Mpox multi-antigen mRNA vaccine candidates by a simplified manufacturing strategy afford efficient protection against lethal orthopoxvirus challenge.

Current unprecedented mpox outbreaks in non-endemic regions represent a global public health concern. Although two live-attenuated vaccinia virus (VACV)-based vaccines have been urgently approved for people at high risk for mpox, a safer and more effective vaccine that can be available for the general public is desperately needed. By utilizing a simplified manufacturing strategy of mixing DNA plasmids before transcription, we developed two multi-antigen mRNA vaccine candidates, which encode four (M1, A29, B6, A35, termed as Rmix4) or six (M1, H3, A29, E8, B6, A35, termed as Rmix6) mpox virus antigens. We demonstrated that those mpox multi-antigen mRNA vaccine candidates elicited similar potent cross-neutralizing immune responses against VACV, and compared to Rmix4, Rmix6 elicited significantly stronger cellular immune responses. Moreover, immunization with both vaccine candidates protected mice from the lethal VACV challenge. Investigation of B-cell receptor (BCR) repertoire elicited by mpox individual antigen demonstrated that the M1 antigen efficiently induced neutralizing antibody responses, and all neutralizing antibodies among the top 20 frequent antibodies appeared to target the same conformational epitope as 7D11, revealing potential vulnerability to viral immune evasion. Our findings suggest that Rmix4 and Rmix6 from a simplified manufacturing process are promising candidates to combat mpox.

Novel (Z)/(E)-1,2,4-triazole derivatives containing oxime ether moiety as potential ergosterol biosynthesis inhibitors: design, preparation, antifungal evaluation, and molecular docking.

Inspired by the highly effective and broad-spectrum antifungal activity of ergosterol biosynthesis inhibitions, a series of novel 1,2,4-triazole derivatives containing oxime ether moiety were constructed for screening the bioactivity against phytopathogenic fungi. The (Z)- and (E)-isomers of target compounds were successfully separated and identified by the spectroscopy and single crystal X-ray diffraction analyses. The bioassay results showed that the (Z)-isomers of target compounds possessed higher antifungal activity than the (E)-isomers. Strikingly, the compound (Z)-5o exhibited excellent antifungal activity against Rhizoctonia solani with the EC50 value of 0.41 µg/mL in vitro and preventive effect of 94.58% in vivo at 200 µg/mL, which was comparable to the positive control tebuconazole. The scanning electron microscopy observation indicated that the compound (Z)-5o caused the mycelial morphology to become wizened and wrinkled. The molecular docking modes of (Z)-5o and (E)-5o with the potential target protein RsCYP51 were especially compared. And the main interactions between ligands and amino acid residues were carefully analyzed to preliminarily explain the mechanism leading to the difference of activity between two isomers. The study provided a new lead molecular skeleton for developing novel triazole fungicides targeting ergosterol biosynthesis.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.

Macrophage deletion of Noc4l triggers endosomal TLR4/TRIF signal and leads to insulin resistance.

In obesity, macrophages drive a low-grade systemic inflammation (LSI) and insulin resistance (IR). The ribosome biosynthesis protein NOC4 (NOC4) mediates 40 S ribosomal subunits synthesis in yeast. Hereby, we reported an unexpected location and function of NOC4L, which was preferentially expressed in human and mouse macrophages. NOC4L was decreased in both obese human and mice. The macrophage-specific deletion of Noc4l in mice displayed IR and LSI. Conversely, Noc4l overexpression by lentivirus treatment and transgenic mouse model improved glucose metabolism in mice. Importantly, we found that Noc4l can interact with TLR4 to inhibit its endocytosis and block the TRIF pathway, thereafter ameliorated LSI and IR in mice.

A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2.

The rapid expansion of the COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health and economic priority. Taking advantage of versatility and rapid development, three SARS-CoV-2 mRNA vaccine candidates have entered clinical trials with a two-dose immunization regimen. However, the waning antibody response in convalescent patients after SARS-CoV-2 infection and the emergence of human re-infection have raised widespread concerns about a possible short duration of SARS-CoV-2 vaccine protection. Here, we developed a nucleoside-modified mRNA vaccine in lipid-encapsulated form that encoded the SARS-CoV-2 RBD, termed as mRNA-RBD. A single immunization of mRNA-RBD elicited both robust neutralizing antibody and cellular responses, and conferred a near-complete protection against wild SARS-CoV-2 infection in the lungs of hACE2 transgenic mice. Noticeably, the high levels of neutralizing antibodies in BALB/c mice induced by mRNA-RBD vaccination were maintained for at least 6.5 months and conferred a long-term notable protection for hACE2 transgenic mice against SARS-CoV-2 infection in a sera transfer study. These data demonstrated that a single dose of mRNA-RBD provided long-term protection against SARS-CoV-2 challenge.

Molecular basis of EphA2 recognition by gHgL from gammaherpesviruses.

The human γ-herpesviruses Kaposi sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are associated with many human malignancies. Viral glycoprotein H (gH) and glycoprotein L (gL) are crucial for the cell tropism by binding to specific receptors. Recently, EphA2 was identified as the specific entry receptor for both KSHV and EBV. Here, we characterized the crystal structures of KSHV gHgL or EBV gHgL in complex with the ligand binding domain (LBD) of EphA2. Both KSHV and EBV gHgL bind to the channel and peripheral regions of LBD primarily using gL. Extensive interactions with more contacts contribute to the higher affinity of KSHV gHgL to LBD than that of EBV gHgL. These binding characteristics were verified using cell-based fusion assays with mutations in key EphA2 residues. Our experiments suggest that multiple animal γ-herpesviruses could use EphA2 as an entry receptor, implying a potential threat to human health.

N-glycosylation of PD-1 promotes binding of camrelizumab.

PD-1 is a highly glycosylated inhibitory receptor expressed mainly on T cells. Targeting of PD-1 with monoclonal antibodies (MAbs) to block the interaction with its ligand PD-L1 has been successful for the treatment of multiple tumors. However, polymorphisms at N-glycosylation sites of PD-1 exist in the human population that might affect antibody binding, and dysregulated glycosylation has been observed in the tumor microenvironment. Here, we demonstrate varied N-glycan composition in PD-1, and show that the binding affinity of camrelizumab, a recently approved PD-1-specific MAb, to non-glycosylated PD-1 proteins from E. coli is substantially decreased compared with glycosylated PD-1. The structure of the camrelizumab/PD-1 complex reveals that camrelizumab mainly utilizes its heavy chain to bind to PD-1, while the light chain sterically inhibits the binding of PD-L1 to PD-1. Glycosylation of asparagine 58 (N58) promotes the interaction with camrelizumab, while the efficiency of camrelizumab to inhibit the binding of PD-L1 is substantially reduced for glycosylation-deficient PD-1. These results increase our understanding of how glycosylation affects the activity of PD-1-specific MAbs during immune checkpoint therapy.

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.

Isolation of Monoclonal Antibodies from Zika Virus-Infected Patient Samples.

The combination of sorting antigen-specific memory B cells with determining immunoglobulin (Ig) genes at the single-cell level enables the isolation of monoclonal antibodies (mAbs) in individuals. This method requires a small amount of blood (usually 10 mL) and is rapid (less than 2 weeks to isolate antigen-specific mAbs). Due to the application of antigens as the bait to capture the specific memory B cells, the majority of isolated mAbs are true binders to the antigen, which increases the isolation efficiency. Here, applying this approach, we describe the characterization of mAbs against Zika virus from a convalescent patient sample. From 10 mL whole blood, we sorted 33 Zika envelope (E) protein-interacting single memory B cells. The Ig genes from 15 cells were determined, and 13 mAbs were found that bind to Zika E protein with varied binding affinities.

Molecular Basis of Binding between Middle East Respiratory Syndrome Coronavirus and CD26 from Seven Bat Species.

Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting humans have occurred since the identification of this virus in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozen spillover infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with equilibrium dissociation constant values ranging from several to hundreds at the micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.IMPORTANCE In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.

Structures of the four Ig-like domain LILRB2 and the four-domain LILRB1 and HLA-G1 complex.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs), also known as CD85 and immunoglobulin-like transcripts (ILTs), play pivotal roles in regulating immune responses. These receptors define an immune checkpoint that immune therapy can target. Through cis or trans interactions with human leukocyte antigen (HLA)-G, the two most abundantly expressed inhibitory LILRs, LILRB1, and LILRB2 (LILRB1/2, also known as CD85j/d and ILT2/4), are involved in immunotolerance in pregnancy and transplantation, autoimmune diseases, and immune evasion by tumors. Although the discrete domains of LILRB1/2 are clear, the assembly mode of the four extracellular Ig-like domains (D1, D2, D3, and D4) remains unknown. Previous data indicate that D1D2 is responsible for binding to HLA class I (HLA-I), but the roles of D3D4 are still unclear. Here, we determined the crystal structure of the four Ig-like domain LILRB2 and four-domain LILRB1 in complex with HLA-G1. The angles between adjacent domains and the staggered assembly of the four domains suggest limited flexibility and limited plasticity of the receptors during ligand binding. The complex structure of four-domain LILRB1 and HLA-G1 supports the model that D1D2 is responsible for HLA-I binding, while D3D4 acts as a scaffold. Accordingly, cis and trans binding models for HLA-I binding to LILRB1/2 are proposed. The geometries of LILRB1/2 in complex with dimeric and monomeric HLA-G1 suggest the accessibility of the dimeric receptor, which in turn, transduces more inhibitory signals. The assembly of LILRB1/2 and its binding to HLA-G1 could aid in the design of immune regulators and benefit immune interference.

Molecular Basis of Arthritogenic Alphavirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.

Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.

The FG Loop of PD-1 Serves as a "Hotspot" for Therapeutic Monoclonal Antibodies in Tumor Immune Checkpoint Therapy.

Programmed cell death 1 (PD-1)/PD-1 ligand-1 (PD-L1)-blocking monoclonal antibodies (mAbs) have taken center stage for tumor immune checkpoint therapy. Identification of the "hotspots" on PD-1 for mAbs will help to develop next-generation oral deliverable agents with long-lasting efficacy. Here, we identified two PD-1-targeting mAbs, GY-5 and GY-14, with PD-1/PD-L1-blocking efficacy. Complex structural information revealed that both mAbs mainly bind to the FG loop of PD-1, which also contributes multiple interactions with PD-L1. The FG loop adopts substantially varied conformations upon binding to different mAbs, providing a novel targetable region for the development of PD-1-specific biologics and small chemical molecules. Glycosylation modifications of PD-1 could be observed in three of the four potential N-linked glycosylation sites. However, the binding of GY-5 and GY-14 to PD-1 was not affected by glycosylation. These findings broaden our understanding of the mechanism of anti-PD-1 mAbs and provide insight into the development of agents targeting PD-1.

Neutralization mechanism of human monoclonal antibodies against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

Glycosylation-independent binding of monoclonal antibody toripalimab to FG loop of PD-1 for tumor immune checkpoint therapy.

Monoclonal antibody (mAb)-based blockade of programmed cell death 1 (PD-1) or its ligand to enable antitumor T-cell immunity has been successful in treating multiple tumors. However, the structural basis of the binding mechanisms of the mAbs and PD-1 and the effects of glycosylation of PD-1 on mAb interaction are not well understood. Here, we report the complex structure of PD-1 with toripalimab, a mAb that is approved by China National Medical Products Administration as a second-line treatment for melanoma and is under multiple Phase 1-Phase 3 clinical trials in both China and the US. Our analysis reveals that toripalimab mainly binds to the FG loop of PD-1 with an unconventionally long complementarity-determining region 3 loop of the heavy chain, which is distinct from the known binding epitopes of anti-PD-1 mAbs with structural evidences. The glycan modifications of PD-1 could be observed in three potential N-linked glycosylation sites, while no substantial influences were detected to the binding of toripalimab. These findings benefit our understanding of the binding mechanisms of toripalimab to PD-1 and shed light for future development of biologics targeting PD-1. Atomic coordinates have been deposited in the Protein Data Bank under accession code 6JBT.

Humoral and cellular immunity against both ZIKV and poxvirus is elicited by a two-dose regimen using DNA and non-replicating vaccinia virus-based vaccine candidates.

The Zika virus (ZIKV) and poxvirus infection are considered as public health emergencies, necessitating the development of effective vaccines. Here, we report novel recombinant DNA-based and non-replicating vaccinia virus (NTV)-based vaccine candidates that express the precursor membrane-envelope (prME) or envelope (E) glycoproteins of ZIKV. After immunization of BABL/c mice with the vaccines using a homologous protocol (DNA/DNA, NTV/NTV) or heterogeneous (DNA/NTV) protocol, a similar level of anti-E IgG and neutralizing antibodies (microneutralization test) were detected in the mice. However, a significantly higher level of E-specific T cell responses was elicited in mice when a heterogeneous prime-boost protocol was used (DNA/NTV) with either the DNA-based or NTV-based vaccines. Furthermore, neutralizing antibodies and a T cell immune response against the vaccinia virus (VV) were detected in mice that were subjected to the prime-boost protocol (DNA/NTV), whereas those subjected to a homologous NTV/NTV protocol had higher levels of anti-VV IgG and neutralizing antibodies. Lastly, a novel H-2d-restricted CD8 T-cell epitope, VRSYCYEASISDMAS, was identified in the ZIKV E protein. These data demonstrate proof of concept of a bivalent vaccine candidate against ZIKV and orthopoxvirus, and support the use of DNA-prME prime and NTV-E boost protocols to protect against ZIKV and orthopoxvirus infections.