Cost-effective serological test to determine exposure to SARS-CoV-2: ELISA based on the receptor-binding domain of the spike protein (Spike-RBDN318-V510) expressed in Escherichia coli

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this aim, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical. Here, we report the bacterial production of the peptide S-RBDN318-V510, which contains the receptor binding domain of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S RBDN318 V510 and a commercial full-length spike protein were compared in two distinct ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBDN318-V510. Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBDN318-V510 and the full length spike protein. Our results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures. S-RBDN318-V510 is amenable to massive production and may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing.

Initial experience in Mexico with convalescent plasma in COVID-19 patients with severe respiratory failure, a retrospective case series

IntroductionHospital mortality due to COVID-19 in Mexico is high (32%) and as of today, effective treatment options are limited. More effective treatments that shorten hospital stay and reduce mortality are needed. Initial reports for the use of convalescent plasma (CP) therapy for COVID-19 appear promising. We describe a case series of eight patients with impending respiratory failure, who underwent CP therapy. MethodsSix male and two female (ages 31 to 79) patients that were admitted to the intensive-care unit for severe COVID-19 were transfused with two doses of CP (250 mL per dose, anti-SARS-CoV-2 IgG titers > 1:100). Donors were six SARS-CoV-2 infected males who remained asymptomatic for > 7 days and were negative for two nasopharyngeal RT-PCR tests. Clinical characteristics, inflammatory and cellular injury markers, chest X-ray findings and viral loads were analyzed before and after CP administration. Viral load association to disease severity was further analyzed on a separate cohort of asymptomatic vs hospitalized patients with COVID-19. ResultsEight patients with respiratory failure were successfully discharged with a median length of stay of 22.5 (IQR 18.25-29.00). After CP therapy, we observed a reduction of C-reactive protein (CRP) (median, 22.80 mg/dL vs. 1.63 mg/dL), and of procalcitonin (median, 0.27 ng/mL vs. 0.13 ng/mL). High-Sensitivity Cardiac Troponin I (hs-cTnI), Brain Natriuretic Peptide (BNP) and Lactate Dehydrogenase (LDH) were lower, and a mild reduction of pulmonary infiltrates by chest X-ray was observed. Lastly, a reduction of viral load was after CP therapy was found. (log, median [IQR], 1.2 [0.70-2.20] vs. 0.25 [0.00-1.78]). We observed no adverse effects. ConclusionsCP could potentially be an effective therapeutic option for patients with severe COVID-19. Clinical benefit needs to be studied further through randomized controlled trials.

Tumor necrosis factor alpha promoter-308G/A polymorphism in Mexican patients with patchy alopecia areata.

BACKGROUND: Alopecia areata (AA) is a chronic inflammatory condition characterized by hair loss, most frequently from the scalp. Its etiopathogenesis is currently unknown, but inflammatory traits and associations with autoimmune diseases suggest that AA shares a similar origin. The tumor necrosis factor alpha (TNFα) gene, located on chromosome 6 within the major histocompatibility complex class III gene, may carry previously described polymorphisms--particularly in the promoter region, such as TNFα-308G/A--known to be risk factors in a wide variety of inflammatory pathologies. In Mexican populations, this polymorphism has been associated with augmented TNFα production and, thus, renders carriers more susceptible to developing autoimmune diseases; however, as yet it has not been associated with AA. OBJECTIVES: To assess a possible association between the presence of TNFα-308G/A and patchy AA. MATERIALS AND METHODS: Blood samples were taken from 59 patients affected by patchy AA and 103 control subjects without AA, all from the northeastern Mexican population. Genomic DNA was isolated using the phenol-chloroform method and samples subjected to polymerase chain reaction-restriction fragment length polymorphism in order to detect the TNFα-308G/A polymorphism. RESULTS: TNFα-308G/A (TNF2) allele [odds ratio (OR) = 3.22, P = 0.026, 95% confidence interval (CI) = 0.99-11.61], when segregated in the heterozygous (TNF1/TNF2) genotype (OR = 3.53, P = 0.023, 95% CI = 1.01-12.89) confers a significant risk for developing AA, compared with the genotype TNF1/TNF1 observed in controls (OR = 0.28, P = 0.023, 95% CI = 0.08-0.99). CONCLUSIONS: Our data suggest that there is a plausible association between the presence of the TNFα-308G/A polymorphism and a higher susceptibility for developing patchy AA. This risk might be due to overproduction of TNFα, which would facilitate an autoimmune response against the hair follicle.