Following severe adverse reactions to the AstraZeneca ChAdOx1-S-nCoV-19 vaccine^1,2, European health authorities have recommended that patients under the age of 55 who received one dose of ChAdOx1-S-nCoV-19 vaccine receive a second dose of Pfizer BNT162b2 vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here, we show that the heterologous ChAdOx1-S-nCoV-19/BNT162b2 combination confers better protection against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection than the homologous BNT162b2/BNT162b2 combination in a real-world observational study of healthcare workers (n=13121). To understand the underlying mechanism, we conducted a longitudinal survey of the anti-spike immunity conferred by each vaccine combination. Both combinations induced strong anti-spike antibody (Ab) responses but sera from heterologous vaccinated individuals displayed a stronger neutralizing activity, regardless of the SARS-CoV-2 variant. This enhanced neutralizing potential was correlated with increased frequencies of switched and activated memory B cells recognizing the SARS-CoV-2 Receptor Binding Domain (RBD). The ChAdOx1-S-nCoV-19 vaccine induced a weaker IgG response but a stronger T cell response than the BNT162b2 vaccine after the priming dose, which could explain the complementarity of both vaccines when used in combination. The heterologous vaccination regimen could therefore be particularly suitable for immune compromised individuals.

Understanding the immune responses elicited by SARS-CoV-2 infection is critical in terms of protection against reinfection and, thus, for public health policy and vaccine development for COVID-19. In this study, using either live SARS-CoV-2 particles or retroviruses pseudotyped with the SARS-CoV-2 S viral surface protein (Spike), we studied the neutralizing antibody (nAb) response in serum samples from a cohort of 140 SARS-CoV-2 qPCR-confirmed infections, including patients with mild symptoms and also more severe forms, including those that required intensive care. We show that nAb titers correlated strongly with disease severity and with anti-spike IgG levels. Indeed, patients from intensive care units exhibited high nAb titers; conversely, patients with milder disease symptoms had heterogeneous nAb titers, and asymptomatic or exclusive outpatient-care patients had no or low nAbs. We found that nAb activity in SARS-CoV-2-infected patients displayed a relatively rapid decline after recovery compared to individuals infected with other coronaviruses. Moreover, we found an absence of cross-neutralization between endemic coronaviruses and SARS-CoV-2, indicating that previous infection by human coronaviruses may not generate protective nAbs against SARS-CoV-2. Finally, we found that the D614G mutation in the spike protein, which has recently been identified as the current major variant in Europe, does not allow neutralization escape. Altogether, our results contribute to our understanding of the immune correlates of SARS-CoV-2-induced disease, and rapid evaluation of the role of the humoral response in the pathogenesis of SARS-CoV-2 is warranted.

EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.

Receptor targeting of vector particles is a key technology to enable cell type-specific in vivo gene delivery. For example, T cells in humanized mouse models can be modified by lentiviral vectors (LVs) targeted to human T-cell markers to enable them to express chimeric antigen receptors (CARs). Here, we provide detailed protocols for the generation of CD4- and CD8-targeted LVs (which takes ~9 d in total). We also describe how to humanize immunodeficient mice with hematopoietic stem cells (which takes 12-16 weeks) and precondition (over 5 d) and administer the vector stocks. Conversion of the targeted cell type is monitored by PCR and flow cytometry of blood samples. A few weeks after administration, ~10% of the targeted T-cell subtype can be expected to have converted to CAR T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. We also discuss how the protocol can be easily adapted to use LVs targeted to other types of receptors and/or for delivery of other genes of interest.

Interferon-induced transmembrane proteins (IFITMs) are a family of interferon-inducible proteins that inhibit a broad range of viruses by interfering with viral-to-cellular membrane fusion. The antiviral activity of IFITMs is highly regulated by several posttranslational modifications and by a number of protein domains that modulate steady-state protein levels, trafficking, and antiviral effectiveness. Taking advantage of the natural diversity existing among IFITMs of different animal species, we have compared 21 IFITMs for their ability to inhibit HIV-1 at two steps, during virus entry into cells (target cell protection) and during the production of novel virion particles (negative imprinting of virion particles' infectivity). We found a high functional heterogeneity among IFITM homologs with respect to both antiviral modalities, with IFITM members that exhibit enhanced viral inhibition, while others have no ability to block HIV-1. These differences could not be ascribed to known regulatory domains and could only be partially explained through differential protein stability, implying the existence of additional mechanisms. Through the use of chimeras between active and inactive IFITMs, we demonstrate that the cross talk between distinct domains of IFITMs is an important contributor of their antiviral potency. Finally, we identified murine IFITMs as natural variants competent for target cell protection, but not for negative imprinting of virion particles' infectivity, suggesting that the two properties may, at least in principle, be uncoupled. Overall, our results shed new light on the complex relationship between IFITMs and viral infection and point to the cross talk between IFITM domains as a novel layer of regulation of their activity. IMPORTANCE IFITMs are broad viral inhibitors capable of interfering with both early and late phases of the replicative cycle of many different viruses. By comparing 21 IFITM proteins issued from different animal species for their ability to inhibit HIV-1, we have identified several that exhibit either enhanced or impaired antiviral behavior. This functional diversity is not driven by differences in known domains and can only be partly explained through differential protein stability. Chimeras between active and inactive IFITMs point to the cross talk between individual IFITM domains as important for optimal antiviral activity. Finally, we show that murine IFITMs are not capable of decreasing the infectivity of newly produced HIV-1 virion particles, although they retain target cell protection abilities, suggesting that these properties may be, in principle, disconnected. Overall, our results shed new light on the complex layers of regulation of IFITM proteins and enrich our current understanding of these broad antiviral factors.

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK⁺/HK2^- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK⁺/HK2^- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

During inflammatory diseases, cancer, and infection, the cGAS/STING pathway is known to recognize foreign or self-DNA in the cytosol and activate an innate immune response. Here, we report that negative-strand RNA paramyxoviruses, Nipah virus (NiV), and measles virus (MeV), can also trigger the cGAS/STING axis. Although mice deficient for MyD88, TRIF, and MAVS still moderately control NiV infection when compared with wild-type mice, additional STING deficiency resulted in 100% lethality, suggesting synergistic roles of these pathways in host protection. Moreover, deletion of cGAS or STING resulted in decreased type I interferon production with enhanced paramyxoviral infection in both human and murine cells. Finally, the phosphorylation and ubiquitination of STING, observed during viral infections, confirmed the activation of cGAS/STING pathway by NiV and MeV. Our data suggest that cGAS/STING activation is critical in controlling paramyxovirus infection and possibly represents attractive targets to develop countermeasures against severe disease induced by these pathogens.