Correlation between levels of anti-cytokine antibodies, recombination activity of the mutant RAG proteins and clinical phenotype in patients with RAG deficiency

RAG mutations cause various phenotypes: SCID, Omenn syndrome (OS), leaky SCID (LS) and combined immunodeficiency (CID). We had previously reported autoantibodies targeting IFN-alpha, IFN-omega in patients with RAG deficiency. However, how the presence of such antibodies correlated with the severity of the clinical phenotype and with the recombination activity of the mutant proteins was unknown. To address this, we have studied anti-cytokine antibodies in 118 patients with RAG defects (SCID, n = 28;OS, n = 29;LS, n = 29;CID, n = 32), and in 42 controls (protocols NCT03394053 and NCT03610802). RAG mutant proteins associated with CID and LS retained 35.6 +/- 4.3 (mean +/- SE) and 29.8 +/- 5.1% recombination activity respectively, compared to wildtype protein, which was significantly higher than the recombination activity of the mutant RAG proteins associated with OS (4.1 +/- 1.5%) and SCID (5.7 +/- 2.1%) (p < 0.0001). Among 32 CID patients, 24 tested positive for anti-IFN-alpha and 21 for anti-IFN-omega antibodies. Among 29 LS patients, 15 had high levels of anti-IFN-alpha and 13 of anti-IFN-omega antibodies. A minority of the CID and LS patients had also high levels of anti-IFN-beta and anti-IL-22 antibodies. By contrast, none of the OS patients tested positive for anti-cytokine antibodies. High levels of anti-IFN-alpha and anti-IFN-omega antibodies correlated with their neutralizing activity as demonstrated in vitro by analysis of STAT1 phosphorylation upon stimulation of healthy donor monocytes in the presence of the appropriate cytokine and patient's or control plasma. Severe viral infections were recorded in 26/41 patients with CID and LS who tested positive and in 7/20 who tested negative for anti-IFN-alpha and/or anti-IFN-omega antibodies (p <0.05). Among those with anti-IFN antibodies, EBV (n = 8), CMV (n = 6), HSV (n = 5), VZV (n = 4) and adenovirus (n = 4) infections were more common. Two patients had COVID-19, which was fatal in one. Presence of the rubella virus was documented in 5 patients with anti-type I IFN antibodies. These results demonstrate that high levels of neutralizing anti-IFN-alpha and anti-IFN-omega antibodies are common in patients with RAG mutations manifesting as CID and LS, but not in those with OS, and that their presence is associated with a high risk of serious viral infections.Copyright © 2023 Elsevier Inc.

Association of anosmia and neutralizing antibody production in adolescents with SARS-CoV-2.

BACKGROUND: To monitor olfactory/gustatory dysfunction and its relationship to SARS-CoV-2 IgG antibody responses in an adolescent population. METHODS: Adolescents with changes in olfactory/gustatory functions were enrolled in a 15-month study. The patients were evaluated with 1) SNOT-22, 2) pediatric smell wheel, and 3) SARS-CoV-2 antibody testing. The relationship between these scores and length of anosmia, and the amount of SARS-CoV-2 IgG antibodies were assessed. A brain MRI was performed in cases of persistent special sensory symptoms. RESULTS: Eighteen patients were identified with smell and/or taste complaints. Most of the patients were female (67%) and median age was 15 years (range 11-17). Twelve patients had prior SARS-CoV-2 PCR testing, with only five patients with a positive result. The median SNOT-22 score was 16 (range 0-52) and the median smell wheel score was 6.5 (range 1-11). Patients with taste difficulty were more likely to have a score less than eight. 78% of the patients tested positive for antibodies and there was a strong negative correlation between smell wheel score and antibody level (Spearman, ρ = -0.798, p = 0.002). Five patients underwent MRI scan, and all resulted as normal olfactory bulb structures. 66% received nasal corticosteroids. 11 patients presented in follow up. CONCLUSIONS: Adolescents presenting to a pediatric ENT clinic during the SARS-CoV-2 pandemic were likely to have prolonged (>6 weeks) symptoms of SARS-CoV-2. The majority do not report positive PCR testing result but do report systemic symptoms including anosmia. This suggests that anosmia may be both a late and prolonged symptom of SARS-CoV-2.

RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq.

As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.