18-month longitudinal SARS COV-2 neutralizing antibody dynamics in haemodialysis patients receiving heterologous 3-dose vaccination (AZD-1222- AZD-1222- BNT162b2) in a lower middle income setting.

BACKGROUND: Patients with chronic kidney disease on haemodialysis (HD) were given priority COVID-19 vaccination due to increased disease risk. The immune response to COVID-19 vaccination in patients on HD was diminished compared to healthy individuals in 2-dose studies. This study aimed to evaluate seroconversion rate, neutralizing antibody (nAB) levels and longitudinal antibody dynamics to 3-dose heterologous vaccination against COVID-19 in a cohort of HD patients compared to healthy controls and assess patient factors associated with antibody levels. METHODS: This study was a case-control longitudinal evaluation of nAB dynamics in 74 HD patients compared to 37 healthy controls in a low/middle income setting. Corresponding samples were obtained from the two cohorts at time-points (TP) 1-1-month post 2nd dose of AZD1222 vaccine, TP2- 4 months post 2nd dose, TP4- 2 weeks post 3rd dose with BNT162b2 vaccine, TP5-5 months post 3rd dose and TP6-12 months post 3rd dose. Additional data is available at TP0- pre 2nd dose and TP3- 6 months post 2nd dose in HC and HD cohorts respectively. Anti-SARS-CoV-2 nAB were detected using Genscript cPassTM pseudoviral neutralization kit. Demographic and clinical details were obtained using an interviewer administered questionnaire. RESULTS: Cohorts were gender matched while mean age of the HD cohort was 54.1yrs (vs HCs mean age, 42.6yrs, p < 0.05). Percentage seroconverted and mean/median antibody level (MAB) in the HD cohort vs HCs at each sampling point were, TP1-83.7% vs 100% (p < 0.05), MAB-450 IU/ml vs 1940 IU/ml (p < 0.0001); TP2-71.4% vs 100%, (p < 0.001), MAB- 235 IU/ml vs 453 IU/ml, (p < 0.05); TP4-95.2% vs 100% (p > 0.05), MAB-1029 IU/ml vs 1538 IU/ml (p < 0.0001); TP5-100% vs 100%, MAB-1542 IU/ml vs 1741IU/ml (p > 0.05); TP6-100% vs 100%, MAB-1961 IU/ml vs 2911 IU/ml (p > 0.05). At TP2, patients aged < 60 years (p < 0.001) were associated with maintaining seropositivity compared to patients > 60 years. CONCLUSION: Two dose vaccination of haemodialysis patients provided poor nAB levels which improved markedly following 3rd dose vaccination, the effect of which was long- lasting with high nAB levels in both patients and controls detectable at 1 year follow-up.

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

CD161 expression defines new human γδ T cell subsets.

γδ T cells are a highly versatile immune lineage involved in host defense and homeostasis, but questions remain around their heterogeneity, precise function and role during health and disease. We used multi-parametric flow cytometry, dimensionality reduction, unsupervised clustering, and self-organizing maps (SOM) to identify novel γδ T cell naïve/memory subsets chiefly defined by CD161 expression levels, a surface membrane receptor that can be activating or suppressive. We used middle-to-old age individuals given immune blockade is commonly used in this population. Whilst most Vδ1+subset cells exhibited a terminal differentiation phenotype, Vδ1- subset cells showed an early memory phenotype. Dimensionality reduction revealed eight γδ T cell clusters chiefly diverging through CD161 expression with CD4 and CD8 expression limited to specific subpopulations. Comparison of matched healthy elderly individuals to bronchiectasis patients revealed elevated Vδ1+ terminally differentiated effector memory cells in patients potentially linking this population with chronic proinflammatory disease.