their bionomic traits, as well as related human factors, can help combat gaps in protection. METHODS: In San José de Chamanga, Esmeraldas, at the Ecuadorian Pacific coast, anopheline mosquitoes were sampled by both human landing collections (HLCs) and indoor-resting aspirations (IAs) and identified using both morphological and molecular methods. Human behaviour observations (HBOs) (including temporal location and bed net use) were documented during HLCs as well as through community surveys to determine exposure to mosquito bites. A cross-sectional evaluation of Plasmodium falciparum and Plasmodium vivax infections was conducted alongside a malaria questionnaire. RESULTS: Among 222 anopheline specimens captured, based on molecular analysis, 218 were Nyssorhynchus albimanus, 3 Anopheles calderoni (n = 3), and one remains unidentified. Anopheline mean human-biting rate (HBR) outdoors was (13.69), and indoors (3.38) (p = 0.006). No anophelines were documented resting on walls during IAs. HBO-adjusted human landing rates suggested that the highest risk of being bitten was outdoors between 18.00 and 20.00 h. Human behaviour-adjusted biting rates suggest that overall, long-lasting insecticidal bed nets (LLINs) only protected against 13.2% of exposure to bites, with 86.8% of exposure during the night spent outside of bed net protection. The malaria survey found 2/398 individuals positive for asymptomatic P. falciparum infections. The questionnaire reported high (73.4%) bed net use, with low knowledge of malaria. CONCLUSION: The exophagic feeding of anopheline vectors in San Jose de Chamanga, when analysed in conjunction with human behaviour, indicates a clear gap in protection even with high LLIN coverage. The lack of indoor-resting anophelines suggests that indoor residual spraying (IRS) may have limited effect. The presence of asymptomatic infections implies the presence of a human reservoir that may maintain transmission.

[Time series of non-work related sickness absence incidence by subgroups of duration (2009-2018)]. / Evolución temporal de la incidencia por incapacidad temporal de origen común según tramos de duración (2009-2018).

OBJECTIVE: We describe time trend incidence (2009-2018) of non-work related Sickness Absence(SA) segmented by duration of episodes in Spain. METHODS: We used SA cases from a health insurance company ("mutua") in Spain. Overall non-work related SA incidence and incidence by duration of episodes (1-3 days, 4-15 days, 16-30 days, 31-90 days and> 90 days) were obtained. A time series ecological study was carried out with an inflection point in 2013. The annual percentage of change and 95% confidence interval were obtained. The analyses were stratified by age and sex. RESULTS: Overall incidence went from 35.3 cases per 100 workers-years in 2009 to 25.2 in 2013. From 2014, there is a sustained rise in the incidence of ITCC, ending 2018 with 34.1 cases per 100 workers-years. The overall incidence is determined mainly by processes less than 16 days in young population. The decrease in 2009-2013 occurred in all the duration segments, especially in 4 to 15 (APC=-11,2; 95% CI=(-14,1 a -8,2)) and more than 90 days (APC =-9,4; 95% CI =(-15,5 a -2,8)), mainly in young people. The rise in 2013-2018 was observed in all the segments, with the largest significant increase in sections of 1-3 days(younger workers: APC =18,9; 95% CI =(14,8 a 23,2)) and in more than 90 days (mainly in older ones). Time trend of SA showed similar pattern in both sexes. CONCLUSIONS: Time trend analysis of SA incidence by duration segments offers a detailed information of SA. These results are useful for professionals in the prevention and management of SA.

OBJETIVO: Describir la evolución temporal (2009-2018) de la incidencia de la incapacidad temporal por contingencia común (ITCC) según tramos de duración en España. MÉTODOS: Se consideraron casos de ITCC de la población afiliada de una mutua laboral. Se obtuvo la incidencia global de la incapacidad temporal por ITCC y por tramos de duración (1-3 días, 4-15 días, 16-30 días, 31-90 días y >90 días). Se realizó un estudio ecológico de series temporales considerando un punto de inflexión en 2013. Se calculó el porcentaje de cambio anual e intervalo de confianza del 95%. Los análisis se estratificaron por edad y sexo. RESULTADOS: La incidencia global pasó de 35,3 casos por cada 100 trabajadores-año en 2009 a 25,2 en 2013. A partir del 2014 se observa un ascenso sostenido de la incidencia de ITCC, finalizando el 2018 con 34,1 casos por cada 100 trabajadores-año. La incidencia global está determinada fundamentalmente por procesos menores a 16 días en población joven. El descenso en 2009-2013 se produjo en todos los tramos de duración, en especial en 4 a 15 (PCA=-11,2; IC 95%=(-14,1 a -8,2)) y más de 90 días (PCA=-9,4; IC 95%=(-15,5 a -2,8)), principalmente en jóvenes. El ascenso en 2013-2018 se observó en todos los tramos, siendo el incremento más significativo en tramos de 1-3 días (trabajadores de menor edad: PCA=18,9; IC 95%=(14,8 a 23,2)) y en más de 90 días (principalmente en mayores). La evolución de la ITCC presentó un patrón similar en ambos sexos. CONCLUSIONES: La descripción temporal de la incidencia de ITCC por tramos de duración ofrece una información detallada de la incapacidad temporal. Estos resultados son útiles para profesionales de la prevención y gestión de la ITCC.

[The analysis of sickness absence incidence by subgroups of duration]. / Segmentación de la incidencia de la incapacidad temporal según su duración como estrategia de análisis.

BACKGROUND: We describe the usefulness of performing an analysis of sickness absence (SA) incidence, segmented by the duration of episodes, in comparison with the more common analysis that considers the overall incidence of SA without segmentation. METHODS: We used data from a health insurance company ("mutua") in Spain during 2011 (230,332 episodes, 752,906 workers) and non-work related SA as a case study. We compared the overall incidence of SA and incidence segmented by duration of episodes: short (≤ 15 days), medium (16-30 and 31-90 days) and long (> 90 days). The analyses were also performed by age, as an example of one of the multiple variables that affect SA incidence. RESULTS: The overall incidence of SA was 30.6%, and declined steadily with increasing age. When SA incidence was analyzed by duration, we observed that: 1) the incidence of the episodes of short duration is the highest; 2) the overall excess observed in younger workers (<25 years) is driven mainly by short duration and 3) the pattern for long-term SA incidence was reversed, being more frequent among those ≥55 years of age relative to the youngest. CONCLUSIONS: Examining SA incidence by duration is more informative than relying on overall incidence of SA.

INTRODUCCIÓN: Se describe la utilidad de realizar un análisis de la incidencia de incapacidad temporal (IT) segmentado según la duración de los episodios, en comparación con el análisis que considera la incidencia global de la IT. MÉTODOS: Se considera un ejemplo utilizando datos de una mutua en España durante 2011 (230.332 episodios, 752.906 trabajadores) y la incapacidad temporal por contingencia común (ITcc). Comparamos la incidencia de ITcc global y la segmentada por la duración de los episodios: corta (≤ 15 días), media (16-30 y 31-90) y larga (> 90 días). Los análisis se realizaron también según edad, como ejemplo de una de las múltiples variables que afectan a la incidencia de la IT. RESULTADOS: La incidencia global de ITcc fue del 30,6%, mostrando una disminución con la edad (36,4% en <25 años frente a 29,3% en ≥55 años (RR = 1,24, IC del 95% = 1,22 - 1,27)). El análisis de la incidencia de ITcc segmentado por la duración de los episodios, permite obtener resultados más específicos: 1) la incidencia de corta duración es la más alta respecto al resto de segmentos (20,1% en la corta frente a 2,8% en larga duración (RR = 7,29, IC 95% = 7,19 ­ 7,40); 2) el exceso global observado en jóvenes se produce mayoritariamente por los episodios cortos (RR = 1,98, IC del 95% = 1,93-2,03, en <25 años frente a ≥55 años), y 3) la incidencia de larga duración cambia este patrón, siendo más frecuente en ≥55 años (5,2%) en relación a jóvenes (1,6%)(RR = 0,31, IC 95% = 0,29-0,34). CONCLUSIONES: El análisis de la incidencia de IT segmentada por duración ofrece una aproximación más precisa que la obtenida del análisis global.

Efeitos da reabilitação pulmonar sobre a qualidade de vida: uma visão das crianças asmáticas e de seus pais / Effects of the pulmonary rehabilitation program on the quality of life of asthmaticchildren and their parents

Introdução: A determinação da qualidade de vida de crianças e adolescentes asmáticos é importante, pois a asma grave ou com sintomasmal controlados, impede a participação desses indivíduos em esportes, prejudica o sono e, conseqüentemente, o rendimento escolar.Entretanto a doença não tem somente um impacto sobre os pacientes, mas também afeta a qualidade de vida de indivíduos ligados a eles.Objetivo: Avaliar a qualidade de vida das crianças asm??ticas e a percepção dos pais/responsáveis quanto à qualidade de vida de seusfilhos antes e após um programa de reabilitação pulmonar (RP). Materiais e Métodos: Foram estudadas 5 crianças, sexo masculino, comidade média de 8,16 ± 1,83 anos e 6 pais/responsáveis, antes e após um programa de reabilitação de 24 sessões. Todas as crianças tinhamdiagnóstico clínico de asma leve e moderada. Foi aplicado o questionário Pediatric Asthma Quality of Life Questionnaire (PAQLQ)para as crianças e um segundo questionário específico para os pais. As pontuações numéricas dos questionários pré e pós RP, foramcomparadas pelo teste de Wilcoxon, sendo considerado um p < 0,05. Resultado: Não houve diferença significativa, na comparação dosresultados pré e pós RP Entretanto, verificou-se melhora absoluta na maioria das questões em ambos os questionários. Conclusão: Osquestionários de qualidade de vida aplicados às crianças asmáticas e aos seus pais/responsáveis, não detectaram variação significativa.Contudo, as variações absolutas em vários itens envolvendo os dois instrumentos sugerem uma melhora clínica na qualidade de vida em ambos os questionários.