The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

SARS-CoV-2 vaccination of convalescents boosts neutralization capacity against Omicron subvariants BA.1, BA.2 and BA.5 and can be predicted by anti-S antibody concentrations in serological assays.

Background: Recent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed. Methods and findings: The study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p<0.0001) with 52.7-, 210.7-, 141.3- and 105.4-fold higher geometric mean of 50% neutralizing titers (NT50) in vaccinated compared to unvaccinated convalescents. 91.4% of the superimmunized individuals showed neutralization of BA.1, 97.2% of BA.2 and 91.5% of BA.5 with a titer ≥ 640. The increase in neutralizing titers was already achieved by one vaccination dose. Neutralizing titers were highest in the first 3 months after the last immunization event. Concentrations of anti-S antibodies in the anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S assays predicted neutralization capacity against B.1 and Omicron subvariants BA.1, BA.2 and BA.5. Conclusions: These findings confirm substantial immune evasion of the Omicron sublineages, which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies.

Serum neutralizing capacity and T-cell response against the omicron BA.1 variant in seropositive children and their parents one year after SARS-CoV-2 infection.

Introduction: Durability of immune protection against reinfection with SARS-CoV-2 remains enigmatic, especially in the pediatric population and in the context of immune-evading variants of concern. Obviously, this knowledge is required for measures to contain the spread of infection and in selecting rational preventive measures. Methods: Here, we investigated the serum neutralization capacity of 36 seropositive adults and 34 children approximately one year after infection with the ancestral Wuhan strain of SARS-CoV-2 by using a pseudovirus neutralization assay. Results: We found that 88.9% of seropositive adult (32/36) and 94.1% of seropositive children (32/34) convalescents retained the neutralizing activity against the SARS-CoV-2 Wuhan strain (WT). Although, the neutralization effect against Omicron BA.1 (B.1.1.529.1) was significantly lower, 70.6% (24/34) of children and 41.7% (15/36) of adults possessed BA.1 cross-neutralizing antibodies. The spike 1 (S1)-specific T cell recall capacity using an activation-induced marker assay was analyzed in 18 adults and 16 children. All participants had detectable S1-specific CD4 T cells against WT, and 72.2% (13/18) adults and 81,3% (13/16) children had detectable S1 WT-specific CD8 T cells. CD4 cross-reactivity against BA.1 was demonstrated in all investigated adults (18/18), and 66.7% (12/18) adult participants had also detectable specific CD8 BA.1 T cells while we detected BA.1 S1 reactive CD4 and CD8 T cells in 81.3% (13/16) children. Discussion: Together, our findings demonstrate that infection with the ancestral strain of SARS-CoV-2 in children as well as in adults induces robust serological as well as T cell memory responses that persist over at least 12 months. This suggests persistent immunological memory and partial cross-reactivity against Omicron BA.1.

Interferon antagonists encoded by SARS-CoV-2 at a glance.

The innate immune system is a powerful barrier against invading pathogens. Interferons (IFNs) are a major part of the cytokine-mediated anti-viral innate immune response. After recognition of a pathogen by immune sensors, signaling cascades are activated that culminate in the release of IFNs. These activate cells in an autocrine or paracrine fashion eventually setting cells in an anti-viral state via upregulation of hundreds of interferon-stimulated genes (ISGs). To evade the anti-viral effect of the IFN system, successful viruses like the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved strategies to counteract both IFN induction and signaling. In fact, more than half of the about 30 proteins encoded by SARS-CoV-2 target the IFN system at multiple levels to escape IFN-mediated restriction. Here, we review recent insights into the molecular mechanisms used by SARS-CoV-2 proteins to suppress IFN production and the establishment of an anti-viral state.

Endogenous IFITMs boost SARS-Coronavirus 1 and 2 replication while overexpression inhibits infection by relocalizing ACE2

Opposing effects of Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) on SARS-CoV-2 infection have been reported. The reasons for this are unclear and the role of IFITMs in infection of other human coronaviruses (hCoVs) remains poorly understood. Here, we show that endogenous expression of IFITM2 and/or IFITM3 is critical for efficient replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 but has little effect on MERS-, NL63- and 229E-CoVs. In contrast, overexpression of IFITMs inhibits all these hCoVs, as well as the corresponding Spike-containing pseudo-particles, except OC43, which is enhanced by IFITM3. We further demonstrate that overexpression of IFITMs impairs cell surface expression of ACE2 representing the entry receptor of SARS-CoVs and hCoV-NL63 but not hCoV-OC43. Our results explain the inhibitory effects of artificial IFITM overexpression on ACE2-tropic SARS-CoVs and show that three hCoVs, including major causative agents of severe respiratory disease, hijack IFITMs for efficient infection of human cells. Graphical abstract Opposing effects of IFITM proteins on infection by human coronaviruses including SARS-CoV-2 have been reported. Here, Xie and colleagues resolved these seeming controversies and show that endogenous expression of IFITMs has little if any inhibitory effects but strongly enhances replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43.

Endogenous IFITMs boost SARS-coronavirus 1 and 2 replication whereas overexpression inhibits infection by relocalizing ACE2.

Opposing effects of interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) on SARS-CoV-2 infection have been reported. The reasons for this are unclear and the role of IFITMs in infection of other human coronaviruses (hCoVs) remains poorly understood. Here, we demonstrate that endogenous expression of IFITM2 and/or IFITM3 is critical for efficient replication of SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 but has little effect on MERS-, NL63-and 229E-hCoVs. In contrast, overexpression of IFITMs inhibits all these hCoVs, and the corresponding spike-containing pseudo-particles, except OC43, which is enhanced by IFITM3. We further demonstrate that overexpression of IFITMs impairs cell surface expression of ACE2 representing the entry receptor of SARS-CoVs and hCoV-NL63 but not hCoV-OC43. Our results explain the inhibitory effects of artificial IFITM overexpression on ACE2-tropic SARS-CoVs and show that three hCoVs, including major causative agents of severe respiratory disease, hijack IFITMs for efficient infection of human cells.

Reduced replication but increased interferon resistance of SARS-CoV-2 Omicron BA.1.

The IFN system constitutes a powerful antiviral defense machinery. Consequently, effective IFN responses protect against severe COVID-19 and exogenous IFNs inhibit SARS-CoV-2 in vitro. However, emerging SARS-CoV-2 variants of concern (VOCs) may have evolved reduced IFN sensitivity. Here, we determined differences in replication and IFN susceptibility of an early SARS-CoV-2 isolate (NL-02-2020) and the Alpha, Beta, Gamma, Delta, and Omicron VOCs in Calu-3 cells, iPSC-derived alveolar type-II cells (iAT2) and air-liquid interface (ALI) cultures of primary human airway epithelial cells. Our data show that Alpha, Beta, and Gamma replicated to similar levels as NL-02-2020. In comparison, Delta consistently yielded higher viral RNA levels, whereas Omicron was attenuated. All viruses were inhibited by type-I, -II, and -III IFNs, albeit to varying extend. Overall, Alpha was slightly less sensitive to IFNs than NL-02-2020, whereas Beta, Gamma, and Delta remained fully sensitive. Strikingly, Omicron BA.1 was least restricted by exogenous IFNs in all cell models. Our results suggest that enhanced innate immune evasion rather than higher replication capacity contributed to the effective spread of Omicron BA.1.

Native and activated antithrombin inhibits TMPRSS2 activity and SARS-CoV-2 infection.

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.

Near-Native Visualization of SARS-CoV-2 Induced Membrane Remodeling and Virion Morphogenesis.

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.

High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

BNT162b2 booster after heterologous prime-boost vaccination induces potent neutralizing antibodies and T cell reactivity against SARS-CoV-2 Omicron BA.1 in young adults.

In light of the decreasing immune protection against symptomatic SARS-CoV-2 infection after initial vaccinations and the now dominant immune-evasive Omicron variants, 'booster' vaccinations are regularly performed to restore immune responses. Many individuals have received a primary heterologous prime-boost vaccination with long intervals between vaccinations, but the resulting long-term immunity and the effects of a subsequent 'booster', particularly against Omicron BA.1, have not been defined. We followed a cohort of 23 young adults, who received a primary heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, over a 7-month period and analysed how they responded to a BNT162b2 'booster'. We show that already after the primary heterologous vaccination, neutralization titers against Omicron BA.1 are recognizable but that humoral and cellular immunity wanes over the course of half a year. Residual responsive memory T cells recognized spike epitopes of the early SARS-CoV-2 B.1 strain as well as the Delta and BA.1 variants of concern (VOCs). However, the remaining antibody titers hardly neutralized these VOCs. The 'booster' vaccination was well tolerated and elicited both high antibody titers and increased memory T cell responses against SARS-CoV-2 including BA.1. Strikingly, in this young heterologously vaccinated cohort the neutralizing activity after the 'booster' was almost as potent against BA.1 as against the early B.1 strain. Our results suggest that a 'booster' after heterologous vaccination results in effective immune maturation and potent protection against the Omicron BA.1 variant in young adults.

Determinants of Spike infectivity, processing, and neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2.

SARS-CoV-2 Omicron rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission and immune evasion are thought to be driven by numerous mutations in the Omicron Spike protein. Here, we systematically introduced BA.1 and/or BA.2 Omicron Spike mutations into the ancestral Spike protein and examined the impacts on Spike function, processing, and susceptibility to neutralization. Individual mutations of S371F/L, S375F, and T376A in the ACE2-receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes to G339D, D614G, N764K, and L981F moderately enhanced it. Most mutations in the N-terminal region and receptor-binding domain reduced the sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine and by therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that have allowed this SARS-CoV-2 variant to dominate the current pandemic.

SARS-CoV-2 Variants of Concern Hijack IFITM2 for Efficient Replication in Human Lung Cells.

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.

Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Boosts Neutralizing Activity Against Seasonal Human Coronaviruses.

BACKGROUND: Most of the millions of people that are vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), have previously been infected by related circulating human coronaviruses (hCoVs) causing common colds and will experience further encounters with these viruses in the future. Whether COVID-19 vaccinations impact neutralization of seasonal coronaviruses is largely unknown. METHODS: We analyzed the capacity of sera derived from 24 individuals before and after heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination to neutralize genuine OC43, NL63, and 229E hCoVs, as well as viral pseudoparticles carrying the SARS-CoV-1, SARS-CoV-2, Middle East Respiratory Syndrome (MERS)-CoV, and hCoV-OC43, hCoV-NL63, and hCoV-229E spike proteins. Genuine hCoVs or spike containing pseudovirions were incubated with different concentrations of sera and neutralization efficiencies were determined by measuring viral RNA yields, intracellular viral nucleocapsid expression, or reporter gene expression in Huh-7 cells. RESULTS: All individuals showed strong preexisting immunity against hCoV-OC43. Neutralization of hCoV-NL63 was more variable and all sera showed only modest inhibitory activity against genuine hCoV-229E. SARS-CoV-2 vaccination resulted in efficient cross-neutralization of SARS-CoV-1 but not of MERS-CoV. On average, vaccination significantly increased the neutralizing activity against genuine hCoV-OC43, hCoV-NL63, and hCoV-229E. CONCLUSIONS: Heterologous COVID-19 vaccination may confer some cross-protection against endemic seasonal coronaviruses.

Omicron: What Makes the Latest SARS-CoV-2 Variant of Concern So Concerning?

Emerging strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, that show increased transmission fitness and/or immune evasion are classified as "variants of concern" (VOCs). Recently, a SARS-CoV-2 variant first identified in November 2021 in South Africa has been recognized as a fifth VOC, termed "Omicron." What makes this VOC so alarming is the high number of changes, especially in the viral Spike protein, and accumulating evidence for increased transmission efficiency and escape from neutralizing antibodies. In an amazingly short time, the Omicron VOC has outcompeted the previously dominating Delta VOC. However, it seems that the Omicron VOC is overall less pathogenic than other SARS-CoV-2 VOCs. Here, we provide an overview of the mutations in the Omicron genome and the resulting changes in viral proteins compared to other SARS-CoV-2 strains and discuss their potential functional consequences.

Serially passaged, conditionally reprogrammed nasal epithelial cells as a model to study epithelial functions and SARS-CoV-2 infection.

Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.