Brunck, M. E. G. | Search bioRxiv

bioRxiv2022

A pseudotyped lentivirus-based assay to titer SARS-CoV-2 neutralizing antibodies in Mexico

Measuring the neutralizing potential of SARS-CoV-2 antigens-exposed sera informs on effective humoral immunity. This is relevant to 1-monitor levels of protection within an asymptomatic population, 2-evaluate the efficacy of existing and novel vaccines against emerging variants, 3-test prospective therapeutic monoclonal neutralizing antibodies (NAbs) and, overall, to contribute to understand SARS-CoV-2 immunity. However, the gold-standard method to titer NAbs is a functional assay of virus-mediated infection, which requires biosafety level 3 (BSL-3) facilities. As these facilities are insufficient in Latin American countries, including Mexico, scant information has been obtained about NAb in these countries during the COVID-19 pandemic. An alternative solution to acquire NAb information locally is to use non-replicative viral particles that display the SARS-CoV-2 Spike (S) protein on their surface, and deliver a reporter gene into target cells upon transduction. Here we present the development of a NAb-measuring assay based on Nanoluc-mediated luminescence measurements from SARS-CoV-2 S-pseudotyped lentiviral particle-infected cells. The successive steps of development are presented, including lentiviral particles production, target cell selection, and TCID50 determination. We applied the optimized assay in a BSL-2 facility to measure NAbs in 15 pre-pandemic, 18 COVID-19 convalescent and 32 BNT162b2 vaccinated serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. The assay highlighted heterogeneity in neutralization curves which are relevant in discussing neutralization potency dynamics. Overall, this is the first report of a BSL-2 safe functional assay to measure SARS-CoV-2 in Mexico and a cornerstone methodology necessary to measure NAb with a functional assay in the context of limited resources settings. ImportanceEvaluating effective humoral immunity against SARS-CoV-2 requires a functional assay with infectious virus. Handling the authentic SARS-CoV-2 virus requires specialized facilities that are not readily available in Latin America, including Mexico. Here we produce non-replicative viral particles pseudotyped with the SARS-CoV-2 S protein that are used as safe surrogate viral particles in an optimized BSL-2 ready neutralization assay. The establishment of this assay is critical to allow the evaluation of effective humoral immunity to SARS-CoV-2 post-infection and to monitor the efficacy of existing or novel vaccines against emerging variants in the Mexican population.

microbiology↗

bioRxiv2021

Insights into the trafficking of human leukocytes to colostrum evidences a modulation of the B lymphocyte compartment in obesity

Breastmilk is a dynamic fluid which initial goal is to provide the most adapted nutrition to the neonate. Additional functions have been recently attributed to breastmilk, with the evidence of a specific microbiota and the presence of a variety of components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to future health of the infant. Obesity is a rising condition worldwide, that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother. We applied a panel of 6 antibodies plus viability marker to the peripheral blood and colostrum obtained from obese (BMI > 30) and lean (BMI < 25) mothers to characterize 10 major leukocyte subpopulations using flow cytometry. While lymphoid cells were otherwise unaffected by their tissue of origin, the phenotypes of granulocyte and monocyte populations significantly contrasted between blood and colostrum, including variations in morphology and surface expression of CD45 and CD16. These differences recapitulated across groups, which suggests a generalized cell-specific phenotype alteration caused by trafficking to colostrum. The B lymphocyte compartment was significantly reduced in obese colostrum and these cells did not exhibit enhanced CD16 shedding in this tissue, unlike B lymphocytes from lean mothers colostrum. This is the first exhaustive characterization of major leukocyte subsets in obese mothers colostrum, and the first report of leukocyte subpopulations from Latin-American womens colostrum. This pioneering study is a steppingstone to further investigate active immunity in human breastmilk.

immunology↗