Rational design of a 'two-in-one' immunogen DAM drives potent immune response against mpox virus.

The global outbreak of the mpox virus (MPXV) in 2022 highlights the urgent need for safer and more accessible new-generation vaccines. Here, we used a structure-guided multi-antigen fusion strategy to design a 'two-in-one' immunogen based on the single-chain dimeric MPXV extracellular enveloped virus antigen A35 bivalently fused with the intracellular mature virus antigen M1, called DAM. DAM preserved the natural epitope configuration of both components and showed stronger A35-specific and M1-specific antibody responses and in vivo protective efficacy against vaccinia virus (VACV) compared to co-immunization strategies. The MPXV-specific neutralizing antibodies elicited by DAM were 28 times higher than those induced by live VACV vaccine. Aluminum-adjuvanted DAM vaccines protected mice from a lethal VACV challenge with a safety profile, and pilot-scale production confirmed the high yield and purity of DAM. Thus, our study provides innovative insights and an immunogen candidate for the development of alternative vaccines against MPXV and other orthopoxviruses.

A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS.

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

An engineered bispecific human monoclonal antibody against SARS-CoV-2.

The global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires effective therapies against coronavirus disease 2019 (COVID-19), and neutralizing antibodies are a promising therapy. A noncompeting pair of human neutralizing antibodies (B38 and H4) blocking SARS-CoV-2 binding to its receptor, ACE2, have been described previously. Here, we develop bsAb15, a bispecific monoclonal antibody (bsAb) based on B38 and H4. bsAb15 has greater neutralizing efficiency than these parental antibodies, results in less selective pressure and retains neutralizing ability to most SARS-CoV-2 variants of concern (with more potent neutralizing activity against the Delta variant). We also selected for escape mutants of the two parental mAbs, a mAb cocktail and bsAb15, demonstrating that bsAb15 can efficiently neutralize all single-mAb escape mutants. Furthermore, prophylactic and therapeutic application of bsAb15 reduced the viral titer in infected nonhuman primates and human ACE2 transgenic mice. Therefore, this bsAb is a feasible and effective strategy to treat and prevent severe COVID-19.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease.

Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice.

Zika virus (ZIKV) persists in the semen of male patients, a first for flavivirus infection. Here, we demonstrate that ZIKV can induce inflammation in the testis and epididymidis, but not in the prostate or seminal vesicle, and can lead to damaged testes after 60 days post-infection in mice. ZIKV induces innate immune responses in Leydig, Sertoli, and epididymal epithelial cells, resulting in the production of pro-inflammatory cytokines/chemokines. However, ZIKV does not induce a rapid and abundant cytokine production in peritubular cell and spermatogonia, suggesting that these cells are vulnerable for ZIKV infection and could be the potential repositories for ZIKV. Our study demonstrates a correlation between ZIKV and testis infection/damage and suggests that ZIKV infection, under certain circumstances, can eventually lead to male infertility.

Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

Defining a de novo non-RBM antibody as RBD-8 and its synergistic rescue of immune-evaded antibodies to neutralize Omicron SARS-CoV-2.

Currently, monoclonal antibodies (MAbs) targeting the SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein are classified into seven classes based on their binding epitopes. However, most of these antibodies are seriously impaired by SARS-CoV-2 Omicron and its subvariants, especially the recent BQ.1.1, XBB and its derivatives. Identification of broadly neutralizing MAbs against currently circulating variants is imperative. In this study, we identified a "breathing" cryptic epitope in the S protein, named as RBD-8. Two human MAbs, BIOLS56 and IMCAS74, were isolated recognizing this epitope with broad neutralization abilities against tested sarbecoviruses, including SARS-CoV, pangolin-origin coronaviruses, and all the SARS-CoV-2 variants tested (Omicron BA.4/BA.5, BQ.1.1, and XBB subvariants). Searching through the literature, some more RBD-8 MAbs were defined. More importantly, BIOLS56 rescues the immune-evaded antibody, RBD-5 MAb IMCAS-L4.65, by making a bispecific MAb, to neutralize BQ.1 and BQ.1.1, thereby producing an MAb to cover all the currently circulating Omicron subvariants. Structural analysis reveals that the neutralization effect of RBD-8 antibodies depends on the extent of epitope exposure, which is affected by the angle of antibody binding and the number of up-RBDs induced by angiotensin-converting enzyme 2 binding. This cryptic epitope which recognizes non- receptor binding motif (non-RBM) provides guidance for the development of universal therapeutic antibodies and vaccines against COVID-19.

Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance. IMPORTANCE: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαßs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αß paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/ß clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/ß clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

dSCOPE: a software to detect sequences critical for liquid-liquid phase separation.

Membrane-based cells are the fundamental structural and functional units of organisms, while evidences demonstrate that liquid-liquid phase separation (LLPS) is associated with the formation of membraneless organelles, such as P-bodies, nucleoli and stress granules. Many studies have been undertaken to explore the functions of protein phase separation (PS), but these studies lacked an effective tool to identify the sequence segments that critical for LLPS. In this study, we presented a novel software called dSCOPE (http://dscope.omicsbio.info) to predict the PS-driving regions. To develop the predictor, we curated experimentally identified sequence segments that can drive LLPS from published literature. Then sliding sequence window based physiological, biochemical, structural and coding features were integrated by random forest algorithm to perform prediction. Through rigorous evaluation, dSCOPE was demonstrated to achieve satisfactory performance. Furthermore, large-scale analysis of human proteome based on dSCOPE showed that the predicted PS-driving regions enriched various protein post-translational modifications and cancer mutations, and the proteins which contain predicted PS-driving regions enriched critical cellular signaling pathways. Taken together, dSCOPE precisely predicted the protein sequence segments critical for LLPS, with various helpful information visualized in the webserver to facilitate LLPS-related research.

Loss of TDP-43 mediates severe neurotoxicity by suppressing PJA1 gene transcription in the monkey brain.

The nuclear loss and cytoplasmic accumulation of TDP-43 (TAR DNA/RNA binding protein 43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previously, we reported that the primate-specific cleavage of TDP-43 accounts for its cytoplasmic mislocalization in patients' brains. This prompted us to investigate further whether and how the loss of nuclear TDP-43 mediates neuropathology in primate brain. In this study, we report that TDP-43 knockdown at the similar effectiveness, induces more damage to neuronal cells in the monkey brain than rodent mouse. Importantly, the loss of TDP-43 suppresses the E3 ubiquitin ligase PJA1 expression in the monkey brain at transcriptional level, but yields an opposite upregulation of PJA1 in the mouse brain. This distinct effect is due to the species-dependent binding of nuclear TDP-43 to the unique promoter sequences of the PJA1 genes. Further analyses reveal that the reduction of PJA1 accelerates neurotoxicity, whereas overexpressing PJA1 diminishes neuronal cell death by the TDP-43 knockdown in vivo. Our findings not only uncover a novel primate-specific neurotoxic contribution to the loss of function theory of TDP-43 proteinopathy, but also underscore a potential therapeutic approach of PJA1 to the loss of nuclear TDP-43.

Transcriptome profiling in swine macrophages infected with African swine fever virus at single-cell resolution.

African swine fever virus (ASFV) is the causative agent of African swine fever, a highly contagious and usually fatal disease in pigs. The pathogenesis of ASFV infection has not been clearly elucidated. Here, we used single-cell RNA-sequencing technology to survey the transcriptomic landscape of ASFV-infected primary porcine alveolar macrophages. The temporal dynamic analysis of viral genes revealed increased expression of viral transmembrane genes. Molecular characteristics in the ASFV-exposed cells exhibited the activation of antiviral signaling pathways with increased expression levels of interferon-stimulated genes and inflammatory- and cytokine-related genes. By comparing infected cells with unexposed cells, we showed that the unfolded protein response (UPR) pathway was activated in low viral load cells, while the expression level of UPR-related genes in high viral load cells was less than that in unexposed cells. Cells infected with various viral loads showed signature transcriptomic changes at the median progression of infection. Within the infected cells, differential expression analysis and coregulated virus­host analysis both demonstrated that ASFV promoted metabolic pathways but inhibited interferon and UPR signaling, implying the regulation pathway of viral replication in host cells. Furthermore, our results revealed that the cell apoptosis pathway was activated upon ASFV infection. Mechanistically, the production of tumor necrosis factor alpha (TNF-α) induced by ASFV infection is necessary for cell apoptosis, highlighting the importance of TNF-α in ASFV pathogenesis. Collectively, the data provide insights into the comprehensive host responses and complex virus­host interactions during ASFV infection, which may instruct future research on antiviral strategies.

Emerging ultrasmall luminescent nanoprobes for in vivo bioimaging.

Photoluminescence (PL) imaging has become a fundamental tool in disease diagnosis, therapeutic evaluation, and surgical navigation applications. However, it remains a big challenge to engineer nanoprobes for high-efficiency in vivo imaging and clinical translation. Recent years have witnessed increasing research efforts devoted into engineering sub-10 nm ultrasmall nanoprobes for in vivo PL imaging, which offer the advantages of efficient body clearance, desired clinical translation potential, and high imaging signal-to-noise ratio. In this review, we present a comprehensive summary and contrastive discussion of emerging ultrasmall luminescent nanoprobes towards in vivo PL bioimaging of diseases. We first summarize size-dependent nano-bio interactions and imaging features, illustrating the unique attributes and advantages/disadvantages of ultrasmall nanoprobes differentiating them from molecular and large-sized probes. We also discuss general design methodologies and PL properties of emerging ultrasmall luminescent nanoprobes, which are established based on quantum dots, metal nanoclusters, lanthanide-doped nanoparticles, and silicon nanoparticles. Then, recent advances of ultrasmall luminescent nanoprobes are highlighted by surveying their latest in vivo PL imaging applications. Finally, we discuss existing challenges in this exciting field and propose some strategies to improve in vivo PL bioimaging and further propel their clinical applications.

Ultrasensitive Urinary Diagnosis of Organ Injuries Using Time-Resolved Luminescent Lanthanide Nano-bioprobes.

Urinary sensing of synthetic biomarkers that are released into urine after specific activation in an in vivo disease environment is an emerging diagnosis strategy to overcome the insensitivity of a previous biomarker assay. However, it remains a great challenge to achieve sensitive and a specific urinary photoluminescence (PL) diagnosis. Herein, we report a novel urinary time-resolved PL (TRPL) diagnosis strategy by exploiting europium complexes of diethylenetriaminepentaacetic acid (Eu-DTPA) as synthetic biomarkers and designing the activatable nanoprobes. Notably, TRPL of Eu-DTPA in the enhancer can eliminate the urinary background PL for ultrasensitive detection. We achieved sensitive urinary TRPL diagnosis of mice kidney and liver injuries by using simple Eu-DTPA and Eu-DTPA-integrated nanoprobes, respectively, which cannot be realized by traditional blood assays. This work demonstrates the exploration of lanthanide nanoprobes for in vivo disease-activated urinary TRPL diagnosis for the first time, which might advance the noninvasive diagnosis of diverse diseases via tailorable nanoprobe designs.

Suppressing UBE2N ameliorates Alzheimer's disease pathology through the clearance of amyloid beta.

INTRODUCTION: Aging is one of the risk factors for the early onset of Alzheimer's disease (AD). We previously discovered that the age-dependent increase in Ubiquitin Conjugating Enzyme E2 N (UBE2N) plays a role in the accumulation of misfolded proteins through K63 ubiquitination, which has been linked to AD pathogenesis. However, the impact of UBE2N on amyloid pathology and clearance has remained unknown. RESULTS: We observed the elevated UBE2N during the amyloid beta (Aß) generation in the brains of 5×FAD, APP/PS1 mice, and patients with AD, in comparison to healthy individuals. UBE2N overexpression exacerbated amyloid deposition in 5×FAD mice and senescent monkeys, whereas knocking down UBE2N via CRISPR/Cas9 reduced Aß generation and cognitive deficiency. Moreover, pharmacological inhibition of UBE2N ameliorated Aß pathology and subsequent transcript defects in 5×FAD mice. DISCUSSION: We have discovered that age-dependent expression of UBE2N is a critical regulator of AD pathology. Our findings suggest that UBE2N could serve as a potential pharmacological target for the advancement of AD therapeutics. HIGHLIGHTS: Ubiquitin Conjugating Enzyme E2 N (UBE2N) level was elevated during amyloid beta (Aß) deposition in AD mouse and patients' brains. UBE2N exacerbated Aß generation in the AD mouse and senescent monkey. Drug inhibition of UBE2N ameliorated Aß pathology and cognitive deficiency.

Huntington's Disease: Complex Pathogenesis and Therapeutic Strategies.

Huntington's disease (HD) arises from the abnormal expansion of CAG repeats in the huntingtin gene (HTT), resulting in the production of the mutant huntingtin protein (mHTT) with a polyglutamine stretch in its N-terminus. The pathogenic mechanisms underlying HD are complex and not yet fully elucidated. However, mHTT forms aggregates and accumulates abnormally in neuronal nuclei and processes, leading to disruptions in multiple cellular functions. Although there is currently no effective curative treatment for HD, significant progress has been made in developing various therapeutic strategies to treat HD. In addition to drugs targeting the neuronal toxicity of mHTT, gene therapy approaches that aim to reduce the expression of the mutant HTT gene hold great promise for effective HD therapy. This review provides an overview of current HD treatments, discusses different therapeutic strategies, and aims to facilitate future therapeutic advancements in the field.

Aberrant splicing of mutant huntingtin in Huntington's disease knock-in pigs.

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disease caused by a CAG repeat expansion in exon1 of the huntingtin gene (HTT). This expansion leads to the production of N-terminal mutant huntingtin protein (mHtt) that contains an expanded polyglutamine tract, which is toxic to neurons and causes neurodegeneration. While the production of N-terminal mHtt can be mediated by proteolytic cleavage of full-length mHtt, abnormal splicing of exon1-intron1 of mHtt has also been identified in the brains of HD mice and patients. However, the proportion of aberrantly spliced exon1 mHTT in relation to normal mHTT exon remains to be defined. In this study, HTT exon1 production was examined in the HD knock-in (KI) pig model, which more closely recapitulates neuropathology seen in HD patient brains than HD mouse models. The study revealed that aberrant spliced HTT exon1 is also present in the brains of HD pigs, but it is expressed at a much lower level than the normally spliced HTT exon products. These findings suggest that careful consideration is needed when assessing the contribution of aberrantly spliced mHTT exon1 to HD pathogenesis, and further rigorous investigation is required.