Human transferrin receptor can mediate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Analysis of the aging-related biomarker in a nonhuman primate model using multilayer omics.

BACKGROUND: Aging is a prominent risk factor for diverse diseases; therefore, an in-depth understanding of its physiological mechanisms is required. Nonhuman primates, which share the closest genetic relationship with humans, serve as an ideal model for exploring the complex aging process. However, the potential of the nonhuman primate animal model in the screening of human aging markers is still not fully exploited. Multiomics analysis of nonhuman primate peripheral blood offers a promising approach to evaluate new therapies and biomarkers. This study explores aging-related biomarker through multilayer omics, including transcriptomics (mRNA, lncRNA, and circRNA) and proteomics (serum and serum-derived exosomes) in rhesus monkeys (Macaca mulatta). RESULTS: Our findings reveal that, unlike mRNAs and circRNAs, highly expressed lncRNAs are abundant during the key aging period and are associated with cancer pathways. Comparative analysis highlighted exosomal proteins contain more types of proteins than serum proteins, indicating that serum-derived exosomes primarily regulate aging through metabolic pathways. Finally, eight candidate aging biomarkers were identified, which may serve as blood-based indicators for detecting age-related brain changes. CONCLUSIONS: Our results provide a comprehensive understanding of nonhuman primate blood transcriptomes and proteomes, offering novel insights into the aging mechanisms for preventing or treating age-related diseases.

Identification of RACK1 as a novel regulator of non-structural protein 4 of chikungunya virus.

Chikungunya virus (CHIKV) is a neglected arthropod-borne and anthropogenic alphavirus. Over the past two decades, the CHIKV distribution has undergone significant changes worldwide, from the original tropics and subtropics regions to temperate regions, which has attracted global attention. However, the interactions between CHIKV and its host remain insufficiently understood, which dampens the need for the development of an anti-CHIKV strategy. In this study, on the basis of the optimal overexpression of non-structural protein 4 (nsP4), we explore host interactions of CHIKV nsP4 using mass spectrometry-based protein-protein interaction approaches. The results reveal that some cellular proteins that interact with nsP4 are enriched in the ubiquitin-proteasome pathway. Specifically, the scaffold protein receptor for activated C kinase 1 (RACK1) is identified as a novel host interactor and regulator of CHIKV nsP4. The inhibition of the interaction between RACK1 and nsP4 by harringtonolide results in the reduction of nsP4, which is caused by the promotion of degradation but not the inhibition of nsP4 translation. Furthermore, the decrease in nsP4 triggered by the RACK1 inhibitor can be reversed by the proteasome inhibitor MG132, suggesting that RACK1 can protect nsP4 from degradation through the ubiquitin-proteasome pathway. This study reveals a novel mechanism by which the host factor RACK1 regulates CHIKV nsP4, which could be a potential target for developing drugs against CHIKV.

Temporal transcriptomic dynamics in developing macaque neocortex.

Despite intense research on mice, the transcriptional regulation of neocortical neurogenesis remains limited in humans and non-human primates. Cortical development in rhesus macaque is known to recapitulate multiple facets of cortical development in humans, including the complex composition of neural stem cells and the thicker supragranular layer. To characterize temporal shifts in transcriptomic programming responsible for differentiation from stem cells to neurons, we sampled parietal lobes of rhesus macaque at E40, E50, E70, E80, and E90, spanning the full period of prenatal neurogenesis. Single-cell RNA sequencing produced a transcriptomic atlas of developing parietal lobe in rhesus macaque neocortex. Identification of distinct cell types and neural stem cells emerging in different developmental stages revealed a terminally bifurcating trajectory from stem cells to neurons. Notably, deep-layer neurons appear in the early stages of neurogenesis, while upper-layer neurons appear later. While these different lineages show overlap in their differentiation program, cell fates are determined post-mitotically. Trajectories analysis from ventricular radial glia (vRGs) to outer radial glia (oRGs) revealed dynamic gene expression profiles and identified differential activation of BMP, FGF, and WNT signaling pathways between vRGs and oRGs. These results provide a comprehensive overview of the temporal patterns of gene expression leading to different fates of radial glial progenitors during neocortex layer formation.

An inoculation site-retained mRNA vaccine induces robust immune responses against SARS-CoV-2 variants.

mRNA-based vaccines and therapeutic agents hold great promise in prevention and treatment of human diseases, yet high percentage of systemic adverse effect in clinic remains a big safety concern. One major potential cause is a high level of leakage of the locally inoculated mRNA vaccine nanoparticles into circulation. We have screened and optimized a core-shell structured lipopolyplex (LPP) formulation for mRNA with a tissue-retention property. Upon intramuscular inoculation, the mRNA-encapsulated LPP nanoparticles were preferentially taken up by the phagocytic antigen-presentation cells, and potently promoted dendritic cell maturation. We applied the new formulation to prepare a prophylactic vaccine for SARS-CoV-2, and observed potent humoral and cellular immune responses from the vaccine in both murine models and non-human primates. More importantly, the vaccine demonstrated a benign safety profile in non-human primates, with limited side effects after repeated treatment with high dosages of LPP/mRNA. Taken together, the inoculation site-retained vaccine formulation serves as a promising vehicle for mRNA vaccines and therapeutic agents.

Optimization and validation of a virus-like particle pseudotyped virus neutralization assay for SARS-CoV-2.

Spike-protein-based pseudotyped viruses were used to evaluate vaccines during the COVID-19 pandemic. However, they cannot be used to evaluate the envelope (E), membrane (M), and nucleocapsid (N) proteins. The first generation of virus-like particle (VLP) pseudotyped viruses contains these four structural proteins, but their titers for wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are relatively low, even lower for the omicron variant, rendering them unsuitable for neutralizing antibody detection. By optimizing the spike glycoprotein signal peptide, substituting the complexed M and E proteins with SARS-COV-1, optimizing the N protein with specific mutations (P199L, S202R, and R203M), and truncating the packaging signal, PS9, we increased the titer of the wild-type VLP pseudotyped virus over 100-fold, and successfully packaged the omicron VLP pseudotyped virus. The SARS-CoV-2 VLP pseudotyped viruses maintained stable titers, even through 10 freeze-thaw cycles. The key neutralization assay parameters were optimized, including cell type, cell number, and viral inoculum. The assay demonstrated minimal variation in both intra- and interassay results, at 11.5% and 11.1%, respectively. The correlation between the VLP pseudotyped virus and the authentic virus was strong (r = 0.9). Suitable for high-throughput detection of various mutant strains in clinical serum. In summary, we have developed a reliable neutralization assay for SARS-CoV-2 based on VLP pseudotyped virus.

XBB.1.16-RBD-based trimeric protein vaccine can effectively inhibit XBB.1.16-included XBB subvariant infection.

The newly identified XBB.1.16-containing sublineages, including XBB.1.5, have become the prevailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant in circulation. Unlike previous Omicron XBB variants (e.g., XBB.1.5 and XBB.1.9) harboring the F486P substitution, XBB.1.16 also carries a T478R substitution in the receptor-binding domain (RBD). Numerous researchers have delved into the high transmissibility and immune evasion of XBB.1.16 subvariant. Therefore, developing a new vaccine targeting XBB.1.16, including variants of concern (VOCs), is paramount. In our study, we engineered a recombinant protein by directly linking the S-RBD sequence of the XBB.1.16 strain of SARS-CoV-2 to the sequences of two heptad repeat sequences (HR1 and HR2) from the SARS-CoV-2 S2 subunit. Named the recombinant RBDXBB.1.16-HR/trimeric protein, this fusion protein autonomously assembles into a trimer. Combined with an MF59-like adjuvant, the RBDXBB.1.16-HR vaccine induces a robust humoral immune response characterized by high titers of neutralizing antibodies against variant pseudovirus and authentic VOCs and cellular immune responses. Additionally, a fourth heterologous RBDXBB.1.16-HR vaccine enhances both humoral and cellular immune response elicited by three-dose mRNA vaccines. These findings demonstrate that the recombinant RBDXBB.1.16-HR protein, featuring the new T478R mutation, effectively induces solid neutralizing antibodies to combat newly emerged XBB variants.

The inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in immunocompromised AG129 mice.

Zika virus, a mosquito-borne arbovirus, has repeatedly caused large pandemics with symptoms worsening from mild and self-limiting diseases to Guillain-Barré syndrome in adults and fetal microcephaly in newborns. In recent years, Zika virus diseases have posed a serious threat to human health. The shortage of susceptible small animal models makes it difficult to study pathogenic mechanisms and evaluate potential therapies for Zika virus infection. Therefore, we chose immunocompromised mice (AG129 mice) deficient in IFN-α/ß and IFN-γ receptors, which can abolish the innate immune system that prevents Zika virus infection early. AG129 mice were infected with the Zika virus, and this mouse model exhibited replication dynamics, tissue tropism, pathological lesion and immune activation of the Zika virus. Our results suggest that the inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in AG129 mice. By testing the potential antiviral drug favipiravir, several critical indicators, including replication dynamics and survival rates, were identified in AG129 mice after Zika virus infection. It is suggested that the model is reliable for drug evaluation. In brief, this model provides a potential platform for studies of the infectivity, virulence, and pathogenesis of the Zika virus. Moreover, the development of an accessible mouse model of Zika virus infection will expedite the research and deployment of therapeutics and vaccines.

Gastrointestinal microbiota and metabolites possibly contribute to distinct pathogenicity of SARS-CoV-2 proto or its variants in rhesus monkeys.

Gastrointestinal (GI) infection is evidenced with involvement in COVID-19 pathogenesis caused by SARS-CoV-2. However, the correlation between GI microbiota and the distinct pathogenicity of SARS-CoV-2 Proto and its emerging variants remains unclear. In this study, we aimed to determine if GI microbiota impacted COVID-19 pathogenesis and if the effect varied between SARS-CoV-2 Proto and its variants. We performed an integrative analysis of histopathology, microbiomics, and transcriptomics on the GI tract fragments from rhesus monkeys infected with SARS-CoV-2 proto or its variants. Based on the degree of pathological damage and microbiota profile in the GI tract, five of SARS-CoV-2 strains were classified into two distinct clusters, namely, the clusters of Alpha, Beta and Delta (ABD), and Proto and Omicron (PO). Notably, the abundance of potentially pathogenic microorganisms increased in ABD but not in the PO-infected rhesus monkeys. Specifically, the high abundance of UCG-002, UCG-005, and Treponema in ABD virus-infected animals positively correlated with interleukin, integrins, and antiviral genes. Overall, this study revealed that infection-induced alteration of GI microbiota and metabolites could increase the systemic burdens of inflammation or pathological injury in infected animals, especially in those infected with ABD viruses. Distinct GI microbiota and metabolite profiles may be responsible for the differential pathological phenotypes of PO and ABD virus-infected animals. These findings improve our understanding the roles of the GI microbiota in SARS-CoV-2 infection and provide important information for the precise prevention, control, and treatment of COVID-19.

A chimeric adenovirus-vectored vaccine based on Beta spike and Delta RBD confers a broad-spectrum neutralization against Omicron-included SARS-CoV-2 variants.

Urgent research into innovative severe acute respiratory coronavirus-2 (SARS-CoV-2) vaccines that may successfully prevent various emerging emerged variants, particularly the Omicron variant and its subvariants, is necessary. Here, we designed a chimeric adenovirus-vectored vaccine named Ad5-Beta/Delta. This vaccine was created by incorporating the receptor-binding domain from the Delta variant, which has the L452R and T478K mutations, into the complete spike protein of the Beta variant. Both intramuscular (IM) and intranasal (IN) vaccination with Ad5-Beta/Deta vaccine induced robust broad-spectrum neutralization against Omicron BA.5-included variants. IN immunization with Ad5-Beta/Delta vaccine exhibited superior mucosal immunity, manifested by higher secretory IgA antibodies and more tissue-resident memory T cells (TRM) in respiratory tract. The combination of IM and IN delivery of the Ad5-Beta/Delta vaccine was capable of synergically eliciting stronger systemic and mucosal immune responses. Furthermore, the Ad5-Beta/Delta vaccination demonstrated more effective boosting implications after two dosages of mRNA or subunit recombinant protein vaccine, indicating its capacity for utilization as a booster shot in the heterologous vaccination. These outcomes quantified Ad5-Beta/Delta vaccine as a favorable vaccine can provide protective immunity versus SARS-CoV-2 pre-Omicron variants of concern and BA.5-included Omicron subvariants.