Signatures of B Cell Receptor Repertoire Following Pneumocystis Infection

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.

Single-Cell TCR Sequencing Reveals the Dynamics of T Cell Repertoire Profiling During Pneumocystis Infection

T cell responses play critical roles in host adaptive immunity against Pneumocystis. However, the dynamics and diversity of the T cell immune repertoire in human immunodeficiency virus (HIV)-negative Pneumocystis pneumonia (PCP) remains unclear. In this study, single-cell RNA and single-cell T cell receptor (TCR) sequencing were applied to cells sorted from lung tissues of mice infected with Pneumocystis. Our findings demonstrated the clonal cells were mainly composed of CD4+ T cells in response to Pneumocystis, which were marked by highly expressed genes associated with T cell activation. Mice infected with Pneumocystis showed reduced TCR diversity in CD4+ T cells and increased diversity in CD8+ T cells compared with uninfected controls. Furthermore, Th17 cells were mostly clonal CD4+ T cells, which exhibited the phenotype of tissue-resident memory-like Th17 cells. In addition, Pneumocystis-infected mice showed biased usage of TCRβ VDJ genes. Taken together, we characterized the transcriptome and TCR immune repertoires profiles of expanded T cell clones, which demonstrate a skewed TCR repertoire after Pneumocystis infection.

PREVALENCE OF PNEUMOCYSTIS JIROVECII IN FREQUENTLY ILL CHILDREN ON ADMISSION TO THE RESPIRATORY DEPARTMENT AND DURING SANATORIUM REHABILITATION

The aim of the study was to determine the frequency of occurrence and to assess the significance of markers of pneumocystosis in frequently ill children (FIC) during inpatient treatment and during rehabilitation in a sanatorium. Materials and methods of research: 115 children with recurrent respiratory diseases were observed: 52 were treated in a hospital and 63 underwent rehabilitation in a sanatorium. Biological samples (oropharyngeal swabs, saliva, feces and blood serum) were examined for pneumocystosis. Laboratory techniques used: a polymerase chain reaction (PCR) to detect DNA pathogens, an indirect immunofluorescence reaction (NRIF) – antigens, and immunoforment analysis (IFA) – specific antibodies of immunoglobulins (Ig) of classes M (IgM) and G (IgG). The material for laboratory research was smears from the posterior pharyngeal wall (PCR, NRIF) and blood serum (IFA). Microbiological studies included seeding, isolation of cultures with quantitative counting of colony-forming units in samples of oropharyngeal swabs, feces. Determination of the functional activity of the oropharynx microbiocenosis by the concentration of short-chain fatty acids in saliva by means of GLC method. Phagocytic activity of blood neutrophils was also investigated. Results: in hospital patients, markers for pneumocystis were found in 60% of cases, including the active form (40%) and pneumocystis (13%); in the sanatorium – 33% of cases, with active infection (26%) and detection of pneumocystis (17%). Inpatients were diagnosed with clinical variants of pneumocystis infection: pneumonia, obstructive bronchitis, acute respiratory infections; schoolchildren have been diagnosed with active pneumocystis infection and carriage. Dysbiotic disorders of the loci of the oropharynx and intestines were identified in all examined subjects, inhibition of the phagocytic activity of blood neutrophils and shifts in the concentration of short-chain fatty acids have been observed. Conclusion: a high proportion of active pneumocystis infection (40% and 27% of cases) was revealed in FIC treated in the hospital and in the sanatorium, which represents a risk group for infection/reinfection with pneumocystosis in the premises.

Acquired Immunodeficiency from Maternal Chemotherapy and Severe Primary Pneumocystis Infection in an Infant

Pneumocystis jirovecii is recognized as an opportunistic pathogen in immunosuppressed patients. We report a case of severe Pneumocystis pneumonia (PCP) in an infant with acquired combined immunodeficiency secondary to maternal chemotherapy exposure during the second and third trimesters of pregnancy. The infant required cardiorespiratory support with veno-venous extracorporeal membrane oxygenation (VV-ECMO) for severe respiratory failure. This case highlights the potential for severe acquired immunodeficiency in this patient cohort and further postnatal surveillance is highly recommended.

Rezafungin Prevention of Pneumocystis Pneumonia and Pneumocystis reactivation Using Different Doses and Durations of Prophylaxis in a Mouse Model

Rezafungin (RZF) is a novel echinocandin in development for prevention of invasive fungal disease caused by Candida, Aspergillus, and Pneumocystis species in patients undergoing blood and marrow transplantation. RZF has a favorable safety and tolerability profile, a low risk of drug-drug interactions and, with its stability, pharmacokinetics that allow for once-weekly dosing with broad distribution to the lung and other fungal target organs. We previously demonstrated RZF efficacy in preventing Pneumocystis infection in an immunosuppressed mouse model. The present study addressed whether Pneumocystis infection in a similar immunosuppressed mouse model could re-activate after 2 to 8 wks of prophylactic therapy using different dosing regimens of RZF. C3H/HeN mice were infected with P. murina intranasally at 2 x 106/50 µl after immunosuppression with dexamethasone. Mice were administered vehicle as a negative control (C/S), trimethoprim/sulfamethoxazole (T/S) as a positive control, caspofungin (CASPO) as a comparator echinocandin or RZF intraperitoneally at time of challenge. Study drug administration was stopped at 2, 4, 6, and 8 weeks, at which time mice were immunosuppressed for an additional 6 weeks to allow any residual P. murina to re-activate. Mice were then euthanized and lungs were processed for analysis of nuclei and asci. All RZF dose regimens at all timepoints significantly reduced both nuclei and asci burdens versus the C/S group (Figure 1). After 4 weeks of RZF prophylaxis (plus 6 wks additional immunosuppression [week 10 timepoint]; Figure 1), both RZF 20 mg/kg groups (3x/wk and 1x/wk) had prevented P. murina organisms from activating an infection. After 6 and 8 weeks of RZF prophylaxis (week 12 and 14 timepoints; Figure 1), no re-activation of infection was present in any of the study groups. After 2 and 4 weeks of prophylaxis (week 8 and 10 timepoints), there was a significant reduction of nuclei and asci counts in all groups of RZF versus CASPO. After 2, 4, and 6 weeks of RZF prophylaxis, there was a significant reduction of nuclei and asci counts between all groups of RZF versus T/S 1x/week. There was a significant benefit in survival between the RZF group at 20 mg/kg/3x/week versus CASPO at week 14 (Figure 2). In this study, prophylaxis with RZF for as little as 4 weeks prevented P. murina organisms from developing infection after cessation of therapy and showed more efficacy than CASPO. These results in the mouse model of Pneumocystis pneumonia provide evidence that RZF can prevent Pneumocystis reactivation and that such regimens hold promise for prophylaxis against Pneumocystis in at-risk patients undergoing blood and marrow transplantation. Disclosures Cushion: Cidara Therapeutics: Consultancy, Research Funding.

Pneumocystis Infection Outbreaks in Organ Transplantation Units in France: A Nation-Wide Survey

The burden of nosocomial Pneumocystis infections in transplantation units in France was evaluated through a retrospective survey. Over 12 years, 16 outbreaks occurred, including 13 among renal transplant recipients (RTRs). We performed Pneumocystis jirovecii genotyping in 5 outbreaks, which suggested that specific strains may have been selected by RTRs.