Elderly individuals affected by COVID-19 are vulnerable to severe respiratory failure for reasons that have remained poorly defined. Here, we show that platelet-released serotonin drives lung pathology in a mouse model of age-associated disease severity after SARS-CoV-2 infection. In middle-aged mice and aged human healthy donors, platelet serotonin release upon activation was enhanced, and increased morbidity with respiratory dysfunction was associated with activated platelets aggregating and promoting fibrin deposition in the lung microvasculature. Pharmacologic or genetic disruption of serotonin uptake, or blockade of serotonin-dependent signaling, attenuated platelet activation and protected against respiratory distress, independently of viral replication and immune responses. Inhibition of fibrin formation similarly reduced disease severity, implicating serotonin-driven platelet procoagulant activity as a key contributor to age-related lung dysfunction. Thus, serotonin-mediated platelet procoagulant activity is a major contributor to respiratory insufficiency during SARS-CoV-2 infection and a potential therapeutic target for preserving pulmonary function, particularly in the elderly.

Vaccination triggers both humoral and cellular immune responses, generating memory T cells that ensure long-term protection. Among these, stem cell-like memory T cells (TSCM) are crucial for durable immunity due to their self-renewal and multipotency. In people with HIV (PWHIV), vaccine-induced responses can be weakened by persistent immune dysfunction. In this study, we longitudinally analyzed T cell memory responses following mRNA-1273 vaccination in PWHIV. Individuals with incomplete immune reconstitution (CD4+ < 500 cells/μL, CD4/CD8 < 0.4) showed reduced frequencies of Spike-specific CD4+ TSCM, lower levels of TCF-1 and higher expression of immune checkpoint molecules. We identified a subset of PD-1+TIGIT+ CD4+ TSCM and TCM cells that phenotypically resemble CD8+ exhausted-like progenitors (TPEX) and are enriched in PWHIV with poor immune recovery. Modulation of the Wnt/mTORs pathway via GSK3β inhibition restored TCF-1 expression and partially rescued antigen responsiveness, highlighting a potential strategy to improve vaccine efficacy in PWHIV.

Despite effective antiviral drugs that have emerged to combat SARS-CoV-2 infections, novel therapeutic strategies are required to better address the ongoing and future evolutions of the virus. Targeting viral proteases, such as the main protease (Mpro), remains a promising approach. Here, we present a rapid and sensitive luminescence-based reporter system, the i-NSP4/5-Gluc2, to assess Mpro activity. This system employs Gaussia luciferase (Gluc) fused to a pro-interleukin 1β (pro-IL-1β) fragment containing a specific Mpro cleavage site. Upon Mpro cleavage, Gluc is released and secreted, generating a luminescent signal outside the cells. By optimizing the system's design and experimental conditions, we achieved high sensitivity and specificity. The i-NSP4/5-Gluc2 system was validated using the Mpro inhibitor Nirmatrelvir and successfully identified potential Mpro inhibitors from a small library of 46 compounds, as proof of concept. Notably, 13 out of 14 new compounds identified by the i-NSP4/5-Gluc2 assay exhibited potent antiviral activity against live SARS-CoV-2, highlighting the system's accuracy and predictive power. This BSL2-compatible, high-throughput approach facilitates rapid and efficient screening of antiviral compounds, accelerating the development of effective therapeutics against SARS-CoV-2 and future viral pandemics.

Recognition of glycans by simple synthetic receptors is a key issue in supramolecular chemistry, endowed with relevant implications in glycobiology and medicine. In this context, glycoproteins featuring N-glycans represent an important biological target, because they are often exploited by enveloped viruses in adhesion and infection processes. However, a direct evidence for their recognition by a synthetic receptor targeting N-glycans is still missing in the literature. Using a combination of glycoengineering and mass spectrometry techniques, we present here the direct evidence of biomimetic recognition of complex-type N-glycans exposed on the receptor-binding domain (RBD) of the wild-type spike protein of SARS-CoV-2 by a biologically active, synthetic receptor.

IntroductionDespite SARS-CoV-2 pandemic has subsided, vaccine response profiling in patients with cancer remains critical. MethodsWe longitudinally assessed humoral and cellular immunity in adults with solid tumours treated with chemotherapy (ChT) or non-ChT regimens after two mRNA vaccine doses plus booster, compared with vaccinated cancer-free controls, naturally infected (convalescent) subjects including both patients with cancer and cancer-free individuals, and unvaccinated/uninfected individuals with or without cancer as a baseline reference. ResultsAnti-Spike IgG titres matched cancer-free controls, but anti-RBD titres and neutralising activity were consistently lower in cancer post-vaccination, most markedly with ChT, and declined faster over 4-6 months. Boosters restored IgG, yet gains were smaller in ChT recipients. Cellular analyses revealed sustained and booster-enhanced Spike-specific B cells in all groups; however, ChT exposure was associated with reduced CD27 expression on these cells, suggesting impaired activation and memory maturation.DiscussionThese findings support tailored immune monitoring and vaccination strategies in oncology and identify CD27 downregulation as a novel B-cell dysfunction detected by high-dimensional immunophenotyping.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

The continuous evolution of SARS-CoV-2 generated highly mutated variants able to escape natural and vaccine-induced primary immunity. The administration of a third mRNA vaccine dose induces a secondary response with increased protection. Here we investigate the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses at single-cell level. We sorted 4100 spike protein specific memory B cells identifying 350 neutralizing antibodies. The third dose increases the antibody neutralization potency and breadth against all SARS-CoV-2 variants as observed with hybrid immunity. However, the B cell repertoire generating this response is different. The increases of neutralizing antibody responses is largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization. Our data show that different immunization regimens induce specific molecular signatures which should be considered while designing new vaccines and immunization strategies.

Abstract Background Coronavirus disease 2019 (COVID-19) emerged in late December 2019 and was declared pandemic in March 2020 by the World Health Organization, causing clinically acute respiratory manifestations and corresponding symptoms, pathological inflammation and multi-organ dysfunctions. The total commitment of the scientific community to develop therapeutics to deal with this global emergency in the shortest possible period was unprecedented. In a very short time, several vaccines were approved by the EMA (European Medicines Agency) and the FDA (Food and Drug Administration). Despite this, it is conceivable that COVID-19 will continue to spread globally through evolving variants in more or less cyclic waves. With these perspectives, it is essential to quickly develop additional therapeutic tools to deal with the next wave of infection. Methods In the present study we describe the development and characterization of neutralizing mouse monoclonal antibodies (mAbs) against the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) protein. Results The mAbs identified are able to specifically detect the RBD of SARS-CoV-2 Spike protein in all tested applications, including enzyme-linked immunosorbent assay (ELISA), flow cytometry (FACS) and bio-layer interferometry. In addition, we show that these mAbs efficiently block entry of both SARS-CoV-2 pseudoparticles carrying the spike protein of the original SARS-CoV-2 strain and a broad set of variants of concern (VOC). Conclusions Here we report a panel of monoclonal antibodies that target RBD and inhibit SARS-CoV-2 variants infection and enable the isolation of novel therapeutic tools to neutralize SARS-CoV-2 virus

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir – an orally available inhibitor of the 3-chymotrypsin-like cysteine protease – has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using a mouse model of SARS-CoV-2 infection, we show that nirmatrelvir administration early after infection blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and to mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

COVID-19 has proven to be particularly serious and life-threatening for patients presenting with pre-existing pathologies. Patients affected by rheumatic musculoskeletal disease (RMD) are likely to have impaired immune responses against SARS-CoV-2 infection due to their compromised immune system and the prolonged use of disease-modifying anti-rheumatic drugs (DMARDs), which include conventional synthetic (cs) DMARDs or biologic and targeted synthetic (b/ts) DMARDs. To provide an integrated analysis of the immune response following SARS-CoV-2 infection in RMD patients treated with different classes of DMARDs we carried out an immunological analysis of the antibody responses toward SARS-CoV-2 nucleocapsid and RBD proteins and an extensive immunophenotypic analysis of the major immune cell populations. We showed that RMD individuals under most DMARD treatments mount a sustained antibody response to the virus, with neutralizing activity. In addition, they displayed a sizable percentage of effector T and B lymphocytes. Among b-DMARDs, we found that anti-TNFα treatments are more favorable drugs to elicit humoral and cellular immune responses as compared to CTLA4-Ig and anti-IL6R inhibitors. This study provides a whole picture of the humoral and cellular immune responses in RMD patients by reassuring the use of DMARD treatments during COVID-19. The study points to TNF-α inhibitors as those DMARDs permitting elicitation of functional antibodies to SARS-CoV-2 and adaptive effector populations available to counteract possible re-infections.

Background The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/ hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. Methods Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. Findings We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. Interpretation We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).