Phage therapy is an experimental therapeutic approach used to target multidrug-resistant bacterial infections. A lack of reliable data with regard to its efficacy and regulatory hurdles hinders a broad application. Here we report, for the first time, a case of vancomycin-resistant Enterococcus faecium abdominal infection in a one-year-old, critically ill, and three times liver transplanted girl, which was successfully treated with intravenous injections (twice per day for 20 days) of a magistral preparation containing two Enterococcus phages. This correlated with a reduction in baseline C-reactive protein (CRP), successful weaning from mechanical ventilation and without associated clinical adverse events. Prior to clinical use, phage genome was sequenced to confirm the absence of genetic determinants conferring lysogeny, virulence or antibiotic resistance, and thus their safety. Using a phage neutralization assay, no neutralizing anti-phage antibodies in the patient’s serum could be detected. Vancomycin-susceptible E. faecium isolates were identified in close relation to phage therapy and, by using whole-genome sequencing, it was demonstrated that vancomycin-susceptible E. faecium emerged from vancomycin-resistant progenitors. Covering a one year follow up, we provide further evidence for the feasibility of bacteriophage therapy that can serve as a basis for urgently needed controlled clinical trials.

The situation of limited data concerning the response to COVID-19 mRNA vaccinations in immunocom-promised children hinders evidence-based recommendations. This prospective observational study investigated humoral and T cell responses after primary BNT162b2 vaccination in secondary immunocompromised and healthy children aged 5–11 years. Participants were categorized as: children after kidney transplantation (KTx, n = 9), proteinuric glomerulonephritis (GN, n = 4) and healthy children (controls, n = 8). Expression of activation-induced markers and cytokine secretion were determined to quantify the T cell response from PBMCs stimulated with peptide pools covering the spike glycoprotein of SARS-CoV-2 Wuhan Hu-1 and Omicron BA.5. Antibodies against SARS-CoV-2 spike receptor-binding domain were quantified in serum. Seroconversion was detected in 56% of KTx patients and in 100% of the GN patients and controls. Titer levels were significantly higher in GN patients and controls than in KTx patients. In Ktx patients, the humoral response increased after a third immunization. No differences in the frequency of antigen-specific CD4+ and CD8+ T cells between all groups were observed. T cells showed a predominant anti-viral capacity in their secreted cytokines; however, this capacity was reduced in KTx patients. This study provides missing evidence concerning the humoral and T cell response in immunocompromised children after COVID-19 vaccination.

Pediatric SARS‐CoV‐2 infection is often mild or asymptomatic and the immune responses of children are understudied compared to adults. Here, we present and evaluate the performance of a two‐panel (16‐ and 17 parameter) flow cytometry‐based approach for immune phenotypic analysis of cryopreserved PBMC samples from children after SARS‐CoV‐2 infection. The panels were optimized based on previous SARS‐CoV‐2 related studies for the pediatric immune system. PBMC samples from seven SARS‐CoV‐2 seropositive children from early 2020 and five age‐matched healthy controls were stained for analysis of T‐cells (panel T), B and innate immune cells (panel B). Performance of the panels was evaluated in two parallel approaches, namely classical manual gating of known subpopulations and unbiased clustering using the R‐based algorithm PhenoGraph. Using manual gating we clearly identified 14 predefined subpopulations of interest for panel T and 19 populations in panel B in low‐volume pediatric samples. PhenoGraph found 18 clusters within the T‐cell panel and 21 clusters within the innate and B‐cell panel that could be unmistakably annotated. Combining the data of the two panels and analysis approaches, we found expected differentially abundant clusters in SARS‐CoV‐2 seropositive children compared to healthy controls, underscoring the value of these two panels for the analysis of immune response to SARS‐CoV‐2. We established a two‐panel flow cytometry approach that can be used with limited amounts of cryopreserved pediatric samples. Our workflow allowed for a rapid, comprehensive, and robust pediatric immune phenotyping with comparable performance in manual gating and unbiased clustering. These panels may be adapted for large multi‐center cohort studies to investigate the pediatric immune response to emerging virus variants in the ongoing and future pandemics.

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN- γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

Almost two years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children <18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44 %), the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow up of household contacts showed incomplete seroconversion in most families, with higher seroconversion rates in families with adult index cases compared to pediatric index cases (OR: 1.79, P=0.047). Most importantly, children showed sustained seroconversion up to nine months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG Spike children vs. adults 90 days PSO: 1.75, P<0.001, 180 days: 1.38, P=0.01, 270 days: 1.54, P=0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroconversion rates in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.