Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS.

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

Contributions of single-particle cryoelectron microscopy toward fighting COVID-19.

Single-particle cryoelectron microscopy (cryo-EM), whose full capabilities have been realized only within the past decade, has had a pivotal role in the fight against COVID-19. This is due to the technique's intrinsic power to depict both structural and dynamic features of molecules; in this case, of the spike protein of SARS-CoV-2. By now, numerous cryo-EM studies have furthered our understanding of spike protein-angiotensin-converting enzyme 2 (ACE2) receptor interactions, which has informed the design of effective vaccines, and have enabled the characterization of neutralizing antibody binding sites, which will lead to the design of novel therapeutics as the virus evolves.

Infection of primary nasal epithelial cells differentiates among lethal and seasonal human coronaviruses.

The nasal epithelium is the initial entry portal and primary barrier to infection by all human coronaviruses (HCoVs). We utilize primary human nasal epithelial cells grown at air-liquid interface, which recapitulate the heterogeneous cellular population as well as mucociliary clearance functions of the in vivo nasal epithelium, to compare lethal [Severe acute respiratory syndrome (SARS)-CoV-2 and Middle East respiratory syndrome-CoV (MERS-CoV)] and seasonal (HCoV-NL63 and HCoV-229E) HCoVs. All four HCoVs replicate productively in nasal cultures, though replication is differentially modulated by temperature. Infections conducted at 33 °C vs. 37 °C (reflective of temperatures in the upper and lower airway, respectively) revealed that replication of both seasonal HCoVs (HCoV-NL63 and -229E) is significantly attenuated at 37 °C. In contrast, SARS-CoV-2 and MERS-CoV replicate at both temperatures, though SARS-CoV-2 replication is enhanced at 33 °C late in infection. These HCoVs also diverge significantly in terms of cytotoxicity induced following infection, as the seasonal HCoVs as well as SARS-CoV-2 cause cellular cytotoxicity as well as epithelial barrier disruption, while MERS-CoV does not. Treatment of nasal cultures with type 2 cytokine IL-13 to mimic asthmatic airways differentially impacts HCoV receptor availability as well as replication. MERS-CoV receptor DPP4 expression increases with IL-13 treatment, whereas ACE2, the receptor used by SARS-CoV-2 and HCoV-NL63, is down-regulated. IL-13 treatment enhances MERS-CoV and HCoV-229E replication but reduces that of SARS-CoV-2 and HCoV-NL63, reflecting the impact of IL-13 on HCoV receptor availability. This study highlights diversity among HCoVs during infection of the nasal epithelium, which is likely to influence downstream infection outcomes such as disease severity and transmissibility.

Acquisition of a multibasic cleavage site does not increase MERS-CoV entry into Calu-3 human lung cells.

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.IMPORTANCEThe highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human-human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.

Mutation of a highly conserved isoleucine residue in loop 2 of several ß-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental for replication.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in non-structural protein 3. Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the glycine-isoleucine-phenylalanine motif. While we previously demonstrated the importance of the glycine residue for CoV replication and pathogenesis, the impact of the isoleucine and phenylalanine residues remains unknown. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that correlated with attenuated replication and pathogenesis of F-A mutant MERS-CoV and SARS-CoV-2 viruses in cell culture and mice. In contrast, the I-A mutant proteins had normal enzyme activity and enhanced ADP-ribose binding. Despite only demonstrating increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 viruses were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. IMPORTANCE: The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-ß promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-ß promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-ß expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-ß production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways­interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)­activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung­derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Identification of a Spike-Specific CD8+ T-Cell Epitope Following Vaccination Against the Middle East Respiratory Syndrome Coronavirus in Humans.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8+ T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8+ T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15).

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

A structural analysis of the nsp9 protein from the coronavirus MERS CoV reveals a conserved RNA binding interface.

Middle East respiratory syndrome coronavirus (MERS CoV) and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) are RNA viruses from the Betacoronavirus family that cause serious respiratory illness in humans. One of the conserved non-structural proteins encoded for by the coronavirus family is non-structural protein 9 (nsp9). Nsp9 plays an important role in the RNA capping process of the viral genome, where it is covalently linked to viral RNA (known as RNAylation) by the conserved viral polymerase, nsp12. Nsp9 also directly binds to RNA; we have recently revealed a distinct RNA recognition interface in the SARS CoV-2 nsp9 by using a combination of nuclear magnetic resonance spectroscopy and biolayer interferometry. In this study, we have utilized a similar methodology to determine a structural model of RNA binding of the related MERS CoV. Based on these data, we uncover important similarities and differences to SARS CoV-2 nsp9 and other coronavirus nsp9 proteins. Our findings that replacing key RNA binding residues in MERS CoV nsp9 affects RNAylation efficiency indicate that recognition of RNA may play a role in the capping process of the virus.

Biphasic MERS-CoV Incidence in Nomadic Dromedaries with Putative Transmission to Humans, Kenya, 2022-2023.

Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in dromedaries in Africa, but camel-to-human transmission is limited. Sustained 12-month sampling of dromedaries in a Kenya abattoir hub showed biphasic MERS-CoV incidence; peak detections occurred in October 2022 and February 2023. Dromedary-exposed abattoir workers (7/48) had serologic signs of previous MERS-CoV exposure.

Structural basis of main proteases of MERS-CoV bound to antineoplastic drug carmofur.

Recurrent epidemics of coronaviruses have posed significant threats to human life and health. The mortality rate of patients infected with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is 35 %. The main protease (Mpro) plays a crucial role in the MERS-CoV life cycle, and Mpro exhibited a high degree of conservation among different coronaviruses. Therefore inhibition of Mpro has become an effective strategy for the development of broad-spectrum anti-coronaviral drugs. The inhibition of SARS-CoV-2 Mpro by the anti-tumor drug carmofur has been revealed, but structural studies of carmofur in complex with Mpro from other types of coronavirus have not been reported. Hence, we revealed the structure of the MERS-CoV Mpro-carmofur complex, analysed the structural basis for the binding of carmofur to MERS-CoV Mpro in detail, and compared the binding patterns of carmofur to Mpros of two different coronaviruses, MERS-CoV and SARS-CoV-2. Considering the importance of Mpros for coronavirus therapy, structural understanding of Mpro inhibition by carmofur could contribute to the design and development of novel antiviral drugs with safe and broad-spectrum efficacy.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

One for all-human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses.

In vitro investigations of host-virus interactions are reliant on suitable cell and tissue culture models. Results are only as good as the model they are generated in. However, choosing cell models for in vitro work often depends on availability and previous use alone. Despite the vast increase in coronavirus research over the past few years, scientists are still heavily reliant on: non-human, highly heterogeneous or not fully differentiated, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors. Complex primary or stem cell models are highly representative of human tissues but are expensive and time-consuming to develop and maintain with limited suitability for high-throughput experiments.Using tissue-specific expression patterns, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the well-characterized human kidney cell line Caki-1 for infection with three human coronaviruses (hCoVs): Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus and Alphacoronavirus hCoV 229E. Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. A panel of 26 custom antibodies shows the location of SARS-CoV-2 proteins during replication using immunocytochemistry. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison for a broad range of respiratory viruses. IMPORTANCE Cell lines remain the backbone of virus research, but results are only as good as their originating model. Despite increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models of: non-human origin, incomplete differentiation, or lacking active interferon responses. We identified the human kidney Caki-1 cell line as a potential target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show the localization of 26 SARS-CoV-2 proteins in Caki-1 cells during natural replication and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.

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.

Antibody-dependent enhancement (ADE) of SARS-CoV-2 in patients exposed to MERS-CoV and SARS-CoV-2 antigens.

This study evaluated the potential for antibody-dependent enhancement (ADE) in serum samples from patients exposed to Middle East respiratory syndrome coronavirus (MERS-CoV). Furthermore, we evaluated the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination on ADE in individuals with a MERS infection history. We performed ADE assay in sera from MERS recovered and SARS-CoV-2-vaccinated individuals using BHK cells expressing FcgRIIa, SARS-CoV-2, and MERS-CoV pseudoviruses (PVs). Further, we analyzed the association of ADE to serum IgG levels and neutralization. Out of 16 MERS patients, nine demonstrated ADE against SARS-CoV-2 PV, however, none of the samples demonstrated ADE against MERS-CoV PV. Furthermore, out of the seven patients exposed to SARS-CoV-2 vaccination after MERS-CoV infection, only one patient (acutely infected with MERS-CoV) showed ADE for SARS-CoV-2 PV. Further analysis indicated that IgG1, IgG2, and IgG3 against SARS-CoV-2 S1 and RBD subunits, IgG1 and IgG2 against the MERS-CoV S1 subunit, and serum neutralizing activity were low in ADE-positive samples. In summary, samples from MERS-CoV-infected patients exhibited ADE against SARS-CoV-2 and was significantly associated with low levels of neutralizing antibodies. Subsequent exposure to SARS-CoV-2 vaccination resulted in diminished ADE activity while the PV neutralization assay demonstrated a broadly reactive antibody response in some patient samples.

Recombination analysis on the receptor switching event of MERS-CoV and its close relatives: implications for the emergence of MERS-CoV.

BACKGROUND: PlMERS-CoV is a coronavirus known to cause severe disease in humans, taxonomically classified under the subgenus Merbecovirus. Recent findings showed that the close relatives of MERS-CoV infecting vespertillionid bats (family Vespertillionidae), named NeoCoV and PDF-2180, use their hosts' ACE2 as their entry receptor, unlike the DPP4 receptor usage of MERS-CoV. Previous research suggests that this difference in receptor usage between these related viruses is a result of recombination. However, the precise location of the recombination breakpoints and the details of the recombination event leading to the change of receptor usage remain unclear. METHODS: We used maximum likelihood-based phylogenetics and genetic similarity comparisons to characterise the evolutionary history of all complete Merbecovirus genome sequences. Recombination events were detected by multiple computational methods implemented in the recombination detection program. To verify the influence of recombination, we inferred the phylogenetic relation of the merbecovirus genomes excluding recombinant segments and that of the viruses' receptor binding domains and examined the level of congruency between the phylogenies. Finally, the geographic distribution of the genomes was inspected to identify the possible location where the recombination event occurred. RESULTS: Similarity plot analysis and the recombination-partitioned phylogenetic inference showed that MERS-CoV is highly similar to NeoCoV (and PDF-2180) across its whole genome except for the spike-encoding region. This is confirmed to be due to recombination by confidently detecting a recombination event between the proximal ancestor of MERS-CoV and a currently unsampled merbecovirus clade. Notably, the upstream recombination breakpoint was detected in the N-terminal domain and the downstream breakpoint at the S2 subunit of spike, indicating that the acquired recombined fragment includes the receptor-binding domain. A tanglegram comparison further confirmed that the receptor binding domain-encoding region of MERS-CoV was acquired via recombination. Geographic mapping analysis on sampling sites suggests the possibility that the recombination event occurred in Africa. CONCLUSION: Together, our results suggest that recombination can lead to receptor switching of merbecoviruses during circulation in bats. These results are useful for future epidemiological assessments and surveillance to understand the spillover risk of bat coronaviruses to the human population.