SARS-CoV-2 infected children form early immune memory responses dominated by nucleocapsid-specific CD8+ T cells and antibodies.

This is the third year of the SARS-CoV-2 pandemic, and yet most children remain unvaccinated. COVID-19 in children manifests as mostly mild or asymptomatic, however high viral titers and strong cellular and humoral responses are observed upon acute infection. It is still unclear how long these responses persist, and if they can protect from re-infection and/or disease severity. Here, we analyzed immune memory responses in a cohort of children and adults with COVID-19. Important differences between children and adults are evident in kinetics and profile of memory responses. Children develop early N-specific cytotoxic T cell responses, that rapidly expand and dominate their immune memory to the virus. Children's anti-N, but not anti-S, antibody titers increase over time. Neutralization titers correlate with N-specific antibodies and CD8+T cells. However, antibodies generated by infection do not efficiently cross-neutralize variants Gamma or Delta. Our results indicate that mechanisms that protect from disease severity are possibly different from those that protect from reinfection, bringing novel insights for pediatric vaccine design. They also underline the importance of vaccination in children, who remain at risk for COVID-19 despite having been previously infected.

Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses.

COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.

Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance.

In vitro drug screening is widely used in the development of new drugs, because they constitute a cost-effective approach to select compounds with more potential for therapy. They are also an attractive alternative to in vivo testing. However, most of these assays are done in two-dimensional culture models, where cells are grown on a polystyrene or glass flat surface. In order to develop in vitro models that would more closely resemble physiological conditions, three-dimensional models have been developed. Here, we introduce two novel fully synthetic scaffolds produced using the polymer polyhydroxybutyrate (PHB): a Solvent-Casting Particle-Leaching (SCPL) membrane; and an electrospun membrane, to be used for 3D cultures of B16 F10 murine melanoma cells and 4T1 murine breast cancer cells. A 2D cell culture system in regular tissue culture plates and a classical 3D model where cells are grown on a commercially available gel derived from Engelbreth-Holm Swarm (EHS) tumor were used for comparison with the synthetic scaffolds. Cells were also collected from in vivo tumors grown as grafts in syngeneic mice. Morphology, cell viability, response to chemotherapy and gene expression analysis were used to compare all systems. In the electrospun membrane model, cells were grown on nanometer-scale fibers and in the SCPL membrane, which provides a foam-like structure for cell growth, pore sizes varied. Cells grown on all 3D models were able to form aggregates and spheroids, allowing for increased cell-cell contact when compared with the 2D system. Cell morphology was also more similar between 3D systems and cells collected from the in vivo tumors. Cells grown in 3D models showed an increase in resistance to dacarbazine, and cisplatin. Gene expression analysis also revealed similarities among all 3D platforms. The similarities between the two synthetic systems to the classic EHS gel model highlight their potential application as cost effective substitutes in drug screening, in which fully synthetic models could represent a step towards higher reproducibility. We conclude PHB synthetic membranes offer a valuable alternative for 3D cultures.