Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo.

RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.

High-throughput screening of mutations affecting SARS-CoV-2 spike functions.

The spike (S) protein of SARS-CoV-2, which is undergoing rapid evolution, plays crucial roles in viral immune escape, infectivity, and transmissibility. To gain clinical insight, Dadonaite et al. developed a novel deep mutational scanning (DMS) platform for mapping the effects of S protein mutations on immune evasion and viral infectivity.

A pan-sarbecovirus vaccine based on RBD of SARS-CoV-2 original strain elicits potent neutralizing antibodies against XBB in non-human primates.

The recently emerged Omicron subvariants XBB and BQ.1.1 have presented striking immune evasion against most monoclonal neutralizing antibodies and convalescent plasma. Therefore, it is essential to develop broad-spectrum COVID-19 vaccines to combat current and future emerging variants. Here, we found that the human IgG Fc-conjugated RBD of the original SARS-CoV-2 strain (WA1) plus a novel STING agonist-based adjuvant CF501 (CF501/RBD-Fc) could induce highly potent and durable broad-neutralizing antibody (bnAb) responses against Omicron subvariants, including BQ.1.1 and XBB in rhesus macaques with NT50s ranging from 2,118 to 61,742 after three doses. A decline of 0.9- to 4.7-fold was observed in the neutralization activity of sera in the CF501/RBD-Fc group against BA.2.2, BA.2.9, BA.5, BA.2.75, and BF.7 relative to D614G after three doses, while a significant decline of NT50 against BQ.1.1 (26.9-fold) and XBB (22.5-fold) relative to D614G. However, the bnAbs were still effective in neutralizing BQ.1.1 and XBB infection. These results suggest that the conservative but nondominant epitopes in RBD could be stimulated by CF501 to generate bnAbs, providing a proof-of-concept for using "nonchangeable against changeables" strategy to develop pan-sarbecovirus vaccines against sarbecoviruses, including SARS-CoV-2 and its variants.

Development of variant-proof severe acute respiratory syndrome coronavirus 2, pan-sarbecovirus, and pan-ß-coronavirus vaccines.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmission rates and striking immune evasion have posed a serious challenge to the application of current first-generation SARS-CoV-2 vaccines. Other sarbecoviruses, such as SARS-CoV and SARS-related coronaviruses (SARSr-CoVs), have the potential to cause outbreaks in the future. These facts call for the development of variant-proof SARS-CoV-2, pan-sarbecovirus or pan-ß-CoV vaccines. Several novel vaccine platforms have been used to develop vaccines with broad-spectrum neutralizing antibody responses and protective immunity to combat the current SARS-CoV-2 and its variants, other sarbecoviruses, as well as other ß-CoVs, in the future. In this review, we discussed the major target antigens and protective efficacy of current SARS-CoV-2 vaccines and summarized recent advances in broad-spectrum vaccines against sarbecoviruses and ß-CoVs.

Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro.

Foot-and-mouth disease virus (FMDV) causes an economically important and highly contagious disease of cloven-hoofed animals such as cattle, swine, and sheep. FMD vaccine is the traditional way to protect against the disease, which can greatly reduce its occurrence. However, the use of FMD vaccines to protect early infection is limited. Therefore, the alternative strategy of applying antiviral agents is required to control the spread of FMDV in outbreak situations. As previously reported, LiCl has obviously inhibition effects on a variety of viruses such as transmissible gastroenteritis virus (TGEV), infectious bronchitis coronavirus (IBV), and pseudorabies herpesvirus and EV-A71 virus. In this study, our findings were the first to demonstrate that LiCl inhibition of the FMDV replication. In this study, BHK-21 cell was dose-dependent with LiCl at various stages of FMDV. Virus titration assay was calculated by the 50% tissue culture infected dose (TCID50 ) with the Reed and Muench method. The cytotoxicity assay of LiCl was performed by the CCK8 kit. The expression level of viral mRNA was measured by RT-qPCR. The results revealed LiCl can inhibit FMDV replication, but it cannot affect FMDV attachment stage and entry stage in the course of FMDV life cycle. Further studies confirmed that the LiCl affect the replication stage of FMDV, especially the early stages of FMDV replication. So LiCl has potential as an effective anti-FMDV drug. Therefore, LiCl may be an effective drug for the control of FMDV. Based on that, the mechanism of the antiviral effect of LiCl on FMDV infection is need to in-depth research in vivo.

The "blownknee" patient's stress fracture of distal tibial component after unilateral TKA: A case report.

RATIONALE: Periprosthetic fractures (PPF) are rare complications of total knee arthroplasty (TKA). The most common PPF after TKA is supracondylar femoral fracture, which is a relatively rare complication that is usually associated with high-energy trauma, with a reported incidence ranging from 0.4 to 1.7% according to the AOANJRR. However, in TKA patients, it is rarer that the stress fracture around the tibial prosthesis occurs due to changes in the lower limb force line, increasing weight-bearing, and changes in walking gait. PATIENT CONCERNS: A 68-year-old woman visited our hospital with "both knees had aggravated pain and deformity for 8 years." TKA was performed first on the left knee and the patient was discharged within 1 week. Three months later, the patient complained of pain in the upper middle 1/3 part of the medial tibia for 2 weeks, which gradually worsened and affected weight-bearing. DIAGNOSES: Physical examination showed that the left knee joint presented varus deformity, and the right valgus deformity, which diagnosed as osteoarthritis of both knees and was so-called "blownknee". The disease was initially diagnosed as osteoarthritis of both knees on first admission and PPF of the tibia in second. INTERVENTIONS: Three operations were performed on this patient. The first was TKA of the left knee, the second was open reduction and internal fixation of the PPF of the tibia 3 months after the first operation, and the third was TKA of the right knee. OUTCOMES: Until now, the patient has had no recurrent PPF, and the fracture is healing from the last X-ray. LESSONS: Clinicians should be aware of the possibility of PPF after TKA, especially in such patients, the most preferred surgical treatment method was open reduction and internal fixation of fractures using locking plates, and if the PPF with loosened implants, Revision TKA, or megaprosthesis was the better choice.

MicroRNA-34a-5p promotes the progression of osteoarthritis secondary to developmental dysplasia of the hip by restraining SESN2-induced autophagy.

Osteoarthritis (OA), a late-stage complication of developmental dysplasia of the hip (DDH), is a key factor leading to further degeneration of joint function. Studies have shown that Sestrin2 (SESN2) is a positive regulator in protecting articular cartilage from degradation. However, the regulatory effects of SESN2 on DDH-OA and its upstream regulators remain obscure. Here, we first identified that the expression of SESN2 significantly decreased in the cartilage of DDH-OA samples, with an expression trend negatively correlated with OA severity. Using RNA sequencing, we identified that the upregulation of miR-34a-5p may be an important factor for the decrease in SESN2 expression. Further exploring the regulation mechanism of miR-34a-5p/SESN2 is of great significance for understanding the mechanism of DDH occurrence and development. Mechanistically, we showed that miR-34a-5p could significantly inhibit the expression of SESN2, thereby promoting the activity of the mTOR signaling pathway. We also found that miR-34a-5p significantly inhibited SESN2-induced autophagy, thereby suppressing the proliferation and migration of chondrocytes. We further validated that knocking down miR-34a-5p in vivo resulted in a significant increase in SESN2 expression and autophagy activity in DDH-OA cartilage. Our study suggests that miR-34a-5p is a negative regulator of DDH-OA, and may provide a new target for the prevention of DDH-OA.

Intranasal G5-BGG/pDNA Vaccine Elicits Protective Systemic and Mucosal Immunity against SARS-CoV-2 by Transfecting Mucosal Dendritic Cells.

Infectious disease pandemics, including the coronavirus disease 2019 pandemic, have heightened the demand for vaccines. Although parenteral vaccines induce robust systemic immunity, their effectiveness in respiratory mucosae is limited. Considering the crucial role of nasal-associated lymphoid tissue (NALT) in mucosal immune responses, in this study, the intranasal complex composed of G5-BGG and antigen-expressing plasmid DNA (pSP), named G5-BGG/pSP complex, is developed to activate NALT and to promote both systemic and mucosal immune defense. G5-BGG/pSP could traverse mucosal barriers and deliver DNA to the target cells because of its superior nasal retention and permeability characteristics. The intranasal G5-BGG/pSP complex elicits robust antigen-specific immune responses, such as the notable production of IgG antibody against several virus variants. More importantly, it induces elevated levels of antigen-specific IgA antibody and a significant expansion of the lung-resident T lymphocyte population. Notably, the intranasal G5-BGG/pSP complex results in antigen expression and maturation of dendritic cells in nasal mucosae. These findings exhibit the potential of G5-BGG, a novel cationic material, as an effective gene carrier for intranasal vaccines to obtain robust systemic and mucosal immunity.

Neutralization of SARS-CoV-2 BA.2.86 and JN.1 by CF501 adjuvant-enhanced immune responses targeting the conserved epitopes in ancestral RBD.

The emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2.86 and JN.1 raise concerns regarding their potential to evade immune surveillance and spread globally. Here, we test sera from rhesus macaques immunized with 3 doses of wild-type SARS-CoV-2 receptor-binding domain (RBD)-Fc adjuvanted with the STING agonist CF501. We find that the sera can potently neutralize pseudotyped XBB.1.5, XBB.1.16, CH.1.1, EG.5, BA.2.86, and JN.1, with 50% neutralization titers ranging from 3,494 to 7,424. We also demonstrate that CF501, but not Alum, can enhance immunogenicity of the RBD from wild-type SARS-CoV-2 to improve induction of broadly neutralizing antibodies (bnAbs) with binding specificity and activity similar to those of SA55, BN03, and S309, thus exhibiting extraordinary broad-spectrum neutralizing activity. Overall, the RBD from wild-type SARS-CoV-2 also contains conservative epitopes. The RBD-Fc adjuvanted by CF501 can elicit potent bnAbs against JN.1, BA.2.86, and other XBB subvariants. This strategy can be adopted to develop broad-spectrum vaccines to combat future emerging and reemerging viral infectious diseases.

An engineered recombinant protein containing three structural domains in SARS-CoV-2 S2 protein has potential to act as a pan-human coronavirus entry inhibitor or vaccine antigen.

The threat to global health caused by three highly pathogenic human coronaviruses (HCoV), SARS-CoV-2, MERS-CoV and SARS-CoV, calls for the development of pan-HCoV therapeutics and vaccines. This study reports the design and engineering of a recombinant protein designated HR1LS. It contains three linked molecules, each consisting of three structural domains, including a heptad repeat 1 (HR1), a central helix (CH), and a stem helix (SH) region, in the S2 subunit of SARS-CoV-2 spike (S) protein. It was found that HR1LS protein automatically formed a trimer able to bind with heptad repeat 2 (HR2) region in the SARS-CoV-2 S2 subunit, thus potently inhibiting HCoV fusion and entry into host cells. Furthermore, immunization of mice with HR1LS, when combined with CF501 adjuvant, resulted in the production of neutralizing antibodies against infection of SARS-CoV-2 and its variants, as well as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63 and MjHKU4r-CoV-1. These results suggest that HR1LS is a promising candidate for further development as a novel HR1-trimer-based pan-HCoV entry inhibitor or vaccine for the treatment and prevention of infection by SARS-CoV-2 and its variants, but also other HCoVs with the potential to cause future emerging and re-emerging infectious coronavirus diseases.

A Perspective on the Roles of Adjuvants in Developing Highly Potent COVID-19 Vaccines.

Several countries have made unremitting efforts to develop an optimal vaccine in the fight against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the increasing occurrence of SARS-CoV-2 variants, current vaccines show decreased neutralizing activities, especially towards the Omicron variant. In this context, adding appropriate adjuvants to COVID-19 vaccines can substantially reduce the number of required doses and improve efficacy or cross-neutralizing protection. We mainly focus on research progress and achievements associated with adjuvanted COVID-19 subunit and inactivated vaccines. We further compare the advantages and disadvantages of different adjuvant formulations in order to provide a scientific reference for designing an effective strategy for future vaccine development.

Identification and functional analysis of circRNAs in the skeletal muscle of juvenile and adult largemouth bass (Micropterus salmoides).

Circular RNA (circRNA) is a novel emerging type of endogenous regulatory non-coding RNA molecules with a covalent closed-loop configuration, which exerts important functions in multiple biological processes. CircRNAs are known to regulate gene expression as functional regulators interacting with miRNAs by sponge, which have been reported to regulate skeletal muscle development. Nevertheless, the information of circRNAs involved in regulating muscle growth and development in fish is largely unknown. Here, we first identified 312 and 511 circRNAs in skeletal muscle of juvenile and adult largemouth bass (LMB) using RNA sequencing, respectively. The differentially expressed circRNAs (DE-circRNAs) analysis showed that there are 44 DE-circRNAs at two different skeletal muscle growth stages. Six circRNAs were chosen randomly and their relative expression levels in juvenile and adult LMB were confirmed by real-time PCR, indicating that these circRNAs were existed authenticity. In addition, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis showed that these hose genes (their linear mRNAs) of DE-circRNAs were mainly enriched in the regulation of actin cytoskeleton signaling pathways. The circRNA-miRNA interaction regulatory networks indicated that one circRNA can regulate one or more miRNA. For instance, more than 30 miRNAs were regulated by two circRNAs (circRNA389 and circRNA399). Of them, the muscle-related miRNAs including the let-7 family, miR-133 and miR-26 and so on were found acting as miRNAs sponge regulated by circRNAs, indicating the roles of circRNAs in regulating muscle growth-related genes expression. Overall, these findings will not only broaden our understanding of circRNAs regulation mechanisms underlying muscle growth and development in LMB but also provides a novel clue for further functional research in carnivorous fish genetic breeding.

Identification and characterization of long non-coding RNAs in juvenile and adult skeletal muscle of largemouth bass (Micropterus salmoides).

Long non-coding RNAs (lncRNAs) are a class of transcriptional RNA molecules, which play critical roles in diverse biological processes. However, little is known about the overall expression pattern and roles of lncRNAs in skeletal muscle of largemouth bass (LMB). Here, we constructed two skeletal muscle RNA libraries to find lncRNAs that may involve in the regulation of skeletal muscle development between juvenile and adult LMB. A total of 16,147 lncRNAs and 4611 differentially expressed lncRNAs were identified. Among these identified lncRNAs, 10 lncRNAs were randomly selected to confirm their expression by real-time qPCR both in libraries, which were consistent with the RNA sequencing results. The target mRNAs of lncRNAs were predicted for GO enrichment analysis. Results showed that these targets associated with growth and development of muscle, such as skeletal muscle fiber development, myoblast proliferation and differentiation. Importantly, correlation analysis of lncRNA-miRNA-mRNA regulatory network revealed that several lncRNAs targeted miRNAs which are closely involved in the regulation of muscle development. It is the first time to identify a number of lncRNA that correlate with skeletal muscle development in LMB. Our results not only provide a comprehensive expression profile of muscle lncRNAs in this species, but also provide a theoretical basis for further elaborating genetic regulation mechanism of muscle growth and development, and pave the way for the future molecular assisted breeding in carnivorous fishes.

A Toxin-Conjugated Recombinant Protein Targeting gp120 and gp41 for Inactivating HIV-1 Virions and Killing Latency-Reversing Agent-Reactivated Latent Cells.

Application of the combination antiretroviral therapy (cART) has reduced AIDS to a manageable chronic infectious disease. However, HIV/AIDS cannot be cured because of the presence of latent reservoirs, thus calling for the development of antiretroviral drugs that can eliminate latency-reversing agent (LRA)-activated HIV-1 virions and latent cells. In this study, we conjugated a small-molecule toxin, DM1, to a gp120-binding protein, mD1.22, a mutated CD4 domain I, and found that mD1.22-DM1 could inactivate HIV-1 virions. However, it could not kill LRA-activated latent cells. We then designed and constructed a dual-targeting protein, DL35D, by linking mD1.22 and the single-chain variable fragment (scFv) of a gp41 NHR-specific antibody, D5, with a 35-mer linker. Subsequently, we conjugated DM1 to DL35D and found that DL35D-DM1 could inhibit HIV-1 infection, inactivate HIV-1 virions, kill HIV-1-infected cells and LRA-reactivated latent cells, suggesting that this toxin-conjugated dual-targeting recombinant protein is a promising candidate for further development as a novel antiviral drug with potential for HIV functional cure. IMPORTANCE Although HIV-1 replication was successfully controlled by antiretroviral drugs, cure strategy for HIV-1/AIDS is still lacking. The long-lived HIV reservoir is considered one of the major obstacles to an HIV/AIDS cure. CD4-PE40 was the first drug that designed to kill HIV-1 infected cells; however, lower efficiency and high immunogenicity have limited its further development. In this study, we designed several dual-targeting recombinant proteins DLDs by linking gp120-binding protein mD1.22 and gp41-binding antibody D5 scFv with different length of linkers. Among them, DL35D with 35-mer linker showed the best anti-HIV-1 activity. We further conjugated the DM1 toxin to DL35D to produce DL35D-DM1, which maintained DL35D's inhibitory and inactivation activity against cell-free HIV-1 strains. Most importantly, DL35D-DM1 could specifically kill HIV-1-infected cells and LRA-reactivated-latent infected cells, suggesting that it is a proper candidate for development as a novel antiviral drug for use in combination with an LRA for HIV functional cure.

Chemically Modified Bovine ß-Lactoglobulin as a Broad-Spectrum Influenza Virus Entry Inhibitor with the Potential to Combat Influenza Outbreaks.

Frequent outbreaks of the highly pathogenic influenza A virus (AIV) infection, together with the lack of broad-spectrum influenza vaccines, call for the development of broad-spectrum prophylactic agents. Previously, 3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin (3HP-ß-LG) was proven to be effective against human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and it has also been used in the clinical control of cervical human papillomavirus (HPV) infections. Here, we show its efficacy in potently inhibiting infection by divergent influenza A and B viruses. Mechanistic studies suggest that 3HP-ß-LG binds, possibly through its negatively charged residues, to the receptor-binding domain in the hemagglutinin 1 (HA1) subunit in the HA of the influenza virus, thus inhibiting the attachment of the HA to sialic acid on host cells. The intranasal administration of 3HP-ß-LG led to the protection of mice against challenges by influenza A(H1N1)/PR8, A(H3N2), and A(H7N9) viruses. Furthermore, 3HP-ß-LG is highly stable when stored at 50 °C for 30 days and it shows excellent safety in vitro and in vivo. Collectively, our findings suggest that 3HP-ß-LG could be successfully repurposed as an intranasal prophylactic agent to prevent influenza virus infections during influenza outbreaks.

Photothermal Regulated Nanozyme of CuFeS2 Nanoparticles for Efficiently Promoting Wound Healing Infected by Multidrug Resistant Bacteria.

Peroxidase-mediated chemokinetic therapy (CDT) can effectively resist bacteria; however, factors such as the high dosage of drugs seriously limit the antibacterial effect. Herein, CuFeS2 nanoparticles (NPs) nanozyme antibacterial system with the photothermal effect and peroxidase-like catalytic activity are proposed as a combined antibacterial agent with biosafety, high-efficiency, and broad-spectrum antibacterial ability. In addition, the as-obtained CuFeS2 NPs with a low doses of Cu+ and Fe3+ can change the permeability of bacterial cell membranes and break the antioxidant balance by consuming intracellular glutathione (GSH), which results in more conducive ROS production. Meanwhile, the photothermal heating can regulate the CuFeS2 NPs close to their optimal reaction temperature (60 °C) to release more hydroxyl radical in low concentrations of H2O2 (100 µM). The proposed CuFeS2 NPs-based antibacterial system achieve more than 99% inactivation efficiency of methicillin-resistant Staphylococcus aureus (106 CFU mL-1 MRSA), hyperspectral bacteria ß-Escherichia coli (106 CFU mL-1 ESBL) and Pseudomonas aeruginosa (106 CFU mL-1 PA), even at low concentration (2 µg mL-1), which is superior to those of the conventional CuO NPs at 4 mg mL-1 reported in the literature. In vivo experiments further confirm that CuFeS2 NPs can effectively treat wounds infected by MRSA and promote the wound healing. This study demonstrates that excellent antibacterial ability and good biocompatibility make CuFeS2 NPs a potential anti-infection nanozyme with broad application prospects.

Effect of Different Adjuvants on Immune Responses Elicited by Protein-Based Subunit Vaccines against SARS-CoV-2 and Its Delta Variant.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become more serious because of the continuous emergence of variants of concern (VOC), thus calling for the development of broad-spectrum vaccines with greater efficacy. Adjuvants play important roles in enhancing the immunogenicity of protein-based subunit vaccines. In this study, we compared the effect of three adjuvants, including aluminum, nanoparticle manganese and MF59, on the immunogenicity of three protein-based COVID-19 vaccine candidates, including RBD-Fc, RBD and S-trimer. We found that the nanoparticle manganese adjuvant elicited the highest titers of SARS-CoV-2 RBD-specific IgG, IgG1 and IgG2a, as well as neutralizing antibodies against infection by pseudotyped SARS-CoV-2 and its Delta variant. What is more, the nanoparticle manganese adjuvant effectively reduced the viral load of the authentic SARS-CoV-2 and Delta variant in the cell culture supernatants. These results suggest that nanoparticle manganese, known to facilitate cGAS-STING activation, is an optimal adjuvant for protein-based COVID-19 subunit vaccines.

A Five-Helix-Based SARS-CoV-2 Fusion Inhibitor Targeting Heptad Repeat 2 Domain against SARS-CoV-2 and Its Variants of Concern.

The prolonged duration of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has resulted in the continuous emergence of variants of concern (VOC, e.g., Omicron) and variants of interest (VOI, e.g., Lambda). These variants have challenged the protective efficacy of current COVID-19 vaccines, thus calling for the development of novel therapeutics against SARS-CoV-2 and its VOCs. Here, we constructed a novel fusion inhibitor-based recombinant protein, denoted as 5-Helix, consisting of three heptad repeat 1 (HR1) and two heptad repeat 2 (HR2) fragments. The 5-Helix interacted with the HR2 domain of the viral S2 subunit, the most conserved region in spike (S) protein, to block homologous six-helix bundle (6-HB) formation between viral HR1 and HR2 domains and, hence, viral S-mediated cell-cell fusion. The 5-Helix potently inhibited infection by pseudotyped SARS-CoV-2 and its VOCs, including Delta and Omicron variants. The 5-Helix also inhibited infection by authentic SARS-CoV-2 wild-type (nCoV-SH01) strain and its Delta variant. Collectively, our findings suggest that 5-Helix can be further developed as either a therapeutic or prophylactic to treat and prevent infection by SARS-CoV-2 and its variants.

A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

A novel STING agonist-adjuvanted pan-sarbecovirus vaccine elicits potent and durable neutralizing antibody and T cell responses in mice, rabbits and NHPs.

The emergence of SARS-CoV-2 variants and potentially other highly pathogenic sarbecoviruses in the future highlights the need for pan-sarbecovirus vaccines. Here, we discovered a new STING agonist, CF501, and found that CF501-adjuvanted RBD-Fc vaccine (CF501/RBD-Fc) elicited significantly stronger neutralizing antibody (nAb) and T cell responses than Alum- and cGAMP-adjuvanted RBD-Fc in mice. Vaccination of rabbits and rhesus macaques (nonhuman primates, NHPs) with CF501/RBD-Fc elicited exceptionally potent nAb responses against SARS-CoV-2 and its nine variants and 41 S-mutants, SARS-CoV and bat SARSr-CoVs. CF501/RBD-Fc-immunized hACE2-transgenic mice were almost completely protected against SARS-CoV-2 challenge, even 6 months after the initial immunization. NHPs immunized with a single dose of CF501/RBD-Fc produced high titers of nAbs. The immunized macaques also exhibited durable humoral and cellular immune responses and showed remarkably reduced viral load in the upper and lower airways upon SARS-CoV-2 challenge even at 108 days post the final immunization. Thus, CF501/RBD-Fc can be further developed as a novel pan-sarbecovirus vaccine to combat current and future outbreaks of sarbecovirus diseases.