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Summary: The rapid emergence of SARS-CoV-2 variants of concern (VoC) and the threat of future zoonotic sarbecovirus spillover emphasizes the need for broadly protective next-generation vaccines and therapeutics. We utilized SARS-CoV-2 spike ferritin nanoparticle (SpFN), and SARS-CoV-2 receptor binding domain ferritin nanoparticle (RFN) immunogens, in an equine model to elicit hyperimmune sera and evaluated its sarbecovirus neutralization and protection capacity. Immunized animals rapidly elicited sera with the potent neutralization of SARS-CoV-2 VoC, and SARS-CoV-1 pseudoviruses, and potent binding against receptor binding domains from sarbecovirus clades 1b, 1a, 2, 3, and 4. Purified equine polyclonal IgG provided protection against Omicron XBB.1.5 virus in the K18-hACE2 transgenic mouse model. These results suggest that SARS-CoV-2-based nanoparticle vaccines can rapidly produce a broad and protective sarbecovirus response in the equine model and that equine serum has therapeutic potential against emerging SARS-CoV-2 VoC and diverse sarbecoviruses, presenting a possible alternative or supplement to monoclonal antibody immunotherapies.

Summary: Pain has become a major symptom of long COVID-19 without effective therapy. Apart from viral infection pathological process, SARS-CoV-2 membranal proteins (envelope [S2E], spike [S2S] and membrane [S2M]) also present pro-inflammatory feature independently. Here, we aim to uncover the neuroinflammatory mechanism of COVID-pain induced by SARS-CoV-2 membranal proteins. We detected the three proteins in both peripheral sensory ganglions and spinal dorsal horn of COVID-19 donors. After intradermal and intrathecal injection, only S2E triggered pain behaviors, accompanied with upregulated-phosphorylation nuclear factor kappa B (NF-κB), which was significantly attenuated by minocycline in mice. We further identified Toll-like receptor 2 (TLR2) among TLRs as the target of S2E to evoke inflammatory responses leading to COVID-pain. This study identified the nociceptive effect of S2E through directly interacting with macrophage/microglia TLR2 and inducing the following NF-κB inflammatory storm. Clearing away S2E and inhibiting macrophage/microglia TLR2 served as perspective therapeutic strategies for COVID-19 pain.

Summary: Clinical research indicates that SARS-CoV-2 infection is linked to several neurological consequences, and the virus is still spreading despite the availability of vaccinations and antiviral medications. To determine how hosts respond to SARS-CoV-2 infection, we employed LC-MS/MS to perform ubiquitinome and proteome analyses of the brain cortexes from K18-hACE2 mice in the presence and absence of SARS-CoV-2 infection. A total of 8,024 quantifiable proteins and 5,220 quantifiable lysine ubiquitination (Kub) sites in 2023 proteins were found. Glutamatergic synapse, calcium signaling pathway, and long-term potentiation may all play roles in the neurological consequences of SARS-CoV-2 infection. Then, we observed possible interactions between 26 SARS-CoV-2 proteins/E3 ubiquitin-protein ligases/deubiquitinases and several differentially expressed mouse proteins or Kub sites. We present the first description of the brain cortex ubiquitinome in K18-hACE2 mice, laying the groundwork for further investigation into the pathogenic processes and treatment options for neurological dysfunction following SARS-CoV-2 infection.

Summary: Although many host factors important for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported, the mechanisms by which the virus interacts with host cells remain elusive. Here, we identified tripartite motif containing (TRIM) 28, TRIM33, euchromatic histone lysine methyltransferase (EHMT) 1, and EHMT2 as proviral factors involved in SARS-CoV-2 infection by CRISPR-Cas9 screening. Our result suggested that TRIM28 may play a role in viral particle formation and that TRIM33, EHMT1, and EHMT2 may be involved in viral transcription and replication. UNC0642, a compound that specifically inhibits the methyltransferase activity of EHMT1/2, strikingly suppressed SARS-CoV-2 growth in cultured cells and reduced disease severity in a hamster infection model. This study suggests that EHMT1/2 may be a therapeutic target for SARS-CoV-2 infection.

Summary: CombiVacS study has demonstrated a strong immune response of the heterologous ChAdOx1-S/BNT162b2 vaccine combination. The primary outcomes of the study were to assess the humoral immune response against SARS-CoV-2, 28 days after a third dose of a mRNA vaccine, in subjects that received a previous prime-boost scheme with ChAdOx1-S/BNT162b2. Secondary outcomes extended the study to 3 and 6 months. The third vaccine dose of mRNA-1273 in naive participants previously vaccinated with ChAdOx1-S/BNT162b2 regimen reached higher neutralizing antibodies titers against the variants of concern Delta and BA.1 lineage of Omicron compared with those receiving a third dose of BNT162b2 at day 28. These differences between BNT162b2 and mRNA-1273 arms were observed against the ancestral variant G614 at day 90. Suboptimal neutralizing response was observed against BQ.1.1, XBB.1.5/XBB.1.9, and JN.1 in a relevant proportion of individuals 180 days after the third dose, even after asymptomatic Omicron breakthrough infections. EudraCT (2021-001978-37); ClinicalTrials.gov (NCT04860739).

Summary: Antibodies represent a primary mediator of protection against respiratory viruses. Serum neutralizing antibodies (NAbs) are often considered a primary correlate of protection. However, detailed antibody profiles including characterization of antibody functions in different anatomic compartments are poorly understood. Here we show that antibody correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge are different in systemic versus mucosal compartments in rhesus macaques. In serum, NAbs were the strongest correlate of protection and linked to spike-specific binding antibodies and other extra-NAb functions that create a larger protective network. In bronchiolar lavage (BAL), antibody-dependent cellular phagocytosis (ADCP) proved the strongest correlate of protection rather than NAbs. Within BAL, ADCP was linked to mucosal spike-specific immunoglobulin (Ig)G, IgA/secretory IgA, and Fcγ-receptor binding antibodies. Our results support a model in which antibodies with different functions mediate protection at different anatomic sites.

Summary: Delta (B.1.617.2) and Omicron (B.1.1.529) variants of SARS-CoV-2 represents unique clinical characteristics. However, their role in altering immunometabolic regulations during acute infection remains convoluted. Here, we evaluated the differential immunopathogenesis of Delta vs. Omicron variants in Golden Syrian hamsters (GSH). The Delta variant resulted in higher virus titers in throat swabs and the lungs and exhibited higher lung damage with immune cell infiltration than the Omicron variant. The gene expression levels of immune mediators and metabolic enzymes, Arg-1 and IDO1 in the Delta-infected lungs were significantly higher compared to Omicron. Further, Delta/Omicron infection perturbed carbohydrates, amino acids, nucleotides, and TCA cycle metabolites and was differentially regulated compared to uninfected lungs. Collectively, our data provide a novel insight into immunometabolic/pathogenic outcomes for Delta vs. Omicron infection in the GSH displaying concordance with COVID-19 patients associated with inflammation and tissue injury during acute infection that offered possible new targets to develop potential therapeutics.

Summary: Prevention of negative COVID-19 infection outcomes is associated with the quality of antibody responses, whose variance by age and sex is poorly understood. Network approaches identified sex and age effects in antibody responses and neutralization potential of de novo infection and vaccination throughout the COVID-19 pandemic. Neutralization values followed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific receptor binding immunoglobulin G (RIgG), spike immunoglobulin G (SIgG) and spike and receptor immunoglobulin G (S, and RIgA) levels based on COVID-19 status. Serum immunoglobulin A (IgA) antibody titers correlated with neutralization only in females 40–60 years old (y.o.). Network analysis found males could improve IgA responses after vaccination dose 2. Complex correlation analyses found vaccination induced less antibody isotype switching and neutralization in older persons, especially in females. Sex-dependent antibody and neutralization decayed the fastest in older males. Shown sex and age characterization can direct studies integrating cell-mediated responses to define yet elusive correlates of protection and inform age and sex precision-focused vaccine design.

Summary: SARS-CoV-2 Omicron BA.2.75 has diversified into multiple subvariants with additional spike mutations and several are expanding in prevalence, particularly CH.1.1 and BN.1. Here, we investigated the viral receptor affinities and neutralization evasion properties of major BA.2.75 subvariants actively circulating in different regions worldwide. We found two distinct evolutionary pathways and three newly identified mutations that shaped the virological features of these subvariants. One phenotypic group exhibited a discernible decrease in viral receptor affinities, but a noteworthy increase in resistance to antibody neutralization, as exemplified by CH.1.1, which is apparently as resistant as XBB.1.5. In contrast, a second group demonstrated a substantial increase in viral receptor affinity but only a moderate increase in antibody evasion, as exemplified by BN.1. We also observed that all prevalent SARS-CoV-2 variants in the circulation presently, except for BN.1, exhibit profound levels of antibody evasion, suggesting this is the dominant determinant of virus transmissibility today.

Summary: Antibodies play a pivotal role in protecting from SARS-CoV-2 infection, but their efficacy is challenged by the continuous emergence of viral variants. In this study, we describe two broadly neutralizing antibodies cloned from the memory B cells of a single convalescent individual after infection with ancestral SARS-CoV-2. Cv2.3194, a resilient class 1 anti-RBD antibody, remains active against Omicron sub-variants up to BA.2.86. Cv2.3132, a near pan-Sarbecovirus neutralizer, targets the heptad repeat 2 membrane proximal region. When combined, Cv2.3194 and Cv2.3132 form a complementary SARS-CoV-2 neutralizing antibody cocktail exhibiting a local dose-dependent synergy. Thus, remarkably robust neutralizing memory B cell antibodies elicited in response to ancestral SARS-CoV-2 infection can withstand viral evolution and immune escape. The cooperative effect of such antibody combination may confer a certain level of protection against the latest SARS-CoV-2 variants.

Summary: SARS-CoV-2 viral entry into host cells depends on the cleavage of spike (S) protein into S1 and S2 proteins. Such proteolytic cleavage by furin results in the exposure of a multibasic motif on S1, which is critical for SARS-CoV-2 viral infection and transmission; however, how such a multibasic motif contributes to the infection of SARS-CoV-2 remains elusive. Here, we demonstrate that the multibasic motif on S1 is critical for its interaction with SLC38A9, an endolysosome-resident arginine sensor. SLC38A9 knockdown prevents S1-induced endolysosome de-acidification and blocks the S protein-mediated entry of pseudo-SARS-CoV-2 in Calu-3, U87MG, Caco-2, and A549 cells. Our findings provide a novel mechanism in regulating SARS-CoV-2 viral entry; S1 present in endolysosome lumen could interact with SLC38A9, which mediates S1-induced endolysosome de-acidification and dysfunction, facilitating the escape of SARS-CoV-2 from endolysosomes and enhancing viral entry.

Summary: The emergence of SARS-CoV-2 variants raises concerns about the efficacy of existing COVID-19 vaccines and therapeutics. Previously, we identified a conserved cryptic class 5 epitope of SARS-CoV-2 receptor binding domain (RBD) by two cross-neutralizing antibodies 7D6 and 6D6. Intriguingly, this site remains resistant to substantial mutations occurred in ever-changing SARS-CoV-2 subvariants. As compared to class 3 antibody S309, 6D6 maintains broad and consistent neutralizing activities against SARS-CoV-2 variants. Furthermore, 6D6 effectively protected hamster from the virulent Beta strain. Sequence alignment of approximately 6 million documented SARS-CoV-2 isolates revealed that 6D6 epitope maintains an exceptionally high conservation rate (99.92%). Structural analysis demonstrated that all 33 mutations accumulated in XBB.1.5 since the original strain do not perturb the binding 6D6 to RBD, in line with the sequence analysis throughout the antigenicity evolution of SARS-CoV-2. These findings suggest the potential of this epitope serving as a critical determinant for vaccines and therapeutic design.

Summary: The studies reported here focus on the impact of pre-existing CD4 T cell immunity on the first encounter with SARS-CoV-2. They leverage PBMC samples from plasma donors collected after a first SARS-CoV-2 infection, prior to vaccine availability and compared to samples collected prior to the emergence of SARS-CoV-2. Analysis of CD4 T cell specificity across the entire SARS-CoV-2 proteome revealed that the recognition of SARS-CoV-2-derived epitopes by CD4 memory cells prior to the pandemic are enriched for reactivity toward non-structural proteins conserved across endemic CoV strains. However, CD4 T cells after primary infection with SARS-CoV-2 focus on epitopes from structural proteins. We observed little evidence for preferential recall to epitopes conserved between SARS-CoV-2 and seasonal CoV, a finding confirmed through use of selectively curated conserved and SARS-unique peptides. Our data suggest that SARS-CoV-2 CD4 T cells elicited by the first infection are primarily established from the naive CD4 T cell pool.

Summary: Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26–32 Y) and older children (12–14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Summary: Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.

Summary: In July/August 2023, the highly mutated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2.86 lineage emerged and its descendant JN.1 is on track to become the dominant SARS-CoV-2 lineage globally. Compared to the spike (S) protein of the parental BA.2.86 lineage, the JN.1 S protein contains one mutation, L455S, which may affect receptor binding and antibody evasion. Here, we performed a virological assessment of the JN.1 lineage employing pseudovirus particles bearing diverse SARS-CoV-2 S proteins. Using this strategy, it was found that S protein mutation L455S confers increased neutralization resistance but reduces ACE2 binding capacity and S protein-driven cell entry efficiency. Altogether, these data suggest that the benefit of increased antibody evasion outweighs the reduced ACE2 binding capacity and further enabled the JN.1 lineage to effectively spread in the human population.

Summary: Monoclonal antibodies (mAb) targeting the SARS-CoV-2 Spike (S) glycoprotein have been exploited for the treatment of severe COVID-19. In this study, we evaluated the immune-regulatory features of two neutralizing anti-S mAbs (nAbs), named J08 and F05, with wild-type (WT) conformation or silenced Fc functions.In the presence of D614G SARS-CoV-2, WT nAbs enhance intracellular viral uptake in immune cells and amplify antiviral type I Interferon and inflammatory cytokine and chemokine production without viral replication, promoting the differentiation of CD16+ inflammatory monocytes and innate/adaptive PD-L1+ and PD-L1+CD80+ plasmacytoid Dendritic Cells. In spite of a reduced neutralizing property, WT J08 nAb still promotes the IL-6 production and differentiation of CD16+ monocytes once binding Omicron BA.1 variant.Fc-mediated regulation of antiviral and inflammatory responses, in the absence of viral replication, highlighted in this study, might positively tune immune response during SARS-CoV-2 infection and be exploited also in mAb-based therapeutic and prophylactic strategies against viral infections.

Summary: A primary reason for the ongoing spread of coronavirus disease 2019 (COVID-19) is the continuous acquisition of mutations by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanism of acquiring mutations is not fully understood. In this study, we isolated SARS-CoV-2 from an immunocompromized patient persistently infected with Omicron strain BF.5 for approximately 4 months to analyze its genome and evaluate drug resistance. Although the patient was administered the antiviral drug remdesivir (RDV), there were no acquired mutations in RDV binding site, and all isolates exhibited susceptibility to RDV. Notably, upon analyzing the S protein sequence of the day 119 isolate, we identified mutations acquired by mutant strains emerging from the BF.5 variant, suggesting that viral genome analysis in persistent COVID-19 patients may be useful in predicting viral evolution. These results suggest mutations in SARS-CoV-2 are acquired during long-term viral replication rather than in response to antiviral drugs.

Summary: Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

Summary: Dysregulated host immune responses contribute to disease severity and worsened prognosis in COVID-19 infection and the underlying mechanisms are not fully understood. In this study, we observed that IL-33, a damage-associated molecular pattern molecule, is significantly increased in COVID-19 patients and in SARS-CoV-2-infected mice. Using IL-33−/− mice, we demonstrated that IL-33 deficiency resulted in significant decreases in bodyweight loss, tissue viral burdens, and lung pathology. These improved outcomes in IL-33−/− mice also correlated with a reduction in innate immune cell infiltrates, i.e., neutrophils, macrophages, natural killer cells, and activated T cells in inflamed lungs. Lung RNA-seq results revealed that IL-33 signaling enhances activation of inflammatory pathways, including interferon signaling, pathogen phagocytosis, macrophage activation, and cytokine/chemokine signals. Overall, these findings demonstrate that the alarmin IL-33 plays a pathogenic role in SARS-CoV-2 infection and provides new insights that will inform the development of effective therapeutic strategies for COVID-19.

Summary: Virucidal filter materials were prepared by electrospinning a solution of 28 wt % poly(vinylidene difluoride) in N,N-dimethylacetamide without and with the addition of 0.25 wt %, 0.75 wt %, 2.0 wt %, or 3.5 wt % Cu(NO3)2 · 2.5H2O as virucidal agent. The fabricated materials had a uniform and defect free fibrous structure and even distribution of copper nanoclusters. X-ray diffraction analysis showed that during the electrospinning process, Cu(NO3)2 · 2.5H2O changed into Cu2(NO3)(OH)3. Electrospun filter materials obtained by electrospinning were essentially macroporous. Smaller pores of copper nanoclusters containing materials resulted in higher particle filtration than those without copper nanoclusters. Electrospun filter material fabricated with the addition of 2.0 wt % and 3.5 wt % of Cu(NO3)2 · 2.5H2O in a spinning solution showed significant virucidal activity, and there was 2.5 ± 0.35 and 3.2 ± 0.30 logarithmic reduction in the concentration of infectious SARS-CoV-2 within 12 h, respectively. The electrospun filter materials were stable as they retained virucidal activity for three months.

Summary: SARS-CoV-2 Omicron variant has evolved into sublineages. Here, we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However, there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population.

Summary: Total plasma protein N-glycosylation (TPNG) changes are a hallmark of many diseases. Here, we analyzed the TPNG of 169 COVID-19 patients and 12 healthy controls, using mass spectrometry, resulting in the relative quantification of 85 N-glycans. We found a COVID-19 N-glycomic signature, with 59 glycans differing between patients and controls, many of them additionally differentiating between severe and mild COVID-19. Tri- and tetra-antennary N-glycans were increased in patients, showing additionally elevated levels of antennary α2,6-sialylation. Conversely, bisection of di-antennary, core-fucosylated, nonsialylated glycans was low in COVID-19, particularly in severe cases, potentially driven by the previously observed low levels of bisection on antibodies of severely diseased COVID-19 patients. These glycomic changes point toward systemic changes in the blood glycoproteome, particularly involvement of acute-phase proteins, immunoglobulins and the complement cascade. Further research is needed to dissect glycosylation changes in a protein- and site-specific way to obtain specific functional leads.

Summary: Both host and viral RNA editing plays a crucial role in host’s response to infection, yet our understanding of host RNA editing remains limited. In this study of in-house generated RNA sequencing (RNA-seq) data of 211 hospitalized COVID-19 patients with PreVOC, Delta, and Omicron variants, we observed a significant differential editing frequency and patterns in long non-coding RNAs (lncRNAs), with Delta group displaying lower RNA editing compared to PreVOC/Omicron patients. Notably, multiple transcripts of UGDH-AS1 and NEAT1 exhibited high editing frequencies. Expression of ADAR1/APOBEC3A/APOBEC3G and differential abundance of repeats were possible modulators of differential editing across patient groups. We observed a shift in crucial infection-related pathways wherein the pathways were downregulated in Delta compared to PreVOC and Omicron. Our genomics-based evidence suggests that lncRNA editing influences stability, miRNA binding, and expression of both lncRNA and target genes. Overall, the study highlights the role of lncRNAs and how editing within host lncRNAs modulates the disease severity.

Summary: This prospective study aimed to determine the prevalence of long COVID in patients hospitalized for SARS-CoV-2 infection from March 2020 to July 2022 and assess the impact of different viral lineages. A total of 2,524 patients were followed up for 12 months, with persistent symptoms reported in 35.2% at one month, decreasing thereafter. Omicron variant patients initially showed higher symptom intensity, but this trend diminished over time. Certain viral lineages, notably Delta lineages AY.126 and AY.43, and Omicron sublineages BA.1.17, BA.2.56, and BA.5.1, consistently correlated with more severe symptoms. Overall, long COVID prevalence and severity were similar across SARS-CoV-2 variants. Specific lineages may influence post-COVID sequelae persistence and severity.

Summary: Multimorbidity entails a higher risk of SARS-CoV-2 infection and COVID-19 complications. We examined vaccine effectiveness (VE) stratified by multimorbidity using a case-control study of territory-wide electronic health records in Hong Kong. Cases of infection (testing positive), hospitalization, and mortality were identified from January to March 2022. Controls were matched by age, sex, outpatient attendance/hospitalization date, and Charlson Comorbidity Index. We demonstrated a consistently good VE among people with increased multimorbidity burden; even more so than among those with minimal such burden. There was also a significantly greater VE after a third dose of BNT162b2 or CoronaVac against infection. The difference in VE between those with multimorbidity and those without was less pronounced for hospitalization, and such difference for COVID-19-related mortality was negligible. In conclusion, VE of both examined vaccines against SARS-CoV-2 infection among people with more complex multimorbidity burden is significant. Further vaccine roll-out should prioritize people with multimorbidity.

Summary: Despite multiple research efforts to characterize coronavirus disease 2019 (COVID-19) in humans, there is no clear data on the specific role of mucosal immunity on COVID-19 disease. Here, we longitudinally profile the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal HCoV-OC43 S proteins in serum and nasopharyngeal swabs from COVID-19 patients. Results showed that specific antibody responses against SARS-CoV-2 and HCoV-OC43 S proteins can be detected in the upper respiratory tract. We found that COVID-19 patients mounted a robust mucosal antibody response against SARS-CoV-2 S with specific secretory immunoglobulin A (sIgA), IgA, IgG, and IgM antibody subtypes detected in the nasal swabs. Additionally, COVID-19 patients showed IgG, IgA, and sIgA responses against HCoV-OC43 S in the local mucosa, whereas no specific IgM was detected. Interestingly, mucosal antibody titers against SARS-CoV-2 peaked at day 7, whereas HCoV-OC43 titers peaked earlier at day 3 post-recruitment, suggesting an immune memory recall to conserved epitopes of beta-HCoVs in the upper respiratory tract.

Summary: The COVID-19 pandemic, driven by the SARS-CoV-2 virus and its variants, highlights the important role of understanding host-viral molecular interactions influencing infection outcomes. Alternative splicing post-infection can impact both host responses and viral replication. We analyzed RNA splicing patterns in immune cells across various SARS-CoV-2 variants, considering immunization status. Using a dataset of 190 RNA-seq samples from our prior studies, we observed a substantial deactivation of alternative splicing and RNA splicing-related genes in COVID-19 patients. The alterations varied significantly depending on the infecting variant and immunization history. Notably, Alpha or Beta-infected patients differed from controls, while Omicron-infected patients displayed a splicing profile closer to controls. Particularly, vaccinated Omicron-infected individuals showed a distinct dynamic in alternative splicing patterns not widely shared among other groups. Our findings underscore the intricate interplay between SARS-CoV-2 variants, vaccination-induced immunity, and alternative splicing, emphasizing the need for further investigations to deepen understanding and guide therapeutic development.

Summary: Conventional neutralizing enzyme-linked immunosorbent assay (ELISA) systems for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mimic the protein-protein interaction between angiotensin-converting enzyme 2 (ACE2) and the receptor-binding domain (RBD). However, an easy and rapidly adaptative ELISA-based system for testing neutralizing antibodies against upcoming SARS-CoV-2 variants is urgently needed. In this study, we closed this gap by developing a tANCHOR-cell-based RBD neutralization assay that avoids time-consuming protein expression and purification followed by coating on ELISA plates. This cell-based assay can be rapidly adopted to monitor neutralizing antibodies (NAbs) against upcoming SARS-CoV-2 variants. We show that the results obtained with the tANCHOR-cell-based assay system strongly correlate with commercially available surrogate assays for testing NAbs. Moreover, this technique can directly measure binding between cell-surface-exposed RBDs and soluble ACE2. With this technique, the degree of antibody escape elicited by emerging SARS-CoV-2 variants in current vaccination regimens can be determined rapidly and reliably.

Summary: Direct acting antivirals (DAAs) represent critical tools for combating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have escaped vaccine-elicited spike-based immunity and future coronaviruses with pandemic potential. Here, we used bioluminescence imaging to evaluate therapeutic efficacy of DAAs that target SARS-CoV-2 RNA-dependent RNA polymerase (favipiravir, molnupiravir) or main protease (nirmatrelvir) against Delta or Omicron VOCs in K18-hACE2 mice. Nirmatrelvir displayed the best efficacy followed by molnupiravir and favipiravir in suppressing viral loads in the lung. Unlike neutralizing antibody treatment, DAA monotherapy regimens did not eradicate SARS-CoV-2 in mice, but combining molnupiravir with nirmatrelvir exhibited superior additive efficacy and led to virus clearance. Furthermore, combining molnupiravir with caspase-1/4 inhibitor mitigated inflammation and lung pathology whereas combining molnupiravir with COVID-19 convalescent plasma demonstrated synergy, rapid virus clearance, and 100% survival. Thus, our study provides insights into in vivo treatment efficacies of DAAs and other effective combinations to bolster COVID-19 therapeutic arsenal.

Summary: Continuing emergence of variants of concern resulting in reduced SARS-CoV-2 vaccine efficacy necessitates additional prevention strategies. The structure of VLPCOV-01, a lipid nanoparticle-encapsulated, self-amplifying RNA COVID-19 vaccine with a comparable immune response to BNT162b2, was revised by incorporating a modified base, 5-methylcytosine, to reduce reactogenicity, and an updated receptor-binding domain derived from the Brazil (gamma) variant. Interim analyses of a phase 1 dose-escalation booster vaccination study with the resulting construct, VLPCOV-02, in healthy, previously vaccinated Japanese individuals (N = 96) are reported (jRCT2051230005). A dose-related increase in solicited local and systemic adverse events was observed, which were generally rated mild or moderate. The most commonly occurring events were tenderness, pain, fatigue, and myalgia. Serum SARS-CoV-2 immunoglobulin titers increased during the 4 weeks post-immunization. VLPCOV-02 demonstrated a favorable safety profile compared with VLPCOV-01, with reduced adverse events and fewer fever events at an equivalent dose. These findings support further study of VLPCOV-02.

Summary: Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NPRBD) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NPRBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NPRBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

Summary: A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

Summary: The 2019 coronavirus pandemic resulted in a massive global healthcare crisis, highlighting the necessity to develop effective and reproducible platforms capable of rapidly and accurately detecting SARS-CoV-2. In this study, we developed an electrolyte-gated indium-gallium-zinc-oxide (IGZO) thin-film transistor with sequential surface modification to realize the low limit of detection (LoD

Summary: Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs.

Summary: SARS-CoV-2 infection in children typically results in asymptomatic or mild disease. There is a paucity of studies on SARS-CoV-2 antiviral immunity in African children. We investigated SARS-CoV-2-specific T cell responses in 71 unvaccinated asymptomatic South African children who were seropositive or seronegative for SARS-CoV-2. SARS-CoV-2-specific CD4+ T cell responses were detectable in 83% of seropositive and 60% of seronegative children. Although the magnitude of the CD4+ T cell response did not differ significantly between the two groups, their functional profiles were distinct, with SARS-CoV-2 seropositive children exhibiting a higher proportion of polyfunctional T cells compared to their seronegative counterparts. The frequency of SARS-CoV-2-specific CD4+ T cells in seronegative children was associated with the endemic human coronavirus (HCoV) HKU1 IgG response. Overall, the presence of SARS-CoV-2-responding T cells in seronegative children may result from cross-reactivity to endemic coronaviruses and could contribute to the relative protection from disease observed in SARS-CoV-2-infected children.

Summary: SARS-CoV-2 infections in children are generally asymptomatic or mild and rarely progress to severe disease and hospitalization. Why this is so remains unclear. Here we explore the potential for protection due to pre-existing cross-reactive seasonal coronavirus antibodies and compare the rate of antibody decline for nucleocapsid and spike protein in serum and oral fluid against SARS-CoV-2 within the pediatric population. No differences in seasonal coronaviruses antibody concentrations were found at baseline between cases and controls, suggesting no protective effect from pre-existing immunity against seasonal coronaviruses. Antibodies against seasonal betacoronaviruses were boosted in response to SARS-CoV-2 infection. In serum, anti-nucleocapsid antibodies fell below the threshold of positivity more quickly than anti-spike protein antibodies. These findings add to our understanding of protection against infection with SARS-CoV-2 within the pediatric population, which is important when considering pediatric SARS-CoV-2 immunization policies.

Summary: Next-generation SARS-CoV-2 vaccines are needed that induce systemic and mucosal immunity. Murine pneumonia virus (MPV), a murine homolog of respiratory syncytial virus, is attenuated by host-range restriction in nonhuman primates and has a tropism for the respiratory tract. We generated MPV vectors expressing the wild-type SARS-CoV-2 spike protein (MPV/S) or its prefusion-stabilized form (MPV/S-2P). Both vectors replicated similarly in cell culture and stably expressed S. However, only S-2P was associated with MPV particles. After intranasal/intratracheal immunization of rhesus macaques, MPV/S and MPV/S-2P replicated to low levels in the airways. Despite its low-level replication, MPV/S-2P induced high levels of mucosal and serum IgG and IgA to SARS-CoV-2 S or its receptor-binding domain. Serum antibodies from MPV/S-2P-immunized animals efficiently inhibited ACE2 receptor binding to S proteins of variants of concern. Based on its attenuation and immunogenicity in macaques, MPV/S-2P will be further evaluated as a live-attenuated vaccine for intranasal immunization against SARS-CoV-2.

Summary: SARS-CoV-2 infection and vaccination elicit potent immune responses. Our study presents a comprehensive multimodal single-cell analysis of blood from COVID-19 patients and healthy volunteers receiving the SARS-CoV-2 vaccine and booster. We profiled immune responses via transcriptional analysis and lymphocyte repertoire reconstruction. COVID-19 patients displayed an enhanced interferon signature and cytotoxic gene upregulation, absent in vaccine recipients. B and T cell repertoire analysis revealed clonal expansion among effector cells in COVID-19 patients and memory cells in vaccine recipients. Furthermore, while clonal αβ T cell responses were observed in both COVID-19 patients and vaccine recipients, expansion of clonal γδ T cells was found only in infected individuals. Our dataset enables side-by-side comparison of immune responses to infection versus vaccination, including clonal B and T cell responses. Our comparative analysis shows that vaccination induces a robust, durable clonal B and T cell responses, without the severe inflammation associated with infection.

Summary: Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is highly variable and could be mediated by a cross-protective pre-immunity. We identified 14 cross-reactive peptides between SARS-CoV-2 and influenza A H1N1, H3N2, and human herpesvirus (HHV)-6A/B with potential relevance. The H1N1 peptide NGVEGF was identical to a peptide in the most critical receptor binding motif in SARS-CoV-2 spike protein that interacts with the angiotensin converting enzyme 2 receptor. About 62%–73% of COVID-19-negative blood donors in Stockholm had antibodies to this peptide in the early pre-vaccination phase of the pandemic. Seasonal flu vaccination enhanced neutralizing capacity to SARS-CoV-2 and T cell immunity to this peptide. Mathematical modeling taking the estimated pre-immunity levels to flu into account could fully predict pre-Omicron SARS-CoV-2 outbreaks in Stockholm and India. This cross-immunity provides mechanistic explanations to the epidemiological observation that influenza vaccination protected people against early SARS-CoV-2 infections and implies that flu-mediated cross-protective immunity significantly dampened the first SARS-CoV-2 outbreaks.

Summary: Vaccines have curtailed the devastation wrought by COVID-19. Nevertheless, emerging variants result in a high incidence of breakthrough infections. Here we assess the impact of vaccination and breakthrough infection on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T cell immunity. We demonstrate that COVID-19 vaccination induces robust spike-specific T cell responses that, within the CD4+ compartment, display comparable IFN-γ responses to SARS-CoV-2 infection without vaccination. Vaccine-induced CD8+ IFN-γ responses however, were significantly greater than those primed by SARS-CoV-2 infection alone. This increased responsiveness is associated with induction of novel HLA-restricted CD8+ T cell epitopes not primed by infection alone (without vaccination). Despite these augmented responses, breakthrough infection still induced de novo T cell responses against additional SARS-CoV-2 CD8+ epitopes that display HLA-associated immunodominance hierarchies consistent with those in unvaccinated COVID-19 convalescent individuals. This study demonstrates the unique modulation of anti-viral T cell responses against multiple viral antigens following consecutive yet distinct priming events in COVID-19 vaccination and breakthrough infection.

Summary: Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and long non-coding RNAs (lncRNAs), including transcripts coding for antiviral factors, such as interferons (IFNs). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-κB pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-κB pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

Summary: The SARS-CoV-2 pandemic was defined by the emergence of new variants formed through virus mutation originating from random errors not corrected by viral proofreading and/or the host antiviral response introducing mutations into the viral genome. While sequencing information hints at cellular RNA editing pathways playing a role in viral evolution, here, we use an in vitro human cell infection model to assess RNA mutation types in two SARS-CoV-2 strains representing the original and the alpha variants. The variants showed both different cellular responses and mutation patterns with alpha showing higher mutation frequency with most substitutions observed being C-U, indicating an important role for apolipoprotein B mRNA editing catalytic polypeptide-like editing. Knockdown of select APOBEC3s through RNAi increased virus production in the original virus, but not in alpha. Overall, these data suggest a deaminase-independent anti-viral function of APOBECs in SARS-CoV-2 while the C-U editing itself might function to enhance genetic diversity enabling evolutionary adaptation.

Summary: Extracellular vesicles (EVs) are entering the clinical arena as novel biologics for infectious diseases, potentially serving as the immunogenic components of next generation vaccines. However, relevant human assays to evaluate the immunogenicity of EVs carrying viral antigens are lacking, contributing to challenges in translating rodent studies to human clinical trials. Here, we engineered EVs to carry SARS-CoV-2 Spike to evaluate the immunogenicity of antigen-carrying EVs using human peripheral blood mononuclear cells (PBMCs). Delivery of Spike EVs to PBMCs resulted in specific immune cell activation as assessed through T cell activation marker expression. Further, Spike EVs were taken up largely by antigen-presenting cells (monocytes, dendritic cells and B cells). Taken together, this human PBMC-based system models physiologically relevant pathways of antigen delivery, uptake and presentation. In summary, the current study highlights the suitability of using human PBMCs for evaluating the immunogenicity of EVs engineered to carry antigens for infectious disease therapeutics.

Summary: Severe COVID-19 outcomes have been reported in people living with HIV (PLWH), yet the underlying pathogenetic factors are largely unknown. We therefore aimed to assess SARS-CoV-2 RNAemia and plasma cytokines in PLWH hospitalized for COVID-19 pneumonia, exploring associations with magnitude and functionality of SARS-CoV-2-specific immune responses.Eighteen unvaccinated PLWH (16/18 on cART; median CD4 T cell count 361.5/μL; HIV-RNA

Summary: To elucidate host response elements that define impending decompensation during SARS-CoV-2 infection, we enrolled subjects hospitalized with COVID-19 who were matched for disease severity and comorbidities at the time of admission. We performed combined single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on peripheral blood mononuclear cells (PBMCs) at admission and compared subjects who improved from their moderate disease with those who later clinically decompensated and required invasive mechanical ventilation or died. Chromatin accessibility and transcriptomic immune profiles were markedly altered between the two groups, with strong signals in CD4+ T cells, inflammatory T cells, dendritic cells, and NK cells. Multiomic signature scores at admission were tightly associated with future clinical deterioration (auROC 1.0). Epigenetic and transcriptional changes in PBMCs reveal early, broad immune dysregulation before typical clinical signs of decompensation are apparent and thus may act as biomarkers to predict future severity in COVID-19.

Summary: COVID-19 pandemic saw emergence of multiple SAR-CoV-2 variants. Exacerbated risk of severe outcome and hospital admissions led us to comprehend differential host-immune kinetics associated with SARS-CoV-2 variants. Longitudinal investigation was conducted through different time periods of Pre-VOC and VOCs (Delta & Omicron) mapping host transcriptome features. Robust antiviral type-1 interferon response marked Omicron infection, which was largely missing during Pre-VOC and Delta waves. SARS-CoV-2-host protein-protein interactions and docking complexes highlighted N protein to interact with HNRNPA1 in Pre-VOC, demonstrating its functional role for enhanced viral replication. Omicron revealed enhanced binding efficiency of LARP1 to N protein, probably potentiating antiviral effects of LARP1. Differential expression of zinc finger protein genes, especially in Omicron, mechanistically support induction of strong IFN (Interferon) response, thereby strengthening early viral clearance. Study highlights eventual adaptation of host to immune activation patterns that interrupt virus evolution with enhanced immune-evasion mutations and counteraction mechanisms, delimiting the next phase of COVID-19 pandemic.

Summary: The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002–2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Summary: The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.

Summary: Mucosal COVID-19 vaccines are needed to block SARS-CoV-2 infection at the mucosal site. Intranasal delivery of a glycosylated Delta variant receptor-binding domain (Delta-RBD) mucosal vaccine elicited potent and balanced systemic antibody titers comparable to those induced by the intramuscular injection of the same vaccine or Omicron-S subunit vaccine, as well as high mucosal IgA antibody responses. It elicited broadly neutralizing antibodies against the original SARS-CoV-2 strain, Delta and Omicron BA1/BA2 variants, completely protecting transgenic mice from lethal challenge with a Delta variant, including complete absence of weight loss. Of note, intramuscular priming with the Omicron-S protein followed by intranasal boosting with the Delta-RBD protein improved the vaccine’s ability to generate broad-spectrum neutralizing antibodies against recent BA5 and XBB Omicron variants. Overall, this vaccine has the potential to prevent the SARS-CoV-2 infection of the respiratory mucosa, while the i.m. priming and i.n. boosting vaccination strategy may offer protection against known and emerging SARS-CoV-2 variants.