A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection.

SARS-CoV-2 has mutated frequently since its first emergence in 2019. Numerous variants, including the currently emerging Omicron variant, have demonstrated high transmissibility or increased disease severity, posing serious threats to global public health. This study describes the identification of an immunodominant non-neutralizing epitope on SARS-CoV-2 receptor-binding domain (RBD). A subunit vaccine against this mutant RBD, constructed by masking this epitope with a glycan probe, did not significantly affect RBD's receptor-binding affinity or antibody-binding affinity, or its ability to induce antibody production. However, this vaccine enhanced the neutralizing activity of this RBD and its protective efficacy in immunized mice. Specifically, this vaccine elicited significantly higher-titer neutralizing antibodies than the prototypic RBD protein against Alpha (B.1.1.7 lineage), Beta (B.1.351 lineage), Gamma (P.1 lineage), and Epsilon (B.1.427 or B.1.429 lineage) variant pseudoviruses containing single or combined mutations in the spike (S) protein, albeit the neutralizing antibody titers against some variants were slightly lower than against original SARS-CoV-2. This vaccine also significantly improved the neutralizing activity of the prototypic RBD against pseudotyped and authentic Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants, although the neutralizing antibody titers were lower than against original SARS-CoV-2. In contrast to the prototypic RBD, the mutant RBD completely protected human ACE2 (hACE2)-transgenic mice from lethal challenge with a prototype SARS-CoV-2 strain and a Delta variant without weight loss. Overall, these findings indicate that this RBD vaccine has broad-spectrum activity against multiple SARS-CoV-2 variants, as well as the potential to be effective and have improved efficacy against Omicron and other pandemic variants. IMPORTANCE Several SARS-CoV-2 variants have shown increased transmissibility, calling for a need to develop effective vaccines with broadly neutralizing activity against multiple variants. This study identified a non-neutralizing epitope on the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, and further shielded it with a glycan probe. A subunit vaccine based on this mutant RBD significantly enhanced the ability of prototypic RBD against multiple SARS-CoV-2 variants, including the Delta and Omicron strains, although the neutralizing antibody titers against some of these variants were lower than those against original SARS-CoV-2. This mutant vaccine also enhanced the protective efficacy of the prototypic RBD vaccine against SARS-CoV-2 infection in immunized animals. In conclusion, this study identified an engineered RBD vaccine against Omicron and other SARS-CoV-2 variants that induced stronger neutralizing antibodies and protection than the original RBD vaccine. It also highlights the need to improve the effectiveness of current COVID-19 vaccines to prevent pandemic SARS-CoV-2 variants.

Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.

A gossypol derivative effectively protects against Zika and dengue virus infection without toxicity.

BACKGROUND: Zika virus (ZIKV) and dengue virus (DENV) cause microcephaly and dengue hemorrhagic fever, respectively, leading to severe problems. No effective antiviral agents are approved against infections of these flaviviruses, calling for the need to develop potent therapeutics. We previously identified gossypol as an effective inhibitor against ZIKV and DENV infections, but this compound is toxic and not suitable for in vivo treatment. RESULTS: In this study, we showed that gossypol derivative ST087010 exhibited potent and broad-spectrum in vitro inhibitory activity against infections of at least ten ZIKV strains isolated from different hosts, time periods, and countries, as well as DENV-1-4 serotypes, and significantly reduced cytotoxicity compared to gossypol. It presented broad-spectrum in vivo protective efficacy, protecting ZIKV-infected Ifnar1-/- mice from lethal challenge, with increased survival and reduced weight loss. Ifnar1-/- mice treated with this gossypol derivative decreased viral titers in various tissues, including the brain and testis, after infection with ZIKV at different human isolates. Moreover, ST087010 potently blocked ZIKV vertical transmission in pregnant Ifnar1-/- mice, preventing ZIKV-caused fetal death, and it was safe for pregnant mice and their pups. It also protected DENV-2-challenged Ifnar1-/- mice against viral replication by reducing the viral titers in the brain, kidney, heart, and sera. CONCLUSIONS: Overall, our data indicate the potential for further development of this gossypol derivative as an effective and safe broad-spectrum therapeutic agent to treat ZIKV and DENV diseases.

Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction.

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.

Molecular Mechanism for Antibody-Dependent Enhancement of Coronavirus Entry.

Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.IMPORTANCE Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.

Rational Design of Zika Virus Subunit Vaccine with Enhanced Efficacy.

Zika virus (ZIKV) infection in pregnant women can lead to fetal deaths and malformations. We have previously reported that ZIKV envelope protein domain III (EDIII) is a subunit vaccine candidate with cross-neutralization activity; however, like many other subunit vaccines, its efficacy is limited. To improve the efficacy of this subunit vaccine, we identified a nonneutralizing epitope on ZIKV EDIII surrounding residue 375, which is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We then shielded this epitope with an engineered glycan probe. Compared to the wild-type EDIII, the mutant EDIII induced significantly stronger neutralizing antibodies in three mouse strains and also demonstrated significantly improved efficacy by fully protecting mice, particularly pregnant mice and their fetuses, against high-dose lethal ZIKV challenge. Moreover, the mutant EDIII immune sera significantly enhanced the passive protective efficacy by fully protecting mice against lethal ZIKV challenge; this passive protection was positively associated with neutralizing antibody titers. We further showed that the enhanced efficacy of the mutant EDIII was due to the shielding of the immunodominant nonneutralizing epitope surrounding residue 375, which led to immune refocusing on the neutralizing epitopes. Taken together, the results of this study reveal that an intrinsic limitation of subunit vaccines is their artificially exposed immunodominant nonneutralizing epitopes, which can be overcome through glycan shielding. Additionally, the mutant ZIKV protein generated in this study is a promising subunit vaccine candidate with high efficacy in preventing ZIKV infections in mice.IMPORTANCE Viral subunit vaccines generally show low efficacy. In this study, we revealed an intrinsic limitation of subunit vaccine designs: artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections.

Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State

Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at 3.3-Å resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, an N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and betacoronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alphacoronavirus S1-CTDs, but its structure differs from that of betacoronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the prefusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that deltacoronavirus spikes are structurally and evolutionally more closely related to alphacoronavirus spikes than to betacoronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by deltacoronaviruses as well as coronaviruses in general. IMPORTANCE In this study, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at a 3.3-Å resolution. This is the first atomic structure of a spike protein from the deltacoronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and betacoronavirus spike proteins. Here, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of PdCoV spike protein and provides many insights into its receptor recognition, membrane fusion, and immune evasion.

A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV.

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. Nanobodies (Nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. In this study, we developed a novel neutralizing Nb (NbMS10) and its human-Fc-fused version (NbMS10-Fc), both of which target the MERS-CoV spike protein receptor-binding domain (RBD). We further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against MERS-CoV infection. Both Nbs can be expressed in yeasts with high yield, bind to MERS-CoV RBD with high affinity, and block the binding of MERS-CoV RBD to the MERS-CoV receptor. The binding site of the Nbs on the RBD was mapped to be around residue Asp539, which is part of a conserved conformational epitope at the receptor-binding interface. NbMS10 and NbMS10-Fc maintained strong cross-neutralizing activity against divergent MERS-CoV strains isolated from humans and camels. Particularly, NbMS10-Fc had significantly extended half-life in vivo; a single-dose treatment of NbMS10-Fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal MERS-CoV challenge. Overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum Nbs against MERS-CoV and has produced Nbs with great potentials as anti-MERS-CoV therapeutics.IMPORTANCE Therapeutic development is critical for preventing and treating continual MERS-CoV infections in humans and camels. Because of their small size, nanobodies (Nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). Here, we have developed novel Nbs that specifically target the receptor-binding domain (RBD) of MERS-CoV spike protein. They bind to a conserved site on MERS-CoV RBD with high affinity, blocking RBD's binding to MERS-CoV receptor. Through engineering a C-terminal human Fc tag, the in vivo half-life of the Nbs is significantly extended. Moreover, the Nbs can potently cross-neutralize the infections of diverse MERS-CoV strains isolated from humans and camels. The Fc-tagged Nb also completely protects humanized mice from lethal MERS-CoV challenge. Taken together, our study has discovered novel Nbs that hold promise as potent, cost-effective, and broad-spectrum anti-MERS-CoV therapeutic agents.

Recombinant Receptor-Binding Domains of Multiple Middle East Respiratory Syndrome Coronaviruses (MERS-CoVs) Induce Cross-Neutralizing Antibodies against Divergent Human and Camel MERS-CoVs and Antibody Escape Mutants.

Middle East respiratory syndrome coronavirus (MERS-CoV) binds to cellular receptor dipeptidyl peptidase 4 (DPP4) via the spike (S) protein receptor-binding domain (RBD). The RBD contains critical neutralizing epitopes and serves as an important vaccine target. Since RBD mutations occur in different MERS-CoV isolates and antibody escape mutants, cross-neutralization of divergent MERS-CoV strains by RBD-induced antibodies remains unknown. Here, we constructed four recombinant RBD (rRBD) proteins with single or multiple mutations detected in representative human MERS-CoV strains from the 2012, 2013, 2014, and 2015 outbreaks, respectively, and one rRBD protein with multiple changes derived from camel MERS-CoV strains. Like the RBD of prototype EMC2012 (EMC-RBD), all five RBDs maintained good antigenicity and functionality, the ability to bind RBD-specific neutralizing monoclonal antibodies (MAbs) and the DPP4 receptor, and high immunogenicity, able to elicit S-specific antibodies. They induced potent neutralizing antibodies cross-neutralizing 17 MERS pseudoviruses expressing S proteins of representative human and camel MERS-CoV strains identified during the 2012-2015 outbreaks, 5 MAb escape MERS-CoV mutants, and 2 live human MERS-CoV strains. We then constructed two RBDs mutated in multiple key residues in the receptor-binding motif (RBM) of RBD and demonstrated their strong cross-reactivity with anti-EMC-RBD antibodies. These RBD mutants with diminished DPP4 binding also led to virus attenuation, suggesting that immunoevasion after RBD immunization is accompanied by loss of viral fitness. Therefore, this study demonstrates that MERS-CoV RBD is an important vaccine target able to induce highly potent and broad-spectrum neutralizing antibodies against infection by divergent circulating human and camel MERS-CoV strains. IMPORTANCE: MERS-CoV was first identified in June 2012 and has since spread in humans and camels. Mutations in its spike (S) protein receptor-binding domain (RBD), a key vaccine target, have been identified, raising concerns over the efficacy of RBD-based MERS vaccines against circulating human and camel MERS-CoV strains. Here, we constructed five vaccine candidates, designated 2012-RBD, 2013-RBD, 2014-RBD, 2015-RBD, and Camel-RBD, containing single or multiple mutations in the RBD of representative human and camel MERS-CoV strains during the 2012-2015 outbreaks. These RBD-based vaccine candidates maintained good functionality, antigenicity, and immunogenicity, and they induced strong cross-neutralizing antibodies against infection by divergent pseudotyped and live MERS-CoV strains, as well as antibody escape MERS-CoV mutants. This study provides impetus for further development of a safe, highly effective, and broad-spectrum RBD-based subunit vaccine to prevent MERS-CoV infection.

A T cell-based SARS-CoV-2 spike protein vaccine provides protection without antibodies.

SARS-CoV-2 spike-based vaccines are used to control the COVID-19 pandemic. However, emerging variants have become resistant to antibody neutralization and further mutations may lead to full resistance. We tested whether T cells alone could provide protection without antibodies. We designed a T cell-based vaccine in which SARS-CoV-2 spike sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies, but strong specific T cell responses. We challenged mice with SARS-CoV-2 wild-type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by the SARS-CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with the SARS-CoV-2 wild-type strain or the Omicron variant were significantly reduced. Importantly, depletion of CD4+ or CD8+ T cells led to significant loss of the protection. Our analyses of spike protein sequences of the variants indicated that fewer than one-third presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in the subunit 1 region. As the subunit 2 region is conservative, the vaccines targeting spike protein are expected to protect against future variants due to the T cell responses.

MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection.

Middle East respiratory syndrome coronavirus (MERS-CoV), a highly pathogenic coronavirus in the same Betacoronavirus genus and Coronaviridae family as SARS-CoV-2, continues to post a threat to human health. Mortality remains high; therefore, there is a need to develop effective vaccines to prevent MERS-CoV infection. The receptor-binding domain (RBD) within the MERS-CoV spike (S) protein is a critical vaccine target. The latest mRNA technology has enabled rapid development of much-needed vaccines with high efficiency and scalable manufacturing capacity. Here, we designed a mRNA vaccine encoding the RBD of MERS-CoV S protein (RBD-mRNA) and evaluated its immunogenicity and protective efficacy in a mouse model. The data showed that nucleoside-modified RBD-mRNA, but not RBD-mRNA lacking the nucleoside modification, was stable and elicited broadly and durable neutralizing antibody and cellular immune responses, which neutralized the original strain and multiple MERS-CoV variants. Among all immunization routes tested, the intradermal route was appropriate for this RBD-mRNA to induce strong B-cell responses and the highest neutralizing antibody titers. Importantly, injection of nucleoside-modified RBD-mRNA through the intradermal route protected immunized mice against challenge with MERS-CoV. This protection correlated with serum neutralizing antibody titers. Overall, we have developed an effective MERS-CoV RBD-based mRNA vaccine (with potential for further development) that prevents infection by divergent strains of MERS-CoV.

An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBDbeta. This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

Inhalation delivery of dexamethasone with iSEND nanoparticles attenuates the COVID-19 cytokine storm in mice and nonhuman primates.

Dexamethasone (DEX) is the first drug to show life-saving efficacy in patients with severe coronavirus disease 2019 (COVID-19), while DEX is associated with serious adverse effects. Here, we report an inhaled, Self-immunoregulatory, Extracellular Nanovesicle-based Delivery (iSEND) system by engineering neutrophil nanovesicles with cholesterols to deliver DEX for enhanced treatment of COVID-19. Relying on surface chemokine and cytokine receptors, the iSEND showed improved targeting to macrophages and neutralized broad-spectrum cytokines. The nanoDEX, made by encapsulating DEX with the iSEND, efficiently promoted the anti-inflammation effect of DEX in an acute pneumonia mouse model and suppressed DEX-induced bone density reduction in an osteoporosis rat model. Relative to an intravenous administration of DEX at 0.1 milligram per kilogram, a 10-fold lower dose of nanoDEX administered by inhalation produced even better effects against lung inflammation and injury in severe acute respiratory syndrome coronavirus 2-challenged nonhuman primates. Our work presents a safe and robust inhalation delivery platform for COVID-19 and other respiratory diseases.

A lung-selective delivery of mRNA encoding broadly neutralizing antibody against SARS-CoV-2 infection.

The respiratory system, especially the lung, is the key site of pathological injury induced by SARS-CoV-2 infection. Given the low feasibility of targeted delivery of antibodies into the lungs by intravenous administration and the short half-life period of antibodies in the lungs by intranasal or aerosolized immunization, mRNA encoding broadly neutralizing antibodies with lung-targeting capability can perfectly provide high-titer antibodies in lungs to prevent the SARS-CoV-2 infection. Here, we firstly identify a human monoclonal antibody, 8-9D, with broad neutralizing potency against SARS-CoV-2 variants. The neutralization mechanism of this antibody is explained by the structural characteristics of 8-9D Fabs in complex with the Omicron BA.5 spike. In addition, we evaluate the efficacy of 8-9D using a safe and robust mRNA delivery platform and compare the performance of 8-9D when its mRNA is and is not selectively delivered to the lungs. The lung-selective delivery of the 8-9D mRNA enables the expression of neutralizing antibodies in the lungs which blocks the invasion of the virus, thus effectively protecting female K18-hACE2 transgenic mice from challenge with the Beta or Omicron BA.1 variant. Our work underscores the potential application of lung-selective mRNA antibodies in the prevention and treatment of infections caused by circulating SARS-CoV-2 variants.

Inactivated vaccine-elicited potent antibodies can broadly neutralize SARS-CoV-2 circulating variants.

A full understanding of the inactivated COVID-19 vaccine-mediated antibody responses to SARS-CoV-2 circulating variants will inform vaccine effectiveness and vaccination development strategies. Here, we offer insights into the inactivated vaccine-induced antibody responses after prime-boost vaccination at both the polyclonal and monoclonal levels. We characterized the VDJ sequence of 118 monoclonal antibodies (mAbs) and found that 20 neutralizing mAbs showed varied potency and breadth against a range of variants including XBB.1.5, BQ.1.1, and BN.1. Bispecific antibodies (bsAbs) based on nonoverlapping mAbs exhibited enhanced neutralizing potency and breadth against the most antibody-evasive strains, such as XBB.1.5, BQ.1.1, and BN.1. The passive transfer of mAbs or their bsAb effectively protected female hACE2 transgenic mice from challenge with an infectious Delta or Omicron BA.2 variant. The neutralization mechanisms of these antibodies were determined by structural characterization. Overall, a broad spectrum of potent and distinct neutralizing antibodies can be induced in individuals immunized with the SARS-CoV-2 inactivated vaccine BBIBP-CorV, suggesting the application potential of inactivated vaccines and these antibodies for preventing infection by SARS-CoV-2 circulating variants.

Advances in mRNA and other vaccines against MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus (CoV). Belonging to the same beta-CoV genus as severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) and SARS-CoV-2, MERS-CoV has a significantly higher fatality rate with limited human-to-human transmissibility. MERS-CoV causes sporadic outbreaks, but no vaccines have yet been approved for use in humans, thus calling for continued efforts to develop effective vaccines against this important CoV. Similar to SARS-CoV-1 and SARS-CoV-2, MERS-CoV contains 4 structural proteins, among which the surface spike (S) protein has been used as a core component in the majority of currently developed MERS-CoV vaccines. Here, we illustrate the importance of the MERS-CoV S protein as a key vaccine target and provide an update on the currently developed MERS-CoV vaccines, including those based on DNAs, proteins, virus-like particles or nanoparticles, and viral vectors. Additionally, we describe approaches for designing MERS-CoV mRNA vaccines and explore the role and importance of naturally occurring pseudo-nucleosides in the design of effective MERS-CoV mRNA vaccines. This review also provides useful insights into designing and evaluating mRNA vaccines against other viral pathogens.

RBD-mRNA vaccine induces broadly neutralizing antibodies against Omicron and multiple other variants and protects mice from SARS-CoV-2 challenge.

Multiple SARS-CoV-2 variants are identified with higher rates of transmissibility or greater disease severity. Particularly, recent emergence of Omicron variant with rapid human-to-human transmission posts new challenges to the current prevention strategies. In this study, following vaccination with an mRNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD-mRNA), we detected serum antibodies that neutralized pseudoviruses expressing spike (S) protein harboring single or multiple mutations, as well as authentic SARS-CoV-2 variants, and evaluated its protection against SARS-CoV-2 infection. The vaccine induced durable antibodies that potently neutralized prototypic strain and B.1.1.7 lineage variant pseudoviruses containing N501Y or D614G mutations alone or in combination with a N439K mutation (B.1.258 lineage), with a L452R mutation (B.1.427 or B.1.429 lineage), or a L452R-E484Q double mutation (B.1.617.1 variant), although neutralizing activity against B.1.1.7 lineage variant containing 10 amino acid changes in the S protein was slightly reduced. The RBD-mRNA-induced antibodies exerted moderate neutralization against authentic B.1.617.2 and B.1.1.529 variants, and pseudotyped B.1.351 and P.1 lineage variants containing K417N/T, E484K, and N501Y mutations, the B.1.617.2 lineage variant harboring L452R, T478K, and P681R mutations, and the B.1.1.529 lineage variant containing 38 mutations in the S protein. Particularly, RBD-mRNA vaccine completely protected mice from challenge with a virulent mouse-adapted SARS-CoV-2 variant. Among these lineages, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 belong to Alpha, Beta, Gamma, Delta, and Omicron variants, respectively. Our observations reveal that RBD-mRNA vaccine is promising and highlights the need to design novel vaccines with improved neutralization against current and future pandemic SARS-CoV-2 variants.

Re-burying Artificially Exposed Surface of Viral Subunit Vaccines Through Oligomerization Enhances Vaccine Efficacy.

Viral subunit vaccines often suffer low efficacy. We recently showed that when taken out of the context of whole virus particles, recombinant subunit vaccines contain artificially exposed surface regions that are non-neutralizing and reduce their efficacy, and thus these regions need to be re-buried in vaccine design. Here we used the envelope protein domain III (EDIII) of Japanese encephalitis virus (JEV), a subunit vaccine candidate, to further validate this important concept for subunit vaccine designs. We constructed monomeric EDIII, dimeric EDIII via a linear space, dimeric EDIII via an Fc tag, and trimeric EDIII via a foldon tag. Compared to monomeric EDIII or linearly linked dimeric EDIII, tightly packed EDIII oligomers via the Fc or foldon tag induce higher neutralizing antibody titers in mice and also protect mice more effectively from lethal JEV challenge. Structural analyses demonstrate that part of the artificially exposed surface areas on recombinant EDIII becomes re-buried in Fc or foldon-mediated oligomers. This study further establishes the artificially exposed surfaces as an intrinsic limitation of subunit vaccines, and suggests that re-burying these surfaces through tightly packed oligomerization is a convenient and effective approach to overcome this limitation.

A Retinol Derivative Inhibits SARS-CoV-2 Infection by Interrupting Spike-Mediated Cellular Entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all-trans retinoic acid (ATRA), a vitamin A (retinol) derivative, showed potent antiviral activity against all SARS-CoV-2 variants in both human cell lines and human organoids of the lower respiratory tract. Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses. IMPORTANCE Retinoids, a group of compounds including vitamin A and its active metabolite all-trans retinoic acid (ATRA), regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis. The ATRA analogues reported to date include more than 4,000 natural and synthetic molecules that are structurally and/or functionally related to ATRA. Here, we found that ATRA showed potent antiviral activity against all SARS-CoV-2 variants by directly binding in a deep hydrophobic pocket of the receptor binding domain (RBD) located on top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the "down" RBDs and locks most of the S trimers in an RBD "all-down" and ACE2-inaccessible inhibitory conformation, suggesting the pharmacological feasibility of using ATRA or its derivatives as a remedy for and prevention of COVID-19 disease.

Infection and chronic disease activate a brain-muscle signaling axis that regulates muscle performance.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show a number of non-neural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood, so we developed three models to investigate the impact of neuroinflammation on muscle performance. We found that bacterial infection, COVID-like viral infection, and expression of a neurotoxic protein associated with Alzheimer' s disease promoted the accumulation of reactive oxygen species (ROS) in the brain. Excessive ROS induces the expression of the cytokine Unpaired 3 (Upd3) in insects, or its orthologue IL-6 in mammals, and CNS-derived Upd3/IL-6 activates the JAK/Stat pathway in skeletal muscle. In response to JAK/Stat signaling, mitochondrial function is impaired and muscle performance is reduced. Our work uncovers a brain-muscle signaling axis in which infections and chronic diseases induce cytokine-dependent changes in muscle performance, suggesting IL-6 could be a therapeutic target to treat muscle weakness caused by neuroinflammation.