The receptor binding domain of SARS-CoV-2 Omicron subvariants targets Siglec-9 to decrease its immunogenicity by preventing macrophage phagocytosis.

The development of a vaccine specific to severe acute respiratory syndrome coronavirus 2 Omicron has been hampered due to its low immunogenicity. Here, using reverse mutagenesis, we found that a phenylalanine-to-serine mutation at position 375 (F375S) in the spike protein of Omicron to revert it to the sequence found in Delta and other ancestral strains significantly enhanced the immunogenicity of Omicron vaccines. Sequence FAPFFAF at position 371-377 in Omicron spike had a potent inhibitory effect on macrophage uptake of receptor-binding domain (RBD) nanoparticles or spike-pseudovirus particles containing this sequence. Omicron RBD enhanced binding to Siglec-9 on macrophages to impair phagocytosis and antigen presentation and promote immune evasion, which could be abrogated by the F375S mutation. A bivalent F375S Omicron RBD and Delta-RBD nanoparticle vaccine elicited potent and broad nAbs in mice, rabbits and rhesus macaques. Our research suggested that manipulation of the Siglec-9 pathway could be a promising approach to enhance vaccine response.

Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Two waves of pro-inflammatory factors are released during the influenza A virus (IAV)-driven pulmonary immunopathogenesis.

Influenza A virus (IAV) infection is a complicated process. After IAVs spread to the lung, extensive pro-inflammatory cytokines and chemokines are released, which largely determine the outcome of infection. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of more than 16,000 immune cells in the pulmonary tissue of infected mice, and demonstrated that two waves of pro-inflammatory factors were released. A group of IAV-infected PD-L1+ neutrophils were the major contributor to the first wave at an earlier stage (day 1-3 post infection). Notably, at a later stage (day 7 post infection) when IAV was hardly detected in the immune cells, a group of platelet factor 4-positive (Pf4+)-macrophages generated another wave of pro-inflammatory factors, which were probably the precursors of alveolar macrophages (AMs). Furthermore, single-cell signaling map identified inter-lineage crosstalk between different clusters and helped better understand the signature of PD-L1+ neutrophils and Pf4+-macrophages. Our data characteristically clarified the infiltrated immune cells and their production of pro-inflammatory factors during the immunopathogenesis development, and deciphered the important mechanisms underlying IAV-driven inflammatory reactions in the lung.

Adenosine deaminase acting on RNA-1 (ADAR1) inhibits hepatitis B virus (HBV) replication by enhancing microRNA-122 processing.

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes.

MicroRNA Expression Profile of Whole Blood Is Altered in Adenovirus-Infected Pneumonia Children.

Human adenovirus (Adv) infection is responsible for most community-acquired pneumonia in infants and children, which results in significant morbidity and mortality in children every year. MicroRNAs (miRNAs) are associated with viral replication and host immune response. Knowing the miRNA expression profile will help understand the role of miRNAs in modulating the host response to adenovirus infection and possibly improve the diagnosis of adenovirus-infected pneumonia. In our study, total RNA extracted from whole blood of adenovirus-infected pneumonia children and healthy controls were analyzed by small RNA deep sequencing. Expression profiles of whole blood microRNAs were altered and distinctly different in adenovirus-infected children. The top 3 upregulated miRNA (hsa-miR-127-3p, hsa-miR-493-5p, and hsa-miR-409-3p) were identified in adenovirus-infected children and provided a clear distinction between infected and healthy individuals. Potential host target genes were predicated and validated by qRT-PCR to study the impact of microRNAs on the host genes. Most of the target genes were involved in the MAPK signaling pathway and innate immune response. These highly upregulated microRNAs may have crucial roles in Adv pathogenesis and are potential biomarkers for adenovirus-infected pneumonia.

Harnessing T-Cells for Enhanced Vaccine Development against Viral Infections.

Despite significant strides in vaccine research and the availability of vaccines for many infectious diseases, the threat posed by both known and emerging infectious diseases persists. Moreover, breakthrough infections following vaccination remain a concern. Therefore, the development of novel vaccines is imperative. These vaccines must exhibit robust protective efficacy, broad-spectrum coverage, and long-lasting immunity. One promising avenue in vaccine development lies in leveraging T-cells, which play a crucial role in adaptive immunity and regulate immune responses during viral infections. T-cell recognition can target highly variable or conserved viral proteins, and memory T-cells offer the potential for durable immunity. Consequently, T-cell-based vaccines hold promise for advancing vaccine development efforts. This review delves into the latest research advancements in T-cell-based vaccines across various platforms and discusses the associated challenges.

BRD7 as key factor in PBAF complex assembly and CD8+ T cell differentiation.

Upon infection, naïve CD8+ T cells differentiate into cytotoxic effector cells to eliminate the pathogen-infected cells. Although many mechanisms underlying this process have been demonstrated, the regulatory role of chromatin remodel system in this process remains largely unknown. Here we showed that BRD7, a component of the polybromo-associated BRG1-associated factor complex (PBAF), was required for naïve CD8+ T cells to differentiate into functional short-lived effector cells (SLECs) in response to acute infections caused by influenza virus or lymphocytic choriomeningitis virus (LCMV). BRD7-deficiency in CD8+ T cells resulted in profound defects in effector population and functions, thereby impairing viral clearance and host recovery. Further mechanical studies indicated that the expression of BRD7 significantly turned to high from naïve CD8+ T cells to effector cells, bridged BRG1 and PBRM1 to the core module of PBAF complex, consequently facilitating the assembly of PBAF complex rather than BAF complex in the effector cells. The PBAF complex changed the chromatin accessibility at the loci of Tbx21 gene and up-regulated its expression, leading to the maturation of effector T cells. Our research confirms BRD7 and the PBAF complex are key in CD8+ T cell development and present a significant target for advancing immune therapies.

SARS-CoV-2 immunity in animal models.

The COVID-19 pandemic, which was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide health crisis due to its transmissibility. SARS-CoV-2 infection results in severe respiratory illness and can lead to significant complications in affected individuals. These complications encompass symptoms such as coughing, respiratory distress, fever, infectious shock, acute respiratory distress syndrome (ARDS), and even multiple-organ failure. Animal models serve as crucial tools for investigating pathogenic mechanisms, immune responses, immune escape mechanisms, antiviral drug development, and vaccines against SARS-CoV-2. Currently, various animal models for SARS-CoV-2 infection, such as nonhuman primates (NHPs), ferrets, hamsters, and many different mouse models, have been developed. Each model possesses distinctive features and applications. In this review, we elucidate the immune response elicited by SARS-CoV-2 infection in patients and provide an overview of the characteristics of various animal models mainly used for SARS-CoV-2 infection, as well as the corresponding immune responses and applications of these models. A comparative analysis of transcriptomic alterations in the lungs from different animal models revealed that the K18-hACE2 and mouse-adapted virus mouse models exhibited the highest similarity with the deceased COVID-19 patients. Finally, we highlighted the current gaps in related research between animal model studies and clinical investigations, underscoring lingering scientific questions that demand further clarification.

Endosomal Escapable and Nuclear Localizing Cationic Polyaspartate-Based CRISPR Activation System for Preventing Respiratory Virus Infection by Specifically Inducing Interferon-λ.

Global pandemics caused by viruses cause widespread panic and economic losses. The lack of specific antivirals and vaccines increases the spreading of viral diseases worldwide. Thus, alternative strategies are required to manage viral outbreaks. Here, we develop a CRISPR activation (CRISPRa) system based on polymeric carriers to prevent respiratory virus infection in a mouse model. A polyaspartate grafted with 2-(diisopropylamino) ethylamine (DIP) and nuclear localization signal peptides (NLS-MTAS fusion peptide) was complexed with plasmid DNA (pDNA) encoding dCas9-VPR and sgRNA targeting IFN-λ. The pH-sensitive DIP and NLS-MTAS groups were favor of endo-lysosomal escape and nuclear localization of pDNA, respectively. They synergistically improved gene transfection efficiency, resulting in significant reporter gene expression and IFN-λ upregulation in lung tissue. In vitro and in vivo prophylactic experiments showed that the non-viral CRISPRa system could prevent infection caused by H1N1 viruses with minimal inflammatory responses, presenting a promising prophylactic approach against respiratory virus infections.

Glycopeptide Antibiotic Teicoplanin Inhibits Cell Entry of SARS-CoV-2 by Suppressing the Proteolytic Activity of Cathepsin L.

Since the outbreak of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), public health worldwide has been greatly threatened. The development of an effective treatment for this infection is crucial and urgent but is hampered by the incomplete understanding of the viral infection mechanisms and the lack of specific antiviral agents. We previously reported that teicoplanin, a glycopeptide antibiotic that has been commonly used in the clinic to treat bacterial infection, significantly restrained the cell entry of Ebola virus, SARS-CoV, and MERS-CoV by specifically inhibiting the activity of cathepsin L (CTSL). Here, we found that the cleavage sites of CTSL on the spike proteins of SARS-CoV-2 were highly conserved among all the variants. The treatment with teicoplanin suppressed the proteolytic activity of CTSL on spike and prevented the cellular infection of different pseudotyped SARS-CoV-2 viruses. Teicoplanin potently prevented the entry of SARS-CoV-2 into the cellular cytoplasm with an IC50 of 2.038 µM for the Wuhan-Hu-1 reference strain and an IC50 of 2.116 µM for the SARS-CoV-2 (D614G) variant. The pre-treatment of teicoplanin also prevented SARS-CoV-2 infection in hACE2 mice. In summary, our data reveal that CTSL is required for both SARS-CoV-2 and SARS-CoV infection and demonstrate the therapeutic potential of teicoplanin for universal anti-CoVs intervention.

Development of Receptor Binding Domain (RBD)-Conjugated Nanoparticle Vaccines with Broad Neutralization against SARS-CoV-2 Delta and Other Variants.

The SARS-CoV-2 Delta (B.1.617.2) strain is a variant of concern (VOC) that has become the dominant strain worldwide in 2021. Its transmission capacity is approximately twice that of the original strain, with a shorter incubation period and higher viral load during infection. Importantly, the breakthrough infections of the Delta variant have continued to emerge in the first-generation vaccine recipients. There is thus an urgent need to develop a novel vaccine with SARS-CoV-2 variants as the major target. Here, receptor binding domain (RBD)-conjugated nanoparticle vaccines targeting the Delta variant, as well as the early and Beta/Gamma strains, are developed. Under both a single-dose and a prime-boost strategy, these RBD-conjugated nanoparticle vaccines induce the abundant neutralizing antibodies (NAbs) and significantly protect hACE2 mice from infection by the authentic SARS-CoV-2 Delta strain, as well as the early and Beta strains. Furthermore, the elicitation of the robust production of broader cross-protective NAbs against almost all the notable SARS-CoV-2 variants including the Omicron variant in rhesus macaques by the third re-boost with trivalent vaccines is found. These results suggest that RBD-based monovalent or multivalent nanoparticle vaccines provide a promising second-generation vaccine strategy for SARS-CoV-2 variants.

A bivalent nanoparticle vaccine exhibits potent cross-protection against the variants of SARS-CoV-2.

Inoculation against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is ongoing worldwide. However, the emergence of SARS-CoV-2 variants could cause immune evasion. We developed a bivalent nanoparticle vaccine that displays the receptor binding domains (RBDs) of the D614G and B.1.351 strains. With a prime-boost or a single-dose strategy, this vaccine elicits a robust neutralizing antibody and full protection against infection with the authentic D614G or B.1.351 strain in human angiotensin-converting enzyme 2 transgene mice. Interestingly, 8 months after inoculation with the D614G-specific vaccine, a new boost with this bivalent vaccine potently elicits cross-neutralizing antibodies for SARS-CoV-2 variants in rhesus macaques. We suggest that the D614G/B.1.351 bivalent vaccine could be used as an initial single dose or a sequential enforcement dose to prevent infection with SARS-CoV-2 and its variants.

Infection of wild-type mice by SARS-CoV-2 B.1.351 variant indicates a possible novel cross-species transmission route.

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.

The interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 replication through competitively blocking the virus entry.

Since the outbreak of coronavirus disease 2019 (COVID-19), it has become a global pandemic. The spike (S) protein of etiologic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specifically recognizes human angiotensin-converting enzyme 2 (hACE2) as its receptor, which is recently identified as an interferon (IFN)-stimulated gene. Here, we find that hACE2 exists on the surface of exosomes released by different cell types, and the expression of exosomal hACE2 is increased by IFNα/ß treatment. In particular, exosomal hACE2 can specifically block the cell entry of SARS-CoV-2, subsequently inhibit the replication of SARS-CoV-2 in vitro and ex vivo. Our findings have indicated that IFN is able to upregulate a viral receptor on the exosomes which competitively block the virus entry, exhibiting a potential antiviral strategy.

Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity.

BACKGROUND: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear. METHOD: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs. RESULTS: We discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo. CONCLUSION: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.

Brd4 Regulates the Homeostasis of CD8+ T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation.

CD8+ T cells are major components of adaptive immunity and confer robust protective cellular immunity, which requires adequate T-cell numbers, targeted migration, and efficient T-cell proliferation. Altered CD8+ T-cell homeostasis and impaired proliferation result in dysfunctional immune response to infection or tumorigenesis. However, intrinsic factors controlling CD8+ T-cell homeostasis and immunity remain largely elusive. Here, we demonstrate the prominent role of Brd4 on CD8+ T cell homeostasis and immune response. By upregulating Myc and GLUT1 expression, Brd4 facilitates glucose uptake and energy production in mitochondria, subsequently supporting naïve CD8+ T-cell survival. Besides, Brd4 promotes the trafficking of naïve CD8+ T cells partially through maintaining the expression of homing receptors (CD62L and LFA-1). Furthermore, Brd4 is required for CD8+ T cell response to antigen stimulation, as Brd4 deficiency leads to a severe defect in clonal expansion and terminal differentiation by decreasing glycolysis. Importantly, as JQ1, a pan-BRD inhibitor, severely dampens CD8+ T-cell immune response, its usage as an anti-tumor agent or latency-reversing agent for human immunodeficiency virus type I (HIV-1) should be more cautious. Collectively, our study identifies a previously-unexpected role of Brd4 in the metabolic regulation of CD8+ T cell-mediated immune surveillance and also provides a potential immunomodulation target.

Host factor SMYD3 is recruited by Ebola virus nucleoprotein to facilitate viral mRNA transcription.

The polymerase complex of Ebola virus (EBOV) is the functional unit for transcription and replication of viral genome. Nucleoprotein (NP) is a multifunctional protein with high RNA binding affinity and recruits other viral proteins to form functional polymerase complex. In our study, we investigated host proteins associated with EBOV polymerase complex using NP as bait in a transcription and replication competent minigenome system by mass spectrometry analysis and identified SET and MYND domain-containing protein 3 (SMYD3) as a novel host protein which was required for the replication of EBOV. SMYD3 specifically interacted with NP and was recruited to EBOV inclusion bodies through NP. The depletion of SMYD3 dramatically suppressed EBOV mRNA production. A mimic of non-phosphorylated VP30, which is a transcription activator, could partially rescue the viral mRNA production downregulated by the depletion of SMYD3. In addition, SMYD3 promoted NP-VP30 interaction in a dose-dependent manner. These results revealed that SMYD3 was a novel host factor recruited by NP to supporting EBOV mRNA transcription through increasing the binding of VP30 to NP. Thus, our study provided a new understanding of mechanism underlying the transcription of EBOV genome, and a novel anti-EBOV drug design strategy by targeting SMYD3.