BackgroundThere is limited information on the functional neutralizing capabilities of breastmilk SARS-CoV-2-specific antibodies and the potential adulteration of breastmilk with vaccine mRNA after SARS-CoV-2 mRNA vaccination.MethodsWe conducted a prospective cohort study of lactating healthcare workers who received the BNT162b2 vaccine and their infants. The presence of SARS-CoV-2 neutralizing antibodies, antibody isotypes (IgG, IgA, IgM) and intact mRNA in serum and breastmilk was evaluated at multiple time points using a surrogate neutralizing assay, ELISA, and PCR, over a 6 week period of the two-dose vaccination given 21 days apart.ResultsThirty-five lactating mothers, median age 34 years (IQR 32-36), were included. All had detectable neutralizing antibodies in the serum immediately before dose 2, with significant increase in neutralizing antibody levels 7 days after this dose [median 168.4 IU/ml (IQR 100.7-288.5) compared to 2753.0 IU/ml (IQR 1627.0-4712.0), p <0.001]. Through the two vaccine doses, all mothers had detectable IgG1, IgA and IgM isotypes in their serum, with a notable increase in all three antibody isotypes after dose 2, especially IgG1 levels. Neutralizing antibodies were detected in majority of breastmilk samples a week after dose 2 [median 13.4 IU/ml (IQR 7.0-28.7)], with persistence of these antibodies up to 3 weeks after. Post the second vaccine dose, all (35/35, 100%) mothers had detectable breastmilk SARS-CoV-2 spike RBD-specific IgG1 and IgA antibody and 32/35 (88.6%) mothers with IgM. Transient, low intact vaccine mRNA levels was detected in 20/74 (27%) serum samples from 21 mothers, and 5/309 (2%) breastmilk samples from 4 mothers within 1 weeks of vaccine dose. Five infants, median age 8 months (IQR 7-16), were also recruited - none had detectable neutralizing antibodies or vaccine mRNA in their serum.ConclusionMajority of lactating mothers had detectable SARS-CoV-2 antibody isotypes and neutralizing antibodies in serum and breastmilk, especially after dose 2 of BNT162b2 vaccination. Transient, low levels of vaccine mRNA were detected in the serum of vaccinated mothers with occasional transfer to their breastmilk, but we did not detect evidence of infant sensitization. Importantly, the presence of breastmilk neutralising antibodies likely provides a foundation for passive immunisation of the breastmilk-fed infant.

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Regulatory T cells (Tregs) are often enriched in tumors, where their immunosuppressive function has a key role in tumor persistence and progression. In colorectal cancer (CRC), however, Tregs are frequently associated with an improved clinical outcome. Tumor-infiltrating Tregs have been shown to exhibit a distinct signature comprising the co-stimulatory molecules (OX40, 4-1BB), cytokine receptors (IL1R2, IL21R, CCR8, CD30), and co-inhibitory molecules (PD-L1, TIGIT). Here, we showed by flow cytometry that circulating CD45RO+ Tregs from patients with CRC (n = 25) have elevated CD30 and OX40 expression compared to healthy subjects (n = 14). We identified co-expression of CD30 and OX40 on circulating CD45RO+ Tregs using single-cell images captured by the DEPArray™ system. The frequency of CD30+OX40+CD45RO+ Tregs was significantly higher in CRC patients than in healthy subjects (P < 0.001). Importantly, receiver operating characteristic analysis confirmed that this CD30+OX40+ Treg subset could strongly discriminate between CRC patients and healthy subjects with the highest accuracy of 92.3%, an AUC of 0.92, a sensitivity of 88%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 82.35%, and a trade-off value of 3.44%, compared to other Treg subsets. Consistently, multiplex-IHC/IF of tumor-infiltrating Tregs revealed a significant association between high densities of CD30+OX40+ Tregs and improved overall survival; no such association was found for other subsets. These data suggest a potential role for CD30+OX40+ Tregs as a diagnostic or prognostic biomarker in CRC.

An accurate depiction of the convalescent COVID-19 immunome will help delineate the immunological milieu crucial for disease resolution and protection. Using mass cytometry, we characterized the immune architecture in patients recovering from mild COVID-19. We identified a virus-specific immune rheostat composed of an effector T (Teff) cell recall response that is balanced by the enrichment of a highly specialized regulatory T (Treg) cell subset. Both components were reactive against a peptide pool covering the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We also observed expansion of IFNγ+ memory CD4+ T cells and virus-specific follicular helper T (TFH) cells. Overall, these findings pinpoint critical immune effector and regulatory mechanisms essential for a potent, yet harmless resolution of COVID-19 infection.

Background:We created a high dimensionality healthy human Immunome atlas by interrogating the peripheral blood mononuclear cells (PBMC) of >200 healthy subjects (cord blood to adult) with 63 unique mechanistic and phenotypic markers per cell by mass cytometry (CyTOF). This database is built with an open source, web-based bioinformatics toolkit, enabling its mining and uploading of datasets for comparison with the EPIC healthy database.Objectives:Here, we demonstrate the platform’s ability to identify the immunological differences of mechanistically important cell subsets in the uploaded data in comparison with EPIC.Methods:CyTOF data from 37 healthy elderly (>60 years old) was uploaded onto the EPIC Discovery tool where down-sampling, normalising and FlowSOM (Flow analysis with Self-Organising Maps) clustering were done with the EPIC database for comparison. Online visualisation outputs include cluster frequency boxplots, correspondence analysis (CA) plot and markers expression heat-map. The CA 2-dimensional plot depicts the global differences in immune cells composition between subjects with proximity between points (subjects) denoting similarity. Kruskal-Wallis test was done to identify age groups differences.Results:Increasing distances on the CA plot with age were observed with the elderly being farthest from the new-borns. Notably, we observed significant changes in naive CD4+IL8+T cells (p<1×10-20), memory CD4+IL17A+T cells (p<1×10-20) and type 2 innate lymphoid cells (ILC2) (Lin-CD7+CD25+CD127+CD161+, p<1×10-17) with increasing age. The naive CD4+IL8+T cells (median: 0.68%, interquartile range: 0.415 to 1.055% of CD45+ PBMC) and ILC2 (0.09%, 0.065 to 0.12%) were lowest and memory IL17A+T cells (0.58%, 0.41 to 0.905%) highest in the elderly. Significantly, the memory IL17A+T cells and ILC2 have been implicated in the pathogenesis of auto-immune conditions1,2.Conclusion:With EPIC, we have created an online tool enabling data uploading for comparison to a healthy database, allowing the holistic characterisation of immunological changes in different clinical scenarios. Using it, we were able to identify mechanistically important differences in immune cells composition in a distinct clinical cohort (elderly) compared to the younger ages. Translationally, the EPIC platform can be utilised similarly to catalyse the discovery process in auto-immune diseases interrogated with the EPIC antibody panels.References:[1]Fasching P, Stradner M, Graninger W, Dejaco C, Fessler J. Therapeutic Potential of Targeting the Th17/Treg Axis in Autoimmune Disorders. Molecules. 2017 Jan 14;22(1). pii: E134.[2]Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nat Immunol. 2016 Jun 21; 17(7): 765-74.Disclosure of Interests:None declared

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Objectives To evaluate the humoral immunogenicity for 6 months after the two-dose COVID-19 mRNA vaccination in adolescents and young adults (AYAs) with childhood-onset rheumatic diseases (cRDs). Methods This monocentric observational study was conducted between August 2020 to March 2022. Humoral immunogenicity was assessed at 2–3 weeks after first vaccine dose and 1, 3, and 6 months after the second dose by the cPass™ SARS-CoV-2 Neutralisation Antibody (nAb) Assay. An inhibition signal of ≥ 30% defined seroconversion threshold and the readings calibrated against the World Health Organisation (WHO) International Standard for SARS-CoV-2 antibodies. Results 169 AYAs with cRDs were recruited (median age 16·8 years (IQR : 14·7–19·5), 52% female, 72% Chinese). Juvenile Idiopathic Arthritis (JIA) (58%) and Systemic Lupus Erythematosus (18%) comprised the major diagnoses. After second vaccine dose, 99% seroconverted with a median nAb titre of 1779·8 IU/ml (IQR : 882·8–2541·9), declining to 935·6 IU/ml (IQR : 261·0–1514·9) and 683·2 IU/ml (IQR : 163·5–1400·5) at the 3- and 6-month timepoints respectively. The diagnosis of JIA (OR 10·1, 95%CI 1·8–58·4, p= 0·010) and treatment with anti-Tumour Necrosis Factor-α (aTNF) (OR 10·1, 95%CI 1·5–70·0, p= 0·019) were independently associated with a &gt; 50% drop of nAb titres at 6 months. Withholding methotrexate or mycophenolate mofetil did not affect the vaccine response or decay rate. The COVID-19 breakthrough infection was estimated at 18·2 cases/1000 patient-months with no clinical risk factors identified. Conclusion Over half of AYAs with cRDs had a significant drop in SARS-CoV-2 nAb at 6-month despite an initial robust humoral response. JIA and aTNF usage are predictors of a faster decay rate.

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