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.

Developments in treatment for middle east respiratory syndrome coronavirus (MERS-CoV).

INTRODUCTION: An important respiratory pathogen that has led to multiple hospital outbreaks both inside and outside of the Arabian Peninsula is the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Given the elevated case fatality rate, there exists a pressing requirement for efficacious therapeutic agents. AREAS COVERED: This is an updated review of the developments in MERS treatment approaches. Using databases like PubMed, Embase, Cochrane, Scopus, and Google Scholar, a thorough search was carried out utilizing keywords like 'MERS,' 'MERS-CoV,' and 'Middle East respiratory syndrome' in conjunction with 'treatment' or 'therapy' from Jan 2012 to Feb 2024. EXPERT OPINION: MERS-CoV is a highly pathogenic respiratory infection that emerged in 2012 and continues to pose a significant public health threat. Despite ongoing efforts to control the spread of MERS-CoV, there is currently no specific antiviral treatment available. While many agents have been tested both in vivo and in vitro, none of them have been thoroughly examined in extensive clinical trials. Only case reports, case series, or cohort studies have been made available as clinical studies. However, there is a limited number of randomized-controlled trials. Because cases are irregular and sporadic, conducting a large prospective randomized trials for establishing an efficacious treatment might be difficult.

The effectiveness of intervention measures on MERS-CoV transmission by using the contact networks reconstructed from link prediction data.

Introduction: Mitigating the spread of infectious diseases is of paramount concern for societal safety, necessitating the development of effective intervention measures. Epidemic simulation is widely used to evaluate the efficacy of such measures, but realistic simulation environments are crucial for meaningful insights. Despite the common use of contact-tracing data to construct realistic networks, they have inherent limitations. This study explores reconstructing simulation networks using link prediction methods as an alternative approach. Methods: The primary objective of this study is to assess the effectiveness of intervention measures on the reconstructed network, focusing on the 2015 MERS-CoV outbreak in South Korea. Contact-tracing data were acquired, and simulation networks were reconstructed using the graph autoencoder (GAE)-based link prediction method. A scale-free (SF) network was employed for comparison purposes. Epidemic simulations were conducted to evaluate three intervention strategies: Mass Quarantine (MQ), Isolation, and Isolation combined with Acquaintance Quarantine (AQ + Isolation). Results: Simulation results showed that AQ + Isolation was the most effective intervention on the GAE network, resulting in consistent epidemic curves due to high clustering coefficients. Conversely, MQ and AQ + Isolation were highly effective on the SF network, attributed to its low clustering coefficient and intervention sensitivity. Isolation alone exhibited reduced effectiveness. These findings emphasize the significant impact of network structure on intervention outcomes and suggest a potential overestimation of effectiveness in SF networks. Additionally, they highlight the complementary use of link prediction methods. Discussion: This innovative methodology provides inspiration for enhancing simulation environments in future endeavors. It also offers valuable insights for informing public health decision-making processes, emphasizing the importance of realistic simulation environments and the potential of link prediction methods.

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.

DNA vaccine prime and replicating vaccinia vaccine boost induce robust humoral and cellular immune responses against MERS-CoV in mice.

As of December 2022, 2603 laboratory-identified Middle East respiratory syndrome coronavirus (MERS-CoV) infections and 935 associated deaths, with a mortality rate of 36%, had been reported to the World Health Organization (WHO). However, there are still no vaccines for MERS-CoV, which makes the prevention and control of MERS-CoV difficult. In this study, we generated two DNA vaccine candidates by integrating MERS-CoV Spike (S) gene into a replicating Vaccinia Tian Tan (VTT) vector. Compared to homologous immunization with either vaccine, mice immunized with DNA vaccine prime and VTT vaccine boost exhibited much stronger and durable humoral and cellular immune responses. The immunized mice produced robust binding antibodies and broad neutralizing antibodies against the EMC2012, England1 and KNIH strains of MERS-CoV. Prime-Boost immunization also induced strong MERS-S specific T cells responses, with high memory and poly-functional (CD107a-IFN-γ-TNF-α) effector CD8+ T cells. In conclusion, the research demonstrated that DNA-Prime/VTT-Boost strategy could elicit robust and balanced humoral and cellular immune responses against MERS-CoV-S. This study not only provides a promising set of MERS-CoV vaccine candidates, but also proposes a heterologous sequential immunization strategy worthy of further development.

Quantitative assay to analyze neutralization and inhibition of authentic Middle East respiratory syndrome coronavirus.

To date, there is no licensed vaccine for Middle East respiratory syndrome coronavirus (MERS-CoV). Therefore, MERS-CoV is one of the diseases targeted by the Coalition for Epidemic Preparedness Innovations (CEPI) vaccine development programs and has been classified as a priority disease by the World Health Organization (WHO). An important measure of vaccine immunogenicity and antibody functionality is the detection of virus-neutralizing antibodies. We have developed and optimized a microneutralization assay (MNA) using authentic MERS-CoV and standardized automatic counting of virus foci. Compared to our standard virus neutralization assay, the MNA showed improved sensitivity when analyzing 30 human sera with good correlation of results (Spearman's correlation coefficient r = 0.8917, p value < 0.0001). It is important to use standardized materials, such as the WHO international standard (IS) for anti-MERS-CoV immunoglobulin G, to compare the results from clinical trials worldwide. Therefore, in addition to the neutralizing titers (NT50 = 1384, NT80 = 384), we determined the IC50 and IC80 of WHO IS in our MNA to be 0.67 IU/ml and 2.6 IU/ml, respectively. Overall, the established MNA is well suited to reliably quantify vaccine-induced neutralizing antibodies with high sensitivity.

Cross-Reactive Antibody Responses to Coronaviruses Elicited by SARS-CoV-2 Infection or Vaccination.

BACKGROUND: The newly emerged SARS-CoV-2 possesses shared antigenic epitopes with other human coronaviruses. We investigated if COVID-19 vaccination or SARS-CoV-2 infection may boost cross-reactive antibodies to other human coronaviruses. METHODS: Prevaccination and postvaccination sera from SARS-CoV-2 naïve healthy subjects who received three doses of the mRNA vaccine (BioNTech, BNT) or the inactivated vaccine (CoronaVac, CV) were used to monitor the level of cross-reactive antibodies raised against other human coronaviruses by enzyme-linked immunosorbent assay. In comparison, convalescent sera from COVID-19 patients with or without prior vaccination history were also tested. Pseudoparticle neutralization assay was performed to detect neutralization antibody against MERS-CoV. RESULTS: Among SARS-CoV-2 infection-naïve subjects, BNT or CV significantly increased the anti-S2 antibodies against Betacoronaviruses (OC43 and MERS-CoV) but not Alphacoronaviruses (229E). The prevaccination antibody response to the common cold human coronaviruses did not negatively impact the postvaccination antibody response to SARS-CoV-2. Cross-reactive antibodies that binds to the S2 protein of MERS-CoV were similarly detected from the convalescent sera of COVID-19 patients with or without vaccination history. However, these anti-S2 antibodies do not possess neutralizing activity in MERS-CoV pseudoparticle neutralization tests. CONCLUSIONS: Our results suggest that SARS-CoV-2 infection or vaccination may potentially modulate population immune landscape against previously exposed or novel human coronaviruses. The findings have implications for future sero-epidemiological studies on MERS-CoV.

Nucleic acid vaccine candidates encapsulated with mesoporous silica nanoparticles against MERS-CoV.

Middle East respiratory coronavirus (MERS-CoV) is a newly emergent, highly pathogenic coronavirus that is associated with 34% mortality rate. MERS-CoV remains listed as priority pathogen by the WHO. Since its discovery in 2012 and despite the efforts to develop coronaviruses vaccines to fight against SARS-CoV-2, there are currently no MERS-CoV vaccine that has been approved. Therefore, there is high demand to continue on the development of prophylactic vaccines against MERS-CoV. Current advancements in vaccine developments can be adapted for the development of improved MERS-CoV vaccines candidates. Nucleic acid-based vaccines, including pDNA and mRNA, are relatively new class of vaccine platforms. In this work, we developed pDNA and mRNA vaccine candidates expressing S.FL gene of MERS-CoV. Further, we synthesized a silane functionalized hierarchical aluminosilicate to encapsulate each vaccine candidates. We tested the nucleic acid vaccine candidates in mice and evaluated humoral antibodies response. Interestingly, we determined that the non-encapsulated, codon optimized S.FL pDNA vaccine candidate elicited the highest level of antibody responses against S.FL and S1 of MERS-CoV. Encapsulation of mRNA with nanoporous aluminosilicate increased the humoral antibody responses, whereas encapsulation of pDNA did not. These findings suggests that MERS-CoV S.FL pDNA vaccine candidate induced the highest level of humoral responses. This study will enhance further optimization of nanosilica as potential carrier for mRNA vaccines. In conclusion, this study suggests MERS-CoV pDNA vaccine candidate as a suitable vaccine platform for further pivotal preclinical testings.

A dual-targeting approach using a human bispecific antibody against the receptor-binding domain of the Middle East Respiratory Syndrome Coronavirus.

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has posed a significant global health concern due to its severe respiratory illness and high fatality rate. Currently, despite the potential for resurgence, there are no specific treatments for MERS-CoV, and only supportive care is available. Our study aimed to address this therapeutic gap by developing a potent neutralizing bispecific antibody (bsAb) against MERS-CoV. Initially, we isolated four human monoclonal antibodies (mAbs) that specifically target the MERS-CoV receptor-binding domain (RBD) using phage display technology and an established human antibody library. Among these four selected mAbs, our intensive in vitro functional analyses showed that the MERS-CoV RBD-specific mAb K111.3 exhibited the most potent neutralizing activity against MERS-CoV pseudoviral infection and the molecular interaction between MERS-CoV RBD and human dipeptidyl peptidase 4. Consequently, we engineered a novel bsAb, K207.C, by utilizing K111.3 as the IgG base and fusing it with the single-chain variable fragment of its non-competing pair, K111.1. This engineered bsAb showed significantly enhanced neutralization potential against MERS-CoV compared to its parental mAb. These findings suggest that K207.C may serve as a potential candidate for effective MERS-CoV neutralization, further highlighting the promise of the bsAb dual-targeting approach in MERS-CoV neutralization.

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

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.