Protocol for the development of mRNA lipid nanoparticle vaccines and analysis of immunization efficiency in mice.

Here, we present a protocol for the development of mRNA-loaded lipid nanoparticle (LNP) vaccines for target antigen sequences of interest. We describe key steps required to design and synthesize mRNA constructs, their LNP encapsulation, and mouse immunization. We then detail quality control assays to determine RNA purity, guidelines to measure RNA immunogenicity using in vitro reporter systems, and a technique to evaluate antigen-specific T cell responses following immunization.

T cell epitope mapping reveals immunodominance of evolutionarily conserved regions within SARS-CoV-2 proteome.

As SARS-CoV-2 variants continue to emerge capable of evading neutralizing antibodies, it has become increasingly important to fully understand the breadth and functional profile of T cell responses to determine their impact on the immune surveillance of variant strains. Here, sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains of SARS-CoV-2 suggested that epitope immunodominance and cross-reactivity are major predictive determinants of T cell immunity. To identify immunodominant epitopes across the viral proteome, we generated a comprehensive map of CD4+ and CD8+ T cell epitopes within non-spike proteins that induced polyfunctional T cell responses in convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by mutations in emerging variants. Conservation analysis across historical human coronaviruses combined with in silico alanine scanning mutagenesis of non-spike proteins underscored the functional importance of mutationally-constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the mutationally-constrained SARS-CoV-2 proteome, potentially providing immune surveillance against emerging variants, and inform the design of next-generation vaccines targeting antigens throughout SARS-CoV-2 proteome for broader and more durable protection.

Author Correction: Sampling the host response to SARS-CoV-2 in hospitals under siege.

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

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8 + T-cells and CD56 dim CD57 + NK cells. Bayesian network analyses revealed nine key regulators of this module, including TBX21 , a central coordinator of exhausted CD8 + T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.

Self-Assembled Metal-Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies.

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 µg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal-organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.