Broad-spectrum coronavirus neutralization induced by hetero RBD-Fc protein vaccine.

In the landscape of infectious diseases, human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 pose significant threats, characterized by severe respiratory illnesses and notable resistance to conventional treatments due to their rapid evolution and the emergence of diverse variants, particularly within SARS-CoV-2. This study investigated the development of broad-spectrum coronavirus vaccines using heterodimeric RBD-Fc proteins engineered through the "Knob-into-Hole" technique. We constructed various recombinant proteins incorporating the receptor-binding domains (RBDs) of different coronaviruses. Heterodimers combining RBDs from SARS-CoV-2 with those of SARS-CoV or MERS-CoV elicited superior neutralizing responses compared to homodimeric proteins in murine models. Additionally, heterotetrameric proteins, specifically D614G_Delta/BA.1_XBB.1.5-RBD and MERS_D614G/BA.1_XBB.1.5-RBD, elicited remarkable breadth and potency in neutralizing all known SARS-CoV-2 variants, SARS-CoV, related sarbecoviruses like GD-Pangolin and WIV1, and even MERS-CoV pseudoviruses. Furthermore, these heterotetrameric proteins also demonstrated enhanced cellular immune responses. These findings underscore the potential of recombinant hetero proteins as a universal vaccine strategy against current and future coronavirus threats.

Pan-beta-coronavirus subunit vaccine prevents SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV challenge.

Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, belonging to the genus beta-CoV, have caused outbreaks or pandemics. SARS-CoV-2 has evolved into many variants with increased resistance to the current vaccines. Spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are important vaccine targets; however, the RBD of the SARS-CoV-2 Omicron variant is highly mutated, rending neutralizing antibodies elicited by ancestral-based vaccines targeting this region ineffective, emphasizing the need for effective vaccines with broad-spectrum efficacy against SARS-CoV-2 variants and other CoVs with pandemic potential. This study describes a pan-beta-CoV subunit vaccine, Om-S-MERS-RBD, by fusing the conserved and highly potent RBD of MERS-CoV into an RBD-truncated SARS-CoV-2 Omicron S protein, and evaluates its neutralizing immunogenicity and protective efficacy in mouse models. Om-S-MERS-RBD formed a conformational structure, maintained effective functionality and antigenicity, and bind efficiently to MERS-CoV receptor, human dipeptidyl peptidase 4, and MERS-CoV RBD or SARS-CoV-2 S-specific antibodies. Immunization of mice with Om-S-MERS-RBD and adjuvants (Alum plus monophosphoryl lipid A) induced broadly neutralizing antibodies against pseudotyped MERS-CoV, SARS-CoV, and SARS-CoV-2 original strain, as well as T-cell responses specific to RBD-truncated Omicron S protein. Moreover, the neutralizing activity against SARS-CoV-2 Omicron subvariants was effectively improved after priming with an Omicron-S-RBD protein. Adjuvanted Om-S-MERS-RBD protein protected mice against challenge with SARS-CoV-2 Omicron variant, MERS-CoV, and SARS-CoV, significantly reducing viral titers in the lungs. Overall, these findings indicated that Om-S-MERS-RBD protein could develop as an effective universal subunit vaccine to prevent infections with MERS-CoV, SARS-CoV, SARS-CoV-2, and its variants. IMPORTANCE: Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential.

Interaction between SARS-CoV PBM and Cellular PDZ Domains Leading to Virus Virulence.

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer.

Structural characteristics of BtKY72 RBD bound to bat ACE2 reveal multiple key residues affecting ACE2 usage of sarbecoviruses.

Two different sarbecoviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2, have caused serious challenges to public health. Certain sarbecoviruses utilize angiotensin-converting enzyme 2 (ACE2) as their cellular receptor, whereas some do not, speculatively due to the two deletions in their receptor-binding domain (RBD). However, it remains unclear whether sarbecoviruses with one deletion in the RBD can still bind to ACE2. Here, we showed that two phylogenetically related sarbecoviruses with one deletion, BtKY72 and BM48-31, displayed a different ACE2-usage range. The cryo-electron microscopy structure of BtKY72 RBD bound to bat ACE2 identified a key residue important for the interaction between RBD and ACE2. In addition, we demonstrated that the mutations involving four types of core residues enabled the sarbecoviruses with deletion(s) to bind to human ACE2 (hACE2) and broadened the ACE2 usage of SARS-CoV-2. Our findings help predict the potential hACE2-binding ability to emerge sarbecoviruses and develop pan-sarbecovirus therapeutic agents. IMPORTANCE: Many sarbecoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), possess the ability to bind to receptor angiotensin-converting enzyme 2 (ACE2) through their receptor-binding domain (RBD). However, certain sarbecoviruses with deletion(s) in the RBD lack this capability. In this study, we investigated two closely related short-deletion sarbecoviruses, BtKY72 and BM48-31, and revealed that BtKY72 exhibited a broader ACE2-binding spectrum compared to BM48-31. Structural analysis of the BtKY72 RBD-bat ACE2 complex identifies a critical residue at position 493 contributing to these differences. Furthermore, we demonstrated that the mutations involving four core residues in the RBD enabled the sarbecoviruses with deletion(s) to bind to human ACE2 and expanded the ACE2 usage spectra of SARS-CoV-2. These findings offer crucial insights for accurately predicting the potential threat of newly emerging sarbecoviruses to human health.

A glimpse into viral warfare: decoding the intriguing role of highly pathogenic coronavirus proteins in apoptosis regulation.

Coronaviruses employ various strategies for survival, among which the activation of endogenous or exogenous apoptosis stands out, with viral proteins playing a pivotal role. Notably, highly pathogenic coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV exhibit a greater array of non-structural proteins compared to low-pathogenic strains, facilitating their ability to induce apoptosis via multiple pathways. Moreover, these viral proteins are adept at dampening host immune responses, thereby bolstering viral replication and persistence. This review delves into the intricate interplay between highly pathogenic coronaviruses and apoptosis, systematically elucidating the molecular mechanisms underpinning apoptosis induction by viral proteins. Furthermore, it explores the potential therapeutic avenues stemming from apoptosis inhibition as antiviral agents and the utilization of apoptosis-inducing viral proteins as therapeutic modalities. These insights not only shed light on viral pathogenesis but also offer novel perspectives for cancer therapy.

Crystal structures of coronaviral main proteases in complex with the non-covalent inhibitor X77.

Main proteases (Mpros) are a class of conserved cysteine hydrolases among coronaviruses and play a crucial role in viral replication. Therefore, Mpros are ideal targets for the development of pan-coronavirus drugs. X77, previously developed against SARS-CoV Mpro, was repurposed as a non-covalent tight binder inhibitor against SARS-CoV-2 Mpro during COVID-19 pandemic. Many novel inhibitors with favorable efficacy have been discovered using X77 as a reference, suggesting that X77 could be a valuable scaffold for drug design. However, the broad-spectrum performance of X77 and underlying mechanism remain less understood. Here, we reported the crystal structures of Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV, and several Mpro mutants from SARS-CoV-2 variants bound to X77. A detailed analysis of these structures revealed key structural determinants essential for interaction and elucidated the binding modes of X77 with different coronaviral Mpros. The potencies of X77 against these investigated Mpros were further evaluated through molecular dynamic simulation and binding free energy calculation. These data provide molecular insights into broad-spectrum inhibition against coronaviral Mpros by X77 and the similarities and differences of X77 when bound to various Mpros, which will promote X77-based design of novel antivirals with broad-spectrum efficacy against different coronaviruses and SARS-CoV-2 variants.

Structural determinants of spike infectivity in bat SARS-like coronaviruses RsSHC014 and WIV1.

The recurrent spillovers of coronaviruses (CoVs) have posed severe threats to public health and the global economy. Bat severe acute respiratory syndrome (SARS)-like CoVs RsSHC014 and WIV1, currently circulating in bat populations, are poised for human emergence. The trimeric spike (S) glycoprotein, responsible for receptor recognition and membrane fusion, plays a critical role in cross-species transmission and infection. Here, we determined the cryo-electron microscopy (EM) structures of the RsSHC014 S protein in the closed state at 2.9 Å, the WIV1 S protein in the closed state at 2.8 Å, and the intermediate state at 4.0 Å. In the intermediate state, one receptor-binding domain (RBD) is in the "down" position, while the other two RBDs exhibit poor density. We also resolved the complex structure of the WIV1 S protein bound to human ACE2 (hACE2) at 4.5 Å, which provides structural basis for the future emergence of WIV1 in humans. Through biochemical experiments, we found that despite strong binding affinities between the RBDs and both human and civet ACE2, the pseudoviruses of RsSHC014, but not WIV1, failed to infect 293T cells overexpressing either human or civet ACE2. Mutagenesis analysis revealed that the Y623H substitution, located in the SD2 region, significantly improved the cell entry efficiency of RsSHC014 pseudoviruses, which is likely accomplished by promoting the open conformation of spike glycoproteins. Our findings emphasize the necessity of both efficient RBD lifting and tight RBD-hACE2 binding for viral infection and underscore the significance of the 623 site of the spike glycoprotein for the infectivity of bat SARS-like CoVs. IMPORTANCE: The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.

The binding and structural basis of fox ACE2 to RBDs from different sarbecoviruses.

Foxes are susceptible to SARS-CoV-2 in laboratory settings, and there have also been reports of natural infections of both SARS-CoV and SARS-CoV-2 in foxes. In this study, we assessed the binding capacities of fox ACE2 to important sarbecoviruses, including SARS-CoV, SARS-CoV-2, and animal-origin SARS-CoV-2 related viruses. Our findings demonstrated that fox ACE2 exhibits broad binding capabilities to receptor-binding domains (RBDs) of sarbecoviruses. We further determined the cryo-EM structures of fox ACE2 complexed with RBDs of SARS-CoV, SARS-CoV-2 prototype (PT), and Omicron BF.7. Through structural analysis, we identified that the K417 mutation can weaken the ability of SARS-CoV-2 sub-variants to bind to fox ACE2, thereby reducing the susceptibility of foxes to SARS-CoV-2 sub-variants. In addition, the Y498 residue in the SARS-CoV RBD plays a crucial role in forming a vital cation-π interaction with K353 in the fox ACE2 receptor. This interaction is the primary determinant for the higher affinity of the SARS-CoV RBD compared to that of the SARS-CoV-2 PT RBD. These results indicate that foxes serve as potential hosts for numerous sarbecoviruses, highlighting the critical importance of surveillance efforts.

Individual virome analysis reveals the general co-infection of mammal-associated viruses with SARS-related coronaviruses in bats.

Bats are the natural reservoir hosts for SARS-related coronavirus (SARSr-CoV) and other highly pathogenic microorganisms. Therefore, it is conceivable that an individual bat may harbor multiple microbes. However, there is limited knowledge on the overall co-circulation of microorganisms in bats. Here, we conducted a 16-year monitoring of bat viruses in south and central China and identified 238 SARSr-CoV positive samples across nine bat species from ten provinces or administrative districts. Among these, 76 individual samples were selected for further metagenomics analysis. We found a complex microenvironment characterized by the general co-circulation of microbes from two different sources: mammal-associated viruses or environment-associated microbes. The later includes commensal bacteria, enterobacteria-related phages, and insect or fungal viruses of food origin. Results showed that 25% (19/76) of the samples contained at least one another mammal-associated virus, notably alphacoronaviruses (13/76) such as AlphaCoV/YN2012, HKU2-related CoV and AlphaCoV/Rf-HuB2013, along with viruses from other families. Notably, we observed three viruses co-circulating within a single bat, comprising two coronavirus species and one picornavirus. Our analysis also revealed the potential presence of pathogenic bacteria or fungi in bats. Furthermore, we obtained 25 viral genomes from the 76 bat SARSr-CoV positive samples, some of which formed new evolutionary lineages. Collectively, our study reveals the complex microenvironment of bat microbiome, facilitating deeper investigations into their pathogenic potential and the likelihood of cross-species transmission.

Nonconserved epitopes dominate reverse preexisting T cell immunity in COVID-19 convalescents.

The herd immunity against SARS-CoV-2 is continuously consolidated across the world during the ongoing pandemic. However, the potential function of the nonconserved epitopes in the reverse preexisting cross-reactivity induced by SARS-CoV-2 to other human coronaviruses is not well explored. In our research, we assessed T cell responses to both conserved and nonconserved peptides shared by SARS-CoV-2 and SARS-CoV, identifying cross-reactive CD8+ T cell epitopes using enzyme-linked immunospot and intracellular cytokine staining assays. Then, in vitro refolding and circular dichroism were performed to evaluate the thermal stability of the HLA/peptide complexes. Lastly, single-cell T cell receptor reservoir was analyzed based on tetramer staining. Here, we discovered that cross-reactive T cells targeting SARS-CoV were present in individuals who had recovered from COVID-19, and identified SARS-CoV-2 CD8+ T cell epitopes spanning the major structural antigens. T cell responses induced by the nonconserved peptides between SARS-CoV-2 and SARS-CoV were higher and played a dominant role in the cross-reactivity in COVID-19 convalescents. Cross-T cell reactivity was also observed within the identified series of CD8+ T cell epitopes. For representative immunodominant peptide pairs, although the HLA binding capacities for peptides from SARS-CoV-2 and SARS-CoV were similar, the TCR repertoires recognizing these peptides were distinct. Our results could provide beneficial information for the development of peptide-based universal vaccines against coronaviruses.

Sarbecovirus disease susceptibility is conserved across viral and host models.

Coronaviruses have caused three severe epidemics since the start of the 21st century: SARS, MERS and COVID-19. The severity of the ongoing COVID-19 pandemic and increasing likelihood of future coronavirus outbreaks motivates greater understanding of factors leading to severe coronavirus disease. We screened ten strains from the Collaborative Cross mouse genetic reference panel and identified strains CC006/TauUnc (CC006) and CC044/Unc (CC044) as coronavirus-susceptible and resistant, respectively, as indicated by variable weight loss and lung congestion scores four days post-infection. We generated a genetic mapping population of 755 CC006xCC044 F2 mice and exposed the mice to one of three genetically distinct mouse-adapted coronaviruses: clade 1a SARS-CoV MA15 (n=391), clade 1b SARS-CoV-2 MA10 (n=274), and clade 2 HKU3-CoV MA (n=90). Quantitative trait loci (QTL) mapping in SARS-CoV MA15- and SARS-CoV-2 MA10-infected F2 mice identified genetic loci associated with disease severity. Specifically, we identified seven loci associated with variation in outcome following infection with either virus, including one, HrS43, that is present in both groups. Three of these QTL, including HrS43, were also associated with HKU3-CoV MA outcome. HrS43 overlaps with a QTL previously reported by our lab that is associated with SARS-CoV MA15 outcome in CC011xCC074 F2 mice and is also syntenic with a human chromosomal region associated with severe COVID-19 outcomes in humans GWAS. The results reported here provide: (a) additional support for the involvement of this locus in SARS-CoV MA15 infection, (b) the first conclusive evidence that this locus is associated with susceptibility across the Sarbecovirus subgenus, and (c) demonstration of the relevance of mouse models in the study of coronavirus disease susceptibility in humans.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

Simultaneous and ultrafast detection of pan-SARS-coronaviruses and influenza A/B viruses by a novel multiplex real-time RT-PCR assay.

Here we report an ultrafast quadruplex RT-qPCR assay with robust diagnostic ability to detect and distinguish pan-SARS-CoVs and influenza A/B viruses within 35 min. This quadruplex RT-qPCR assay comprised of one novel RNA-based internal control targeting human ß2-microglobulin (B2M) for process accuracy and three newly-designed primers-probe sets targeting the envelope protein (E) of pan-SARS-CoV, matrix protein (MP) of influenza A virus and non-structural (NS) region of influenza B virus. This quadruplex assay exhibited a sensitivity comparable to its singleplex counterparts and a slightly higher to that of the Centers for Disease Control and Prevention-recommended SARS-CoV-2 and influenza A/B assays. The novel assay showed no false-positive amplifications with other common respiratory viruses, and its 95 % limits of detection for pan-SARS-CoV and influenza A/B virus was 4.26-4.52 copies/reaction. Moreover, the assay was reproducible with less than 1 % coefficient of variation and adaptable testing different clinical and environmental samples. Our ultrafast quadruplex RT-qPCR assay can serve as an attractive tool for effective differentiation of influenza A/B virus and SARS-CoV-2, but more importantly prognose the reemergence/emergence of SARS and novel coronaviruses or influenza viruses from animal spillover.

Natural evidence of coronaviral 2'-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding.

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2'-O-MTase activity is essential for efficient viral replication and immune evasion. However, the K-D-K-E catalytic motif of 2'-O-MTase is highly conserved across numerous viruses, including flaviviruses, vaccinia viruses, coronaviruses, and extends even to mammals. Here, we observed a stronger 2'-O-MTase activity in SARS-CoV-2 compared to SARS-CoV, despite the presence of a consistently active catalytic center. We further identified critical residues (Leu-36, Asn-138 and Ile-153) which served as determinants of discrepancy in 2'-O-MTase activity between SARS-CoV-2 and SARS-CoV. These residues significantly enhanced the RNA binding affinity of 2'-O-MTase and boosted its versatility toward RNA substrates. Of interest, a triple substitution (Leu36 → Ile36, Asn138 → His138, Ile153 → Leu153, from SARS-CoV-2 to SARS-CoV) within nsp16 resulted in a proportional reduction in viral 2'-O-methylation and impaired viral replication. Furthermore, it led to a significant upregulation of type I interferon (IFN-I) and proinflammatory cytokines both in vitro and vivo, relying on the cooperative sensing of melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). In conclusion, our findings demonstrated that alterations in residues other than K-D-K-E of 2'-O-MTase may affect viral replication and subsequently influence pathogenesis. Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

Characterization of nsp1 Binding to the Viral RNA Leader Sequence of Severe Acute Respiratory Syndrome Coronavirus.

Nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus (SCOV1 and SCOV2) acts as a host shutoff protein by blocking the translation of host mRNAs and triggering their decay. Surprisingly, viral RNA, which resembles host mRNAs containing a 5'-cap and a 3'-poly(A) tail, escapes significant translation inhibition and RNA decay, aiding viral propagation. Current literature proposes that, in SCOV2, nsp1 binds the viral RNA leader sequence, and the interaction may serve to distinguish viral RNA from host mRNA. However, a direct binding between SCOV1 nsp1 and the corresponding RNA leader sequence has not been established yet. Here, we show that SCOV1 nsp1 binds to the SCOV1 RNA leader sequence but forms multiple complexes at a high concentration of nsp1. These complexes are marginally different from complexes formed with SCOV2 nsp1. Finally, mutations of the RNA stem-loop did not completely abolish RNA binding by nsp1, suggesting that an RNA secondary structure is more important for binding than the sequence itself. Understanding the nature of binding of nsp1 to viral RNA will allow us to understand how this viral protein selectively suppresses host gene expression.

Variation in structural motifs within SARS-related coronavirus spike proteins.

SARS-CoV-2 is the third known coronavirus (CoV) that has crossed the animal-human barrier in the last two decades. However, little structural information exists related to the close genetic species within the SARS-related coronaviruses. Here, we present three novel SARS-related CoV spike protein structures solved by single particle cryo-electron microscopy analysis derived from bat (bat SL-CoV WIV1) and civet (cCoV-SZ3, cCoV-007) hosts. We report complex glycan trees that decorate the glycoproteins and density for water molecules which facilitated modeling of the water molecule coordination networks within structurally important regions. We note structural conservation of the fatty acid binding pocket and presence of a linoleic acid molecule which are associated with stabilization of the receptor binding domains in the "down" conformation. Additionally, the N-terminal biliverdin binding pocket is occupied by a density in all the structures. Finally, we analyzed structural differences in a loop of the receptor binding motif between coronaviruses known to infect humans and the animal coronaviruses described in this study, which regulate binding to the human angiotensin converting enzyme 2 receptor. This study offers a structural framework to evaluate the close relatives of SARS-CoV-2, the ability to inform pandemic prevention, and aid in the development of pan-neutralizing treatments.

Therapeutic nanobodies against SARS-CoV-2 and other pathogenic human coronaviruses.

Nanobodies, single-domain antibodies derived from variable domain of camelid or shark heavy-chain antibodies, have unique properties with small size, strong binding affinity, easy construction in versatile formats, high neutralizing activity, protective efficacy, and manufactural capacity on a large-scale. Nanobodies have been arisen as an effective research tool for development of nanobiotechnologies with a variety of applications. Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, have caused serious outbreaks or a global pandemic, and continue to post a threat to public health worldwide. The viral spike (S) protein and its cognate receptor-binding domain (RBD), which initiate viral entry and play a critical role in virus pathogenesis, are important therapeutic targets. This review describes pathogenic human CoVs, including viral structures and proteins, and S protein-mediated viral entry process. It also summarizes recent advances in development of nanobodies targeting these CoVs, focusing on those targeting the S protein and RBD. Finally, we discuss potential strategies to improve the efficacy of nanobodies against emerging SARS-CoV-2 variants and other CoVs with pandemic potential. It will provide important information for rational design and evaluation of therapeutic agents against emerging and reemerging pathogens.

Bat species assemblage predicts coronavirus prevalence.

Anthropogenic disturbances and the subsequent loss of biodiversity are altering species abundances and communities. Since species vary in their pathogen competence, spatio-temporal changes in host assemblages may lead to changes in disease dynamics. We explore how longitudinal changes in bat species assemblages affect the disease dynamics of coronaviruses (CoVs) in more than 2300 cave-dwelling bats captured over two years from five caves in Ghana. This reveals uneven CoV infection patterns between closely related species, with the alpha-CoV 229E-like and SARS-related beta-CoV 2b emerging as multi-host pathogens. Prevalence and infection likelihood for both phylogenetically distinct CoVs is influenced by the abundance of competent species and naïve subadults. Broadly, bat species vary in CoV competence, and highly competent species are more common in less diverse communities, leading to increased CoV prevalence in less diverse bat assemblages. In line with the One Health framework, our work supports the notion that biodiversity conservation may be the most proactive measure to prevent the spread of pathogens with zoonotic potential.

A Cocktail of Lipid Nanoparticle-mRNA Vaccines Broaden Immune Responses against ß-Coronaviruses in a Murine Model.

Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging ß-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-ß-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-ß-coronaviruses. Overall, these results present a potential pan-ß-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

pH inactivation of SARS-CoV-2 and SARS-CoV in virus spiked protein A eluates from a mAb purification process.

Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses. Small scale virus clearance studies are routinely performed using model enveloped viruses such as murine leukemia virus to assess inactivation at the pH range used in the downstream manufacturing process. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide and this can also inactivate viruses. The susceptibility of SARS-CoV-2 and SARS-CoV to low pH conditions using protein A eluate derived material from a monoclonal antibody production process as well as high pH cleaning conditions was addressed. SARS-CoV-2 was effectively inactivated at pH 3.0, moderately inactivated at pH 3.4, but not inactivated at pH 3.8. Low pH was less effective at inactivating SARS-CoV. Both viruses were inactivated at a high pH of ca.13.4. These studies provide important information regarding the effectiveness of viral clearance and inactivation steps of novel coronaviruses when compared to other enveloped viruses.