Development of a high throughput SARS-CoV-2 antibody testing pathway using dried blood spot specimens

Background: Serologic assays for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have roles in seroepidemiology, convalescent plasma-testing, antibody durability and vaccine studies. Currently, SARS-CoV-2 serology is performed using serum/plasma collected by venepuncture. Dried bloodspot (DBS) testing offers significant advantages; as it is minimally invasive, avoids venepuncture with specimens being mailed to the laboratory. Methods: A pathway utilising a newborn screening laboratory infrastructure was developed using an Enzyme-Linked Immunosorbent assay (ELISA) to detect IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in DBS specimens. Paired plasma and DBS specimens from SARS-CoV-2 antibody positive and negative subjects and PCR positive subjects were tested. DBS specimen stability, effect of blood volume and punch location were also evaluated. Results: DBS from antibody-negative (n=85) and positive (n=35) subjects and PCR positive subjects (n=11) had a mean (SD; range) optical density (OD) of 0.14 (0.046; 0.03-0.27), 0.98 (0.41; 0.31-1.64) and 1.12 (0.37; 0.49-1.54), respectively. An action value OD >0.28 correctly assigned all cases. The weighted Deming regression for comparison of the DBS and the plasma assay yielded: y=0.004041+1.005x, r=0.991, Sy/x 0.171, n=82. Extraction efficiency of antibodies from DBS was >99%. DBS were stable for at least 28 days at ambient room temperature and humidity. Conclusions: SARS-CoV-2 IgG RBD antibodies can be reliably detected in DBS. DBS serological testing offers lower costs than either point of care or serum/plasma assays that require patient travel, phlebotomy and hospital/clinic resources; the development of a DBS assay may be particularly important for resource poor settings

CD4(+)CD25(+)FOXP3(+) Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy

Characterization of the human Treg population.

<p>(A) CD4⁺CD25⁻, CD4⁺CD25^int and CD4⁺CD25^hi cells were identified as shown. CD4⁺CD25^hi cells are those where CD25 expression was higher than that on the CD4⁻ population. (B) FOXP3 expression within each population was assessed by intracellular staining. (C) Freshly isolated CD4⁺CD25⁻ T cells (2×10⁴ cells/well) were cultured alone (CD25-/low) or with CD4⁺CD25^hi T cells at various ratios, and stimulated with plate-bound anti-CD3 and soluble anti-CD28. Proliferation was assessed by (³H)-thymidine incorporation. The results represent the average of triplicate wells with standard deviation, from a representative assay.</p

CRC patients are able to mount CD4⁺ T cell responses to the recall antigens PPD and HA.

<p>Whole PBMC from 11 age-matched controls and 14 CRC patients were assessed for responses to the antigens PPD and HA, measured by IFN-γ ELISPOT. Purified PBMC were added at 3.5×10⁵ cells/well, and incubated for 18 hours in the presence of either 1 µg/ml PPD, 1 µg/ml HA or with no antigen. Wells were set up in duplicate. Fewer than 5 spot forming cells/10⁶ PBMC was considered negative.</p

Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort (Nature, (2020), 583, 7814, (90-95), 10.1038/s41586-020-2265-1)

An amendment to this paper has been published and can be accessed via a link at the top of the paper