Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis

AIM:To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.METHODS:The trinitrobenzene sulfonate(TNBS) model of induced colitis was used in Balb/c mice.Subsequently after intravenous adeno-associated virusmediated regulatory T-cell epitopes(Tregitope) delivery,acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS(0.75 mg in 20% ethanol) 8 d later.Control groups included mice not treated with TNBS(healthy control group) and mice treated by TNBS only(diseased group).At the time of sacrifice colon weight,the disease activity index and histology damage score were determined.Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3(Foxp3).Thymus,mesenteric lymph nodes,liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.RESULTS:The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice.In contrast,the mice treated with TNBS alone(no Tregitope) developed colitis,and lost 4% of their initial body weight at the time of sacrifice(P < 0.01).The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group(P < 0.01 and P < 0.001,respectively).Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells.For both TNBS treated groups CD4+ T cell infiltrates were observed in the sub-epithelial layer and the lamina propria.CD4+ T cell infiltrates were also present in the muscularis mucosa layer of the diseased control mice,but were absent in the Tregitope 167 treated group.Numerous Foxp3 positive cells were detected in the lamina propria and sub-epithelium of the colon sections from mice treated with Tregitope 167.Furthermore,the Foxp3 and glycoprotein A repetitions predominant markers were significantly increased in the CD4+ T lymphocyte population in the thymus of the mice pre-treated with adeno-associated virus serotype 5(cytomegalovirus promoter-Tregitope 167),as cytomegalovirus promoter compared to lymphocyte populations in the thymus of diseased and the healthy control mice(P < 0.05 and P < 0.001,respectively).CONCLUSION:This study identifies adeno-associated virus-mediated delivery of regulatory T-cell epitope 167 as a novel anti-inflammatory approach with the capacity to decrease intestinal inflammation and induce longterm remission in inflammatory bowel disease.

Mutations in Spike Gene of SARS-CoV-2 that Are Associated with a Higher Viral Load: A Clinical Case Study

Background: Quantitative PCR (qPCR) can be used to detect and quantify a load of a pathogen. It is a good indicator of the degree of transmissibility. While performing routine qPCR, we observed an unusually short cycle threshold (Ct) value of SARS-CoV-2 for a clinical specimen obtained in Bamako, Mali. This prompted us to sequence the short-cycle SARS-CoV-2 sample to identify potential mutations in the Spike gene (S gene) gene. Methods: Post-infection, Quantitative Reverse Transcription (qRT-PCR) was performed over a defined time course to estimate the Ct of the SARS-CoV-2 specimen collected from the patient. Sanger sequencing was done on the entire fragment of the S gene to identify mutations. Findings: Sanger sequencing revealed mutations in the lineage of interest, designated B.1.525 by Pango, and also known as “Eta” using the nomenclature defined by WHO. This variant was originally found in Nigeria and Italy. The four novel mutations identified in Eta (D228N, Y451N, I1172M, and C1250F) were otherwise observed with a low frequency worldwide. Although the initial Ct was 10 in the case study patient, he did not exhibit severe symptoms of SARS-CoV-2, for example, pneumonia. However, we observed a longer viral clearance period than usual, of 3 weeks. We note that as compared to SARS-CoV-2 samples obtained during the first peaks of SARS-CoV-2 infection in Mali, when the infection was at its peak in March 2020 (Ct = 30.4), circulating strains evaluated at the time the Eta sample was obtained demonstrated a lower mean Ct (Ct = 24). Conclusions: The short cycle threshold associated with this variant, and the temporal association with a decrease in the mean Ct in the region of Bamako, may indicate higher levels of transmissibility due to a circulating variant. This variant is a lineage of interest designated B.1.525 by Pango or Eta by WHO.